Shaker cognate l: Biological Overview | References
Gene name - Shaker cognate l
Cytological map position - 76B7-76B8
Function - voltage-gated potassium channel
Keywords - neuromuscular junction, A-type K+ channels regulate action potential waveform, back-propagation and firing frequency, Eliminating Shal invokes Kruppel-dependent homeostatic rebalancing of ion channel gene expression including enhanced slo, Shab, and Shaker, photoperiod, contributes to currents in the motoneurons, locomotion, SIDL regulates LL-motif-dependent targeting of K(+) channels, shal and shaker, are reciprocally, transcriptionally coupled to maintain A-type channel expression
Symbol - Shal
FlyBase ID: FBgn0005564
Genetic map position - chr3L:19,566,851-19,584,568
NCBI classification - Ion channel; BTB_POZ
Cellular location - surface transmembrane
|Recent literature||Gur, B., Sporar, K., Lopez-Behling, A. and Silies, M. (2019). Distinct expression of potassium channels regulates visual response properties of lamina neurons in Drosophila melanogaster. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. PubMed ID: 31823004
The computational organization of sensory systems depends on the diversification of individual cell types with distinct signal-processing capabilities. The Drosophila visual system, for instance, splits information into channels with different temporal properties directly downstream of photoreceptors in the first-order interneurons of the OFF pathway, L2 and L3. However, the biophysical mechanisms that determine this specialization are largely unknown. This study shows that the voltage-gated Ka channels Shaker and Shal contribute to the response properties of the major OFF pathway input L2. L3 calcium response kinetics postsynaptic to photoreceptors resemble the sustained calcium signals of photoreceptors, whereas L2 neurons decay transiently. Based on a cell-type-specific RNA-seq data set and endogenous protein tagging, this study identified Shaker and Shal as the primary candidates to shape L2 responses. Using in vivo two-photon imaging of L2 calcium signals in combination with pharmacological and genetic perturbations of these Ka channels, it was shown that the wild-type Shaker and Shal function is to enhance L2 responses and cell-autonomously sharpen L2 kinetics. These results reveal a role for Ka channels in determining the signal-processing characteristics of a specific cell type in the visual system.
|Werner, J., Arian, J., Bernhardt, I., Ryglewski, S. and Duch, C. (2020). Differential localization of voltage-gated potassium channels during Drosophila metamorphosis. J Neurogenet: 1-18. PubMed ID: 31997675
Neuronal excitability is determined by the combination of different ion channels and their sub-neuronal localization. This study utilizes protein trap fly strains with endogenously tagged channels to analyze the spatial expression patterns of the four Shaker-related voltage-gated potassium channels, Kv1-4, in the larval, pupal, and adult Drosophila ventral nerve cord. All four channels (Shaker, Kv1; Shab, Kv2; Shaw, Kv3; and Shal, Kv4) each show different spatial expression patterns in the Drosophila ventral nerve cord and are predominantly targeted to different sub-neuronal compartments. Shaker is abundantly expressed in axons, Shab also localizes to axons but mostly in commissures, Shaw expression is restricted to distinct parts of neuropils, and Shal is found somatodendritically, but also in axons of identified motoneurons. During early pupal life expression of all four Shaker-related channels is markedly decreased with an almost complete shutdown of expression at early pupal stage 5 (approximately 30% through metamorphosis). Re-expression of Kv1-4 channels at pupal stage 6 starts with abundant channel localization in neuronal somata, followed by channel targeting to the respective sub-neuronal compartments until late pupal life. The developmental time course of tagged Kv1-4 channel expression corresponds with previously published data on developmental changes in single neuron physiology, thus indicating that protein trap fly strains are a useful tool to analyze developmental regulation of potassium channel expression. This study took advantage of the large diameter of the giant fiber (GF) interneuron to map channel expression onto the axon and axon terminals of an identified interneuron. Shaker, Shaw, and Shal but not Shab channels localize to the non-myelinated GF axonal membrane and axon terminals. This study constitutes a first step toward systematically analyzing sub-neuronal potassium channel localization in Drosophila. Functional implications as well as similarities and differences to Kv1-4 channel localization in mammalian neurons are discussed.
|Eadaim, A., Hahm, E. T., Justice, E. D. and Tsunoda, S. (2020). Cholinergic Synaptic Homeostasis Is Tuned by an NFAT-Mediated alpha7 nAChR-K(v)4/Shal Coupled Regulatory System. Cell Rep 32(10): 108119. PubMed ID: 32905767
Homeostatic synaptic plasticity (HSP) involves compensatory mechanisms employed by neurons and circuits to preserve signaling when confronted with global changes in activity that may occur during physiological and pathological conditions. Cholinergic neurons, which are especially affected in some pathologies, have recently been shown to exhibit HSP mediated by nicotinic acetylcholine receptors (nAChRs). In Drosophila central neurons, pharmacological blockade of activity induces a homeostatic response mediated by the Drosophila α7 (Dα7) nAChR, which is tuned by a subsequent increase in expression of the voltage-dependent K(v)4/Shal channel. This study shows that an in vivo reduction of cholinergic signaling induces HSP mediated by Dα7 nAChRs, and this upregulation of Dα7 itself is sufficient to trigger transcriptional activation, mediated by nuclear factor of activated T cells (NFAT), of the K(v)4/Shal gene, revealing a receptor-ion channel system coupled for homeostatic tuning in cholinergic neurons.
Firing rate homeostasis (FRH) stabilizes neural activity. A pervasive and intuitive theory argues that a single variable, calcium, is detected and stabilized through regulatory feedback. A prediction is that ion channel gene mutations with equivalent effects on neuronal excitability should invoke the same homeostatic response. In agreement, this study demonstrates robust FRH following either elimination of Kv4/Shal protein or elimination of the Kv4/Shal conductance. However, the underlying homeostatic signaling mechanisms are distinct. Eliminating Shal protein invokes Kruppel-dependent rebalancing of ion channel gene expression including enhanced slo, Shab, and Shaker. By contrast, expression of these genes remains unchanged in animals harboring a CRISPR-engineered, Shal pore-blocking mutation where compensation is achieved by enhanced IKDR. These different homeostatic processes have distinct effects on homeostatic synaptic plasticity and animal behavior. It is proposed that FRH includes mechanisms of proteostatic feedback that act in parallel with activity-driven feedback, with implications for the pathophysiology of human channelopathies (Kulik, 2019).
Firing Rate Homeostasis is a form of homeostatic control that stabilizes spike rate and information coding when neurons are confronted by pharmacological, genetic or environmental perturbation. FRH has been widely documented within invertebrate neurons and neural circuits as well as the vertebrate spinal cord, cortical pyramidal neurons and cardiomyocytes. In many of these examples, the genetic deletion of an ion channel is used to induce a homeostatic response. The mechanisms of FRH correct for the loss of the ion channel and precisely restore neuronal firing properties to normal, wild-type levels). To date, little is understood about the underlying molecular mechanisms (Kulik, 2019).
FRH induced by an ion channel gene deletion is truly remarkable. The corrective response is not limited to the de novo expression of an ion channel gene with properties that are identical to the deleted channel, as might be expected for more generalized forms genetic compensation. Instead, the existing repertoire of channels expressed by a neuron can be 'rebalanced' to correct for the deletion of an ion channel. How is it possible to precisely correct for the absence of an essential voltage-gated ion channel? The complexity of the problem seems immense given that many channel types functionally cooperate to achieve the cell-type-specific voltage trajectory of an action potential (Kulik, 2019).
Theoretical work argues that different mixtures of ion channels can achieve similar firing properties in a neuron. These observations have led to a pervasive and intuitively attractive theory that a single physiological variable, calcium, is detected and stabilized through regulatory feedback control of ion channel gene expression. Yet, many questions remain unanswered. There are powerful cell biological constraints on ion channel transcription, translation, trafficking and localization in vivo. How do these constraints impact the expression of FRH? Is calcium the only intracellular variable that is sensed and controlled by homeostatic feedback? There remain few direct tests of this hypothesis. Why are homeostatic signaling systems seemingly unable to counteract disease-relevant ion channel mutations, including those that have been linked to risk for diseases such as epilepsy and autism (Kulik, 2019)?
This study has taken advantage of the molecular and genetic power of Drosophila to explore FRH in a single, genetically identified neuron subtype. Specifically, two different conditions are compared that each eliminate the Shal/Kv4 ion channel conductance and, therefore, are expected to have identical effects on neuronal excitability. Robust FRH is demonstrated following elimination of the Shal protein and, independently, by eliminating the Shal conductance using a pore blocking mutation that is knocked-in to the endogenous Shal locus. Thus, consistent with current theory, FRH can be induced by molecularly distinct perturbations to a single ion channel gene. However, these two different perturbations were found to induce different homeostatic responses, arguing for perturbation-specific effects downstream of a single ion channel gene (Kulik, 2019).
Taken together, these data contribute to a revised understanding of FRH in several ways. First, altered activity cannot be the sole determinant of FRH. Two functionally identical manipulations that eliminate the Shal conductance, each predicted to have identical effects on neuronal excitability, lead to molecularly distinct homeostatic responses. Second, homeostatic signaling systems are sensitive to the type of mutation that affects an ion channel gene. This could have implications for understanding why FRH appears to fail in the context of human disease caused by ion channel mutations, including epilepsy, migraine, autism and ataxia. Finally, the data speak to experimental and theoretical studies arguing that the entire repertoire of ion channels encoded in the genome is accessible to the mechanisms of homeostatic feedback, with a very large combinatorial solution space. The data are consistent with the existence of separable proteostatic and activity-dependent homeostatic signaling systems, potentially acting in concert to achieve cell-type-specific and perturbation-specific FRH (Kulik, 2019).
These data advance mechanistic understanding of FRH in several ways. First, it was demonstrated that FRH can be induced and fully expressed in single, genetically identified neurons. Since changes in the activity of a single motoneuron are unlikely to dramatically alter the behavior of the larvae, these data argue strongly for cell autonomous mechanisms that detect the presence of the ion channel perturbation and induce a corrective, homeostatic response. Second, this study demonstrates that FRH functions to preserve the waveform of individual action potentials. This argues for remarkable precision in the homeostatic response. Third, new evidence is provided that the transcription factor Krüppel is essential for FRH, and selectively controls the homeostatic enhancement of IKCa,the voltage-gated sodium current (INa) and the voltage-gated calcium current (ICa) without altering the baseline ion channel current. Finally, it was demonstrated that different mechanisms of FRH are induced depending upon how the Shal current is eliminated, and these differential expression mechanisms can have perturbation-specific effects on animal behavior (Kulik, 2019).
The existence is proposed of parallel homeostatic mechanisms, responsive to differential disruption of the Shal gene. Different compensatory responses were observed depending upon whether the Shal protein is eliminated or the Shal conductance is eliminated. The following evidence supports the functional equivalence of these manipulations. First, the ShalW362F mutation completely eliminates somatically recorded fast activating and inactivating potassium current IKA. Second, a dramatic reduction in IKA was demonstrated when Shal-RNAi is driven by MN1-GAL4 in a single, identified neuron. Notably, the current-voltage relationship observed for Shal-RNAi is identical to that previously published for the ShalW362F protein null mutation, being of similar size and voltage trajectory including a + 50 mV shift in voltage activation (Bergquist, 2010). This remaining, voltage-shifted, IKA-like conductance is attributed to the compensatory up-regulation of the Shaker channel on axonal membranes (Bergquist, 2010; Parrish, 2014) an effect that does not occur in the ShalW362F mutant. Thus, it seems reasonable to assume that Shal protein elimination and Shal conductance blockade initially create identical effects on neuronal excitability by eliminating Shal function. Subsequently, these perturbations trigger divergent compensatory responses. But, it is acknowledged that direct information is lacking about the immediate effects of the two perturbations (Kulik, 2019).
This study defines FRH as the restoration of neuronal firing rate in the continued presence of a perturbation. This definition is important because it necessitates that the underlying molecular mechanisms of FRH must have a quantitatively accurate ability to adjust ion channel conductances such that firing rate is precisely restored. Mechanistically, a prior example of FRH involves an evolutionarily conserved regulation of sodium channel translation by the translational repressor Pumillio. This work, originally pursued in Drosophila, was extended to mouse central neurons where it was shown that Pumilio-dependent bi-directional changes in the sodium current occur in response to altered synaptic transmission, initiated by application of either AMPA antagonist NBQX or GABA antagonist Gabazine. These data highlight the emerging diversity of molecular mechanisms that can be induced and participate in the execution of FRH (Kulik, 2019).
It is necessary to compare the current results with prior genetic studies of the Shal channel in Drosophila. A prior report, examining the effects of partial Shal knockdown in larval motoneurons, observed a trend toward an increase in the sustained potassium current, but concluded no change (Schaefer, 2010). However, the small sample size for potassium current measurements in that study (n = 3 cells) and the incomplete Shal knockdown that was achieved, likely conspired to prevent documentation of the significant increase in IKDR that was observed (IKCA was not measured in Schaefer, 2010). A second prior study examined over-expression of a pore-blocked Shal transgene in cultured Drosophila embryonic neurons, revealing elevated firing rate and a broadened action potential. This was interpreted as evidence against the existence of FRH. However, neuronal precursors were cultured from 5 hr embryos, prior to establishment of neuronal cell fate and prior to the emergence of IKA currents in vivo, which occurs ~10 hr later in development. It remains unclear whether these cultured neurons are able to achieve a clear cell identity, which may be a prerequisite for the expression of homeostatic plasticity. Another possibility concerns the time-course of FRH, which remains uncertain. Finally, over-expression of the transgene itself might interfere with the mechanisms of FRH, emphasizing the importance of the scarless, CRISPR-mediated gene knock-in approach that was employed (Kulik, 2019).
It is clear from studies in a diversity of systems that FRH can be induced by perturbations that directly alter neuronal activity without genetic or pharmacological disruption of ion channels or neurotransmitter receptors. For example, monocular deprivation induces an immediate depression of neuronal activity in the visual cortex, followed by restoration of normal firing rates. Research on the lobster stomatogastric system ranging from experiments in isolated cell culture to de-centralized ganglia have documented the existence of FRH that is consistent with an activity-dependent mechanism. It is equally clear that FRH can be induced by the deletion of an ion channel gene, including observations in systems as diverse as invertebrate and vertebrate central and peripheral neurons and muscle. But, it has remained unknown whether FRH that is induced by changes in neural activity is governed by the same signaling process that respond to ion channel gene mutations. The current data speak to this gap in knowledge (Kulik, 2019).
Changes in neural activity cannot be solely responsible for FRH. This study compared two different conditions that each completely eliminate the Shal ion channel conductance and, therefore, are expected to have identical effects on neuronal excitability. Robust FRH was demonstrated in both conditions. However, two separate mechanisms account for FRH. Shal-RNAi induces a transcription-dependent homeostatic signaling program. There is enhanced expression of Krüppel and a Krüppel-dependent increase in the expression of the slo channel gene and enhanced IKCA current. By contrast, the ShalW362F mutant does not induce a change in the expression of Krüppel, slo or any of five additional ion channel genes. Instead, a change was observed in the IKDR conductance, the origin of which has not yet been identified, but which appears to be independent of a change in ion channel gene transcription (Kulik, 2019).
The existence is proposed of two independent homeostatic signaling systems, induced by separate perturbations to the Shal channel gene. First, it is proposed that Shal-RNAi and the Shal null mutation trigger a homeostatic response that is sensitive to the absence of the Shal protein. In essence, this might represent an ion channel-specific system that achieves channel proteostasis, a system that might normally be invoked in response to errors in ion channel turnover. It is speculated that many, if not all ion channels could have such proteostatic signaling systems in place. In support of this idea, the induction of Kr is specific to loss of Shal, not occurring in eight other ion channel mutant backgrounds, each of which is sufficient to alter neural activity, including eag, para, Shaker, Shab, Shawl, slo, cac and hyperkinetic. Each of these channel mutations is well established to alter neuronal activity. But, Kr responds only to loss of Shal (Kulik, 2019).
Next, it is proposed that eliminating the Shal conductance in the ShalW362F mutant background induces a separable mechanism of FRH that is independent of ion channel transcription. While the mechanisms of this homeostatic response remain unknown, it is tempting to speculate that this mechanism is activity dependent, consistent with data from other systems. Finally, it remains possible that these homeostatic signaling systems are somehow mechanistically linked. If so, this might provide a means to achieve the precision of FRH. For example, changes in ion channel gene expression might achieve a crude re-targeting of set point firing rates, followed by engagement of activity-dependent processes that fine tune the homeostatic response. Notably, distinct, interlinked negative feedback signaling has been documented in cell biological systems, suggesting a common motif in cell biological regulation (Kulik, 2019).
An interesting prediction of this model is that activity-dependent mechanisms of FRH could be constrained by the action of the channel-specific homeostatic system. For example, loss of Shal induces a Shal-specific gene expression program and activity-dependent homeostatic signaling would be constrained to modulate the Shal-specific response. As such, the homeostatic outcome could be unique for mutations in each different ion channel gene. Given this complexity, it quickly becomes possible to understand experimental observations in non-isogenic animal populations where many different combinations of ion channels are observed to achieve similar firing rates in a given cell. The combined influence of dedicated proteostatic and activity-dependent homeostatic signaling could achieve such complexity, but with an underlying signaling architecture that is different from current theories that focus on a single calcium and activity-dependent feedback processor (Kulik, 2019).
Finally, although the existence is proposed of proteostatic feedback induced by the Shal null mutant and pan-neuronal RNAi, other possibilities certainly exist for activity-independent FRH, inclusive of mechanisms that are sensitive to channel mRNA. For example, the transcriptional compensation that was documented could be considered a more general form of 'genetic compensation'. Yet, the data differ in one important respect, when compared to prior reports of genetic compensation. In most examples of genetic compensation, gene knockouts induce compensatory expression of a closely related gene. For example, it was observed that knockout of β-actin triggers enhanced expression of other actin genes. The compensatory effects that were observed involve re-organization of the expression profiles for many, unrelated ion channel genes. Somehow, these divergent conductances are precisely adjusted to cover for the complete absence of the somato-dendritic A-type potassium conductance. Thus, a more complex form of genetic compensation is favored based upon homeostatic, negative feedback regulation (Kulik, 2019).
How does Kr-dependent control of IKCa participate in FRH? IKCa is a rapid, transient potassium current. Therefore, it makes intuitive sense that elevated IKCa could simply substitute for the loss of the fast, transient IKA current mediated by Shal. If so, this might be considered an instance of simple genetic compensation. But, if this were the case, then blocking the homeostatic increase in IKCa should lead to enhanced firing rates. This is not what was observe. Instead, average firing rates decrease when Kr is eliminated in the background of Shal-RNAi. Thus, the Kr-dependent potentiation of IKCa seems to function as a form of positive feedback, accelerating firing rate in order to achieve precise FRH, rather than simply substituting for the loss of Shal. Consistent with this possibility, acute pharmacological inhibition of IKCa decreases, rather than increases, average firing rate. However, it should also be emphasized that the role of IKCa channels in any neuron are quite complex, with context-specific effects that can either increase or decrease neuronal firing rates. Indeed, it has been argued that BK channels can serve as dynamic range compressors, dampening the activity of hyperexcitable neurons and enhancing the firing of hypoexcitable neurons. This broader interpretation is also consistent with the observed Kr-dependent increase IKCa during FRH (Kulik, 2019).
In the stomatogastic nervous system of the crab, single-cell RT-PCR has documented positive correlations between channel mRNA levels, including transcript levels for IKCa and Shal. The molecular mechanisms responsible for the observed correlations remain unknown, but it seems possible that these correlations reflect a developmental program of channel co-regulation. Upon homeostatic challenge, the steady-state positive correlations are supplanted by homeostatic compensation, notably enhanced IKCa in the presence of 4-AP. The pressing challenge is to define molecular mechanisms that cause the observed correlations and compensatory changes in ion channel expression during homeostatic plasticity. The Kr-dependent control of IKCa following loss of Shal is one such mechanism. Clearly, there is additional complexity, as highlighted by the differential response to Shal null and Shal pore blocking mutations and the pumilio-dependent control of sodium channel translation in flies and mice (Kulik, 2019).
Why do ion channel mutations frequently cause disease? If activity-dependent homeostatic signaling is the primary mechanism of FRH, then any ion channel mutation that alters channel function should be detected by changes in neural activity and firing rates restored. One possibility is that FRH is effective for correcting for an initial perturbation, but the persistent engagement of FRH might become deleterious over extended time. Alternatively, each solution could effectively correct firing rates, but have additional maladaptive consequences related to disease pathology. While this remains to be documented in disease, this study has shown that loss of Shal protein throughout the CNS causes deficits in animal behavior that are not observed in animals harboring a pore-blocking channel mutation. Indeed, if one considers that FRH can include altered expression of a BK channel, the potential for maladaptive consequences is high. Altered BK channel function has been repeatedly linked to neurological disease including idiopathic generalized epilepsy, non-kinesigenic dyskinesia and Alzheimer's disease. Thus, there are potentially deleterious ramifications of altering BK channel expression if a homeostatic signaling process is engaged throughout the complex circuitry of the central nervous system. Although the phenotype of maladaptive compensation that was observe is clear, a block in synaptic homeostasis and impaired animal motility, there is much to be learned about the underlying cause. Ultimately, defining the rules that govern FRH could open new doors toward disease therapies that address these maladaptive effects of compensatory signaling (Kulik, 2019).
As in mammals, Drosophila circadian clock neurons display rhythms of activity with higher action potential firing rates and more positive resting membrane potentials during the day. This rhythmic excitability has been widely observed but, critically, its regulation remains unresolved. This study has characterized and modeled the changes underlying these electrical activity rhythms in the lateral ventral clock neurons (LNvs). Currents mediated by the voltage-gated potassium channels Shaw (Kv3) and Shal (Kv4) oscillate in a circadian manner. Disruption of these channels, by expression of dominant negative (DN) subunits, leads to changes in circadian locomotor activity and shortens lifespan. LNv whole-cell recordings then show that changes in Shaw and Shal currents drive changes in action potential firing rate and that these rhythms are abolished when the circadian molecular clock is stopped. A whole-cell biophysical model using Hodgkin-Huxley equations can recapitulate these changes in electrical activity. Based on this model and by using dynamic clamp to manipulate clock neurons directly, it is possible to rescue the pharmacological block of Shaw and Shal, restore the firing rhythm, and thus demonstrate the critical importance of Shaw and Shal. Together, these findings point to a key role for Shaw and Shal in controlling circadian firing of clock neurons and show that changes in clock neuron currents can account for this. Moreover, with dynamic clamp it is possible to switch the LNvs between morning-like and evening-like states of electrical activity. It is concluded that changes in Shaw and Shal underlie the daily oscillation in LNv firing rate (Smith, 2019).
Sleep is highly conserved across animal species. Both wake- and sleep-promoting neurons are implicated in the regulation of wake-sleep transition at dusk in Drosophila However, little is known about how they cooperate and whether they act via different mechanisms. This study demonstrated that in female Drosophila, sleep onset was specifically delayed by blocking the Shaker cognate L channels Shal, also known as voltage-gated K(+) channel 4, Kv4) in wake-promoting cells, including large ventral lateral neurons (l-LNvs) and pars intercerebralis (PI), but not in sleep-promoting dorsal neurons (DN1s). Delayed sleep onset was also observed in males by blocking Kv4 activity in wake-promoting neurons. Electrophysiological recordings show that Kv4 channels contribute A-type currents (IA) in LNvs and PI cells, but are much less conspicuous in DN1s. Interestingly, blocking Kv4 in wake-promoting neurons preferentially increased firing rates at dusk around ZT13, when the resting membrane potentials (RMPs) and firing rates were at lower levels. Furthermore, pigment-dispersing factor (PDF) is essential for the regulation of sleep onset by Kv4 in l-LNvs, and downregulation of PDF receptor (PDFR) in PI neurons advanced sleep onset, indicating Kv4 controls sleep onset via regulating PDF/PDFR signaling in wake-promoting neurons. It is proposed that Kv4 acts as a sleep onset controller by suppressing membrane excitability in a clock-dependent manner to balance the wake-sleep transition at dusk. These results have important implications for the understanding and treatment of sleep disorders such as insomnia (Feng, 2018b).
This is the first demonstration of the function of Kv4 in sleep regulation. This study has shown that Kv4 is important for night-time sleep in Drosophila, and is especially crucial to normal sleep onset. Pan-neuronal expression of DNKv4 leads to decreased night-time sleep, indicating a general sleep-promoting function of Kv4. The increased night-time sleep in Shal495-null mutant reveals a compensatory overexpression of Shaker. A transcription factor named Kruppel (Kr) was identified to be a central regulator of this process, consistent with the conclusion that the compensatory modulation occurs at the transcriptional level. However, the Kr expression is suggested to be a result from detecting Kv4 or Shaker/Kv1 conductance, since 4-Aminopyridine (4-AP) also increased Shaker RNA expression. In this study, no significant increase was seen in Shaker RNA expression in DNKv4 mutants suggesting that other transcription factor(s), rather than Kr, may be involved in regulating Shal/Kv4 and Shaker balance (Feng, 2018b).
Previous studies have shown that cyclin A and GABAA receptors (or RDL) in circadian neurons also regulate sleep latency. A molecule named WAKE interacts with RDL, and the cycling manner of WAKE promotes excitability oscillation of l-LNvs. Moreover, DN1 neurons may regulate wake-sleep transition at dusk in a clock-dependent manner. The results verified that Kv4-mediated IA in l-LNvs, and function-loss of Kv4 preferentially upregulated membrane excitability at dusk, although Kv4 expression was not rhythmic. Moreover, resting membrane potential (RMP) values exhibit circadian oscillation in l-LNvs, with depolarized RMPs at dawn. Thus, less Kv4 channels would be available for opening when RMPs are depolarized. This supports data showing that blocking Kv4 did not significantly increased firing rate when circadian neurons were hyperexcitable at dawn. Although RMPs and firing rate also exhibit circadian oscillation in DN1s, blocking Kv4 activity has no effects on sleep latency. It was further demonstrated that this may be caused by the near absence of Kv4-mediated IA in DN1s. Thus, Kv4 controls wake-sleep transition at dusk in subsets wake-promoting cells, but not in DN1s. The phases of Ca2+ waves recorded with a fine temporal resolution were approximately coincident with membrane excitability and RMPs oscillations in l-LNvs, but with slight (∼1-2 h) delay in peaks. In this study, DNKv4 preferentially reduced excitability at dusk without causing daily excitability oscillation shifts, and presumably would reduce Ca2+ peaks as well. But how Kv4 exactly regulates intracellular Ca2+ waves and coordination of electrical excitability and Ca2+ waves need further study (Feng, 2018b).
PI exerts its effects as downstream of clock neurons and is one of the key targets (direct or indirect) of PDF neurons, although PI cells do not express molecular clock machinery. Previous studies have suggested that EGFR/ERK signal in insulin-producing PI neurons plays important roles in regulating the consolidation and maintenance of sleep in Drosophila, indicating that PI cells act as wake-promoting neurons. These results are the first to demonstrate that the neuronal activities and PDF/PDFR signaling in subsets of PI cells (50y-GAL4 driver) participate in the regulation of sleep onset (Feng, 2018b).
In this study, defects in sleep initiation were presumed to be due to the hyperactivity of specific circadian neurons caused by blocking Kv4 activity. dTrpA1 experiments further supported this notion. However, a recent study concluded that reducing the Shaker/Kv1 current would decrease, rather than increase, the action potential discharge in dFB (dorsal FB; Pimentel, 2016). Depleting Shaker from dFB neurons could shift the interspike interval distribution toward longer values, making it more difficult to generate next spike. It is supposed that the opposite effect may be due to diverse types of Kv channels and different discharge and biophysical properties of neurons. For example, Kv4 is required for maintaining excitability in cultured neurons, but not in groups of neurons from dissected brain in this study. Whether driving DNKv4 expression in other sleep-promoting or wake-promoting neurons would cause parallel or opposite effects on sleep needs to be investigated. Neurons in EB, FB, or a subset MBNs driven by 201y-GAL4 could be tested in further studies (Feng, 2018b).
Previous work has provided a strong link between circadian periods with habitual sleep timing in human, and CRY1 is suggested to be associated with a familial form of delayed sleep phase disorder. However, no significant change was detected in circadian periods by blocking Kv4 in LNvs and DN1s, indicating that delayed sleep onset in the mutants was not due to changes in circadian periods. Blocking Kv4 in PI cells disrupted rhythmic activities in most flies, and a lengthened circadian period was detected in the rhythmic ones. Because PI is a circadian output region and does not express clock machinery, it is not likely that intrinsic circadian rhythm disorder contributes to the sleep onset phenotype caused by blocking Kv4 (Feng, 2018b).
Studies have provided evidence that links types of potassium channels to sleep phenotypes in Drosophila and human. For example, insomnia has been delineated in patients with neurologic disorders of Voltage-gated K+ channels (Kv) autoimmunity (or named Kv antibody syndrome). Sleep onset insomnia, defined as inability to fall asleep at the desired time, is observed in patients with neurodegenerative diseases and psychiatric disorders. Abnormal sleep timing and pattern have also been observed in Drosophila disease models. This study may provide a potential alternative therapy of sleep onset insomnia by targeting Kv4 channels (Feng, 2018b).
Accumulation of amyloid-beta (Abeta) is widely believed to be an early event in the pathogenesis of Alzheimer's disease (AD). Kv4 is an A-type K(+) channel, and a previous report shows the degradation of Kv4, induced by the Abeta42 accumulation, may be a critical contributor to the hyperexcitability of neurons in a Drosophila AD model. This study used well-established courtship memory assay to investigate the contribution of the Kv4 channel to short-term memory (STM) deficits in the Abeta42-expressing AD model. Abeta42 over-expression in Drosophila was found to lead to age-dependent courtship STM loss, which can be also induced by driving acute Abeta42 expression post-developmentally. Interestingly, mutants with eliminated Kv4-mediated A-type K(+) currents (IA) by transgenically expressing dominant-negative subunit (DNKv4) phenocopied Abeta42 flies in defective courtship STM. Kv4 channels in mushroom body (MB) and projection neurons (PNs) were found to be required for courtship STM. Furthermore, the STM phenotypes can be rescued, at least partially, by restoration of Kv4 expression in Abeta42 flies, indicating the STM deficits could be partially caused by Kv4 degradation. In addition, IA is significantly decreased in MB neurons (MBNs) but not in PNs, suggesting Kv4 degradation in MBNs, in particular, plays a critical role in courtship STM loss in Abeta42 flies. These data highlight causal relationship between region-specific Kv4 degradation and age-dependent learning decline in the AD model, and provide a mechanism for the disturbed cognitive function in AD (Feng, 2018a).
In vertebrate neurons, the axon initial segment (AIS) is specialized for action potential initiation. It is organized by a giant 480 Kd variant of ankyrin G (AnkG) that serves as an anchor for ion channels and is required for a plasma membrane diffusion barrier that excludes somatodendritic proteins from the axon. An unusually long exon required to encode this 480Kd variant is thought to have been inserted only recently during vertebrate evolution, so the giant ankyrin-based AIS scaffold has been viewed as a vertebrate adaptation for fast, precise signaling. This study re-examined AIS evolution through phylogenomic analysis of ankyrins and by testing the role of ankyrins in proximal axon organization in a model multipolar Drosophila neuron (ddaE). Giant isoforms of ankyrin were found in all major bilaterian phyla, and evidence is presented in favor of a single common origin for giant ankyrins and the corresponding long exon in a bilaterian ancestor. This finding raises the question of whether giant ankyrin isoforms play a conserved role in AIS organization throughout the Bilateria. This possibility was examined by looking for conserved ankyrin-dependent AIS features in Drosophila ddaE neurons via live imaging. ddaE neurons were found to have an axonal diffusion barrier proximal to the cell body that requires a giant isoform of the neuronal ankyrin Ank2. Furthermore, the potassium channel shal concentrates in the proximal axon in an Ank2-dependent manner. These results indicate that the giant ankyrin-based cytoskeleton of the AIS may have evolved prior to the radiation of extant bilaterian lineages, much earlier than previously thought (Jegla, 2016).
Accumulation of amyloid-β (Aβ) is widely believed to be an early event in the pathogenesis of Alzheimer's disease (AD). Kv4 is an A-type K+ channel, and previous work has shown that degradation of Kv4, induced by the β42 accumulation, may be a critical contributor to the hyperexcitability of neurons in a Drosophila AD model. This study used well-established courtship memory assay to investigate the contribution of the Kv4 channel to short-term memory (STM) deficits in the Aβ42-expressing AD model. Aβ42 over-expression in Drosophila leads to age-dependent courtship STM loss, which can be also induced by driving acute Aβ42 expression post-developmentally. Interestingly, mutants with eliminated Kv4-mediated A-type K+ currents (IA) by transgenically expressing dominant-negative subunit (DNKv4) phenocopied Aβ42 flies in defective courtship STM. Kv4 channels in mushroom body (MB) and projection neurons (PNs) were found to be required for courtship STM. Furthermore, the STM phenotypes can be rescued, at least partially, by restoration of Kv4 expression in Aβ42 flies, indicating the STM deficits could be partially caused by Kv4 degradation. In addition, IA is significantly decreased in MB neurons (MBNs) but not in PNs, suggesting Kv4 degradation in MBNs, in particular, plays a critical role in courtship STM loss in Aβ42 flies. These data highlight causal relationship between region-specific Kv4 degradation and age-dependent learning decline in the AD model, and provide a mechanism for the disturbed cognitive function in AD (Feng, 2018).
Ion channel gene expression can vary substantially among neurons of a given type, even though neuron-type-specific firing properties remain stable and reproducible. The mechanisms that modulate ion channel gene expression and stabilize neural firing properties are unknown. In Drosophila, this study demonstrates that loss of the Shal potassium channel induces the compensatory rebalancing of ion channel expression including, but not limited to, the enhanced expression and function of Shaker and slowpoke. Using genomic and network modeling approaches combined with genetic and electrophysiological assays, this study demonstrates that the transcription factor Kruppel is necessary for the homeostatic modulation of Shaker and slowpoke expression. Remarkably, Kruppel induction is specific to the loss of Shal, not being observed in five other potassium channel mutants that cause enhanced neuronal excitability. Thus, homeostatic signaling systems responsible for rebalancing ion channel expression can be selectively induced after the loss or impairment of a specific ion channel (Parrish, 2014).
This study provides evidence that the cell fate regulator Kr is a critical player in the compensatory control of potassium channel gene expression. It is speculated that the induction of Kr drives a pattern of gene expression, first used to establish neuronal identity in the embryo and then, postembryonically, to rebalance ion channel expression in the face of persistent or acute perturbation of the Shal channel. Surprisingly, Kr is induced after the loss of Shal, but not other potassium channel gene mutations that have been shown to cause neural hyperexcitability. It is concluded that Shal function is specifically coupled to a homeostatic feedback system that includes the Kr-dependent transcriptional response. As such, these data imply the existence of discoverable 'rules' that define how individual neurons will respond to mutations in ion channel genes. Recent work underscores the possibility that the regulation of ion channel expression can be conserved from Drosophila to mammalian central neurons. In Drosophila, the translational regulator Pumilio was shown to be necessary and sufficient for the modulation of sodium channel transcription after persistent changes to synaptic transmission in the CNS. More recent data indicate that Pumilio-2 regulates NaV1.6 translation in rat visual cortical pyramidal neurons in a manner consistent with that observed in Drosophila. In mammalian neurons, Kr-like genes (KLF) respond to neuronal activity and are studied intensively in the context of axonal regeneration, but a role in ion channel expression or homeostatic rebalancing has yet to be defined (Parrish, 2014).
Kr and its homologs are potent regulators of neuronal cell fate. KLF4 and KLF5, in particular, have been shown to both maintain and reprogram embryonic stem cell fate. This study has provided evidence that Kr protein levels diminish to nearly undetectable levels in the postembryonic CNS. Kr expression is then induced to achieve potassium channel regulation. It is tempting to speculate that the rebalancing of ion channel expression postembryonically is a reinduction of the embryonic mechanisms that initially specify neuronal active properties (Parrish, 2014).
Expression of appropriate ion channels is essential to allow developing neurons to form functional networks. Previous studies have identified LIM-homeodomain (HD) transcription factors (TFs), expressed by developing neurons, that are specifically able to regulate ion channel gene expression. This study used the technique of DNA adenine methyltransferase identification (DamID) to identify putative gene targets of four such TFs that are differentially expressed in Drosophila motoneurons. Analysis of targets for Islet (Isl), Lim3, Hb9/ExEx, and Even-skipped (Eve) identifies both ion channel genes and genes predicted to regulate aspects of dendritic and axonal morphology. Significantly, some ion channel genes are bound by more than one TF, consistent with the possibility of combinatorial regulation. One such gene is Shaker (Sh), which encodes a voltage-dependent fast K(+) channel (Kv1.1). DamID reveals that Sh is bound by both Isl and Lim3. Body wall muscle was used as a test tissue because in conditions of low Ca(2+), the fast K(+) current is carried solely by Sh channels (unlike neurons in which a second fast K(+) current, Shal, also contributes). Ectopic expression of isl, but not Lim3, is sufficient to reduce both Sh transcript and Sh current level. By contrast, coexpression of both TFs is additive, resulting in a significantly greater reduction in both Sh transcript and current compared with isl expression alone. These observations provide evidence for combinatorial activity of Isl and Lim3 in regulating ion channel gene expression (Wolfram, 2014).
Potassium currents play key roles in regulating motoneuron activity, including functional specializations that are important for locomotion. The thoracic and abdominal segments in the Drosophila larval ganglion have repeated arrays of motoneurons that innervate body-wall muscles used for peristaltic movements during crawling. Although abdominal motoneurons and their muscle targets have been studied in detail, owing, in part, to their involvement in locomotion, little is known about the cellular properties of motoneurons in thoracic segments. The goal of this study was to compare firing properties among thoracic motoneurons and the potassium currents that influence them. Whole-cell, patch-clamp recordings performed from motoneurons in two thoracic and one abdominal segment revealed both transient and sustained voltage-activated K(+) currents, each with Ca(++)-sensitive and Ca(++)-insensitive [A-type, voltage-dependent transient K(+) current (I(Av))] components. Segmental differences in the expression of voltage-activated K(+) currents were observed. In addition, it was demonstrated that Shal contributes to I(Av) currents in the motoneurons of the first thoracic segment (Srinivasan, 2012).
Long-term synaptic changes, which are essential for learning and memory, are dependent on homeostatic mechanisms that stabilize neural activity. Homeostatic responses have also been implicated in pathological conditions, including nicotine addiction. Although multiple homeostatic pathways have been described, little is known about how compensatory responses are tuned to prevent them from overshooting their optimal range of activity. This study found that prolonged inhibition of nicotinic acetylcholine receptors (nAChRs), the major excitatory receptors in the Drosophila CNS, resulted in a homeostatic increase in the Drosophila alpha7 (Dalpha7)-nAChR. This response then induced an increase in the transient A-type K(+) current carried by Shaker cognate L (Shal; also known as voltage-gated K(+) channel 4, Kv4) channels. Although increasing Dalpha7-nAChRs boosted miniature excitatory postsynaptic currents, the ensuing increase in Shal channels served to stabilize postsynaptic potentials. These data identify a previously unknown mechanism for fine tuning the homeostatic response (Ping, 2011b).
Rhythmic behaviors, such as walking and breathing, involve the coordinated activity of central pattern generators in the CNS, sensory feedback from the PNS, to motoneuron output to muscles. Unraveling the intrinsic electrical properties of these cellular components is essential to understanding this coordinated activity. This study examined the significance of the transient A-type K(+) current (I(A)), encoded by the highly conserved Shal/K(v)4 gene, in neuronal firing patterns and repetitive behaviors. While I(A) is present in nearly all neurons across species, elimination of I(A) has been complicated in mammals because of multiple genes underlying I(A), and/or electrical remodeling that occurs in response to affecting one gene. In Drosophila, the single Shal/K(v)4 gene encodes the predominant I(A) current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNK(v)4), it was shown that I(A) is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNK(v)4 neurons display multiple defects during prolonged stimuli. DNK(v)4 neurons display shortened latency to firing, a lower threshold for repetitive firing, and a progressive decrement in AP amplitude to an adapted state. Recording was performed from identified motoneurons, and Shal/K(v)4 channels were shown to be similarly required for maintaining excitability during repetitive firing. Larval crawling, and adult climbing and grooming, all behaviors that rely on repetitive firing, were examined. All are defective in the absence of Shal/K(v)4 function. Further, knock-out of Shal/K(v)4 function specifically in motoneurons significantly affects the locomotion behaviors tested. Based on these results, Shal/K(v)4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency. This study shows that Shal/K(v)4 channels play a key role in repetitively firing neurons during prolonged input/output, and suggests that their function and regulation are important for rhythmic behaviors (Ping, 2011a).
Motoneurons in most organisms conserve a division into low-threshold and high-threshold types that are responsible for generating powerful and precise movements. Drosophila 1b and 1s motoneurons may be analogous to low-threshold and high-threshold neurons, respectively, based on data obtained at the neuromuscular junction, although there is little information available on intrinsic properties or recruitment during behavior. Therefore in situ whole cell patch-clamp recordings were used to compare parameters of 1b and 1s motoneurons in Drosophila larvae. Resting membrane potential, voltage threshold, and delay-to-spike distinguish 1b from 1s motoneurons. The longer delay-to-spike in 1s motoneurons is a result of the shal-encoded A-type K(+) current. Functional differences between 1b and 1s motoneurons are behaviorally relevant because a higher threshold and longer delay-to-spike are observed in MNISN-1s in pairwise whole cell recordings of synaptically evoked activity during bouts of fictive locomotion (Schaefer, 2010).
Shal K(+) (K(v)4) channels in mammalian neurons have been shown to be localized exclusively to somato-dendritic regions of neurons, where they function as key determinants of dendritic excitability. To gain insight into the mechanisms underlying dendritic localization of K(v)4 channels, Drosophila melanogaster was used as a model system. Shal K(+) channels were shown to display a conserved somato-dendritic localization in vivo in Drosophila. From a yeast-2-hybrid screen,the novel interactor, SIDL (for Shal Interactor of Di-Leucine Motif), was observed as the first target protein reported to bind the highly conserved di-leucine motif (LL-motif) implicated in dendritic targeting. SIDL was shown to be expressed primarily in the nervous system, co-localizes with GFP-Shal channels in neurons, and interacts specifically with the LL-motif of Drosophila and mouse Shal channels. Shal-SIDL interaction was disrupted by mutating the LL-motif on Shal channels, and Shal K(+) channels were then mislocalized to some, but not all, axons in vivo. These results suggest that there are multiple mechanisms underlying Shal K(+) channel targeting, one of which depends on the LL-motif. The identification of SIDL may provide the first step for future investigation into the molecular machinery regulating the LL-motif-dependent targeting of K(+) channels (Diao, 2010).
Homeostatic control of neural function can be mediated by the regulation of ion channel expression, neurotransmitter receptor abundance, or modulation of presynaptic release. These processes can be implemented through cell autonomous or intercellular signaling. It remains unknown whether different forms of homeostatic regulation can be coordinated to achieve constant neural function. One way to approach this question is to confront a simple neural system with conflicting perturbations and determine whether the outcome reflects a coordinated, homeostatic response. This study demonstrates that two A-type potassium channel genes, shal and shaker, are reciprocally, transcriptionally coupled to maintain A-type channel expression. This homeostatic control of A-type channel expression was shown to prevent target-dependent, homeostatic modulation of synaptic transmission. Thus, this study uncovered a homeostatic mechanism that reciprocally regulates A-type potassium channels, and a hierarchical relationship was defined between cell-intrinsic control of ion channel expression and target-derived homeostatic control of synaptic transmission (Bergquist, 2010).
An electrophysiology-based forward genetic screen identified three potassium channel mutations, including mutations in shal and Drosophila KV3.2, that block the expression of synaptic homeostasis following inhibition of postsynaptic glutamate receptor function. This study focused on how mutations in a single potassium channel, shal, lead to a blockade of synaptic homeostasis. It was first demonstrated that loss of shal induces a compensatory increase in shaker expression, and vice versa, suggesting homeostatic maintenance of A-type channel abundance in Drosophila motoneurons. The compensatory increase in shaker expression is remarkable, however, because it does not replace the A-type current recorded at the motoneuron soma. Rather, increased Shaker functions to restrict neurotransmitter release from the motoneuron terminal, decreasing baseline release and blocking any further homeostatic enhancement of presynaptic release. There are several implications. First, the data demonstrate that the unique subcellular localization of each ion channel will determine how any compensatory change in ion channel abundance affects neural activity and synaptic transmission. Second, it appears that cell-autonomous control of intrinsic excitability can occlude the expression of subsequent intercellular homeostatic signaling. This suggests a hierarchical control of cell-intrinsic excitability compared to circuit level homeostatic regulation. This also calls into question the concept of a master, homeostatic sensor of neuronal activity. Finally, a form of compensation was defined that may largely preserve neuronal output properties without restoring cellular excitation at the level of the cell soma (Bergquist, 2010).
This study has demonstrated that compensatory increase in Shaker expression is necessary and sufficient to block the subsequent expression of synaptic homeostasis following postsynaptic GluR inhibition. In a shal mutant, a ~250% increase in shaker expression was detected. If this increase in Shaker expression is presented in any of three different ways -- 1) genetically by introducing shaker mutations, 2) transgenically through neuron-specific dsRNA knockdown of shaker, or 3) pharmacologically -- then synaptic homeostasis is restored in the shal mutant. Furthermore, acute block of Shaker by 4-AP following PhTx provides evidence that increased Shaker levels block the expression of synaptic homeostasis, not the induction of this form of homeostatic plasticity. Finally, it was demonstrated that exogenous overexpression of a Shaker transgene (EKO) is sufficient to block synaptic homeostasis in an otherwise wild type background. Thus, the compensatory increase in Shaker expression in the shal mutant blocks subsequent expression of synaptic homeostasis (Bergquist, 2010).
Numerous experiments are provided that argue against the possibility that loss of Shaker rescues synaptic homeostasis through a non-specific potentiation of synaptic transmission. First, neuronal expression of shaker RNAi in the shal mutant background reduces shaker transcript (~70% reduction) and restores synaptic homeostasis without potentiating baseline transmission. Second, pharmacological inhibition of Shaker was performed using 4-AP concentrations that have a minimal effect on baseline synaptic transmission (~27% change), yet synaptic homeostasis is restored. Finally, synaptic homeostasis is also blocked in the KV3.2 mutant, but there is no change in shaker expression nor does presynaptic knockdown of shaker in the KV3.2 mutant rescue synaptic homeostasis. It is concluded that the increased shaker expression is specific to the shal mutant and that reducing shaker expression or function in the shal mutant is sufficient to reveal the expression of synaptic homeostasis in the shal mutant (Bergquist, 2010).
Why does increased expression of Shaker, at or near the synaptic terminal block the expression of synaptic homeostasis? It is presumed that increased expression of Shaker in the shal mutant causes a decrease in action potential width. Unfortunately, it is not possible to record the presynaptic action potential from the synaptic terminal because the terminal is embedded within the muscle and is otherwise surrounded by the muscle basal lamina. There are several possible ways that a narrower action potential could block expression of synaptic homeostasis. One possibility is that synaptic homeostasis requires an increase in action potential duration and this is prevented by increased Shaker expression. If so, it is unlikely that Shaker is the direct target of this homeostatic signaling system because homeostatic compensation is observed in the shaker mutant background. Alternatively, a narrower action potential could prevent recruitment of newly inserted presynaptic calcium channels. Genetic data indicate that synaptic homeostasis involves a change in calcium influx at a fixed number of active zones and this could be achieved by an increase in the number of presynaptic calcium channels (Bergquist, 2010).
The transcriptional coupling of shaker and shal would seem to be a homeostatic mechanism since both channels encode A-type potassium currents. However, these channels localize to different subcellular compartments. Thus, increased Shaker expression should not homeostatically restore wild-type motoneuron excitability since the somatic A-current remains absent. Rather, increased Shaker seems to inhibit presynaptic neurotransmitter release and may thereby guard against inappropriately enhanced glutamatergic transmission. This effect differs from current homeostatic hypotheses because baseline neural activity is not re-established, but neural output is constrained within reasonable limits (Bergquist, 2010).
The importance of channel localization during homeostatic compensation is also highlighted by recent studies in vertebrate central neurons. It was recently demonstrated that KV4.2 knockout animals lack dendritically recorded A-type currents in hippocampal neurons. The absence of a dendritic A-type current potentiates back propagating action potentials and enhances LTP. Thus, at the level of the neuronal dendrite, this is an example of failed homeostatic compensation. However, this study also documents a compensatory increase in somatically recorded KV1-type currents. It seems plausible that the observed compensatory increase in somatic KV1-type currents could counteract increased dendritic excitability and, thereby, homeostatically restrain neural output. This possibility is supported by data from additional studies examining KV4.2 knockouts in other neuronal cell types. In these studies, neuronal firing properties measured at the soma are largely normal in the KV4.2 knockout despite the absence of the dendritic A-type current (Bergquist, 2010).
This study demonstrates that shal and shaker, which encode A-type potassium channels, are reciprocally, homeostatically coupled. What drives the compensatory change in ion channel expression following loss of a given ion channel? One possibility, suggested by prior research in other systems is that the neuron senses a persistent change in cellular activity and initiates a homeostatic response that modulates the expression of other ion channels. The data are consistent with an activity-dependent model. Knockdown of shaker expression (65% of the wild type level) leads to a 223% increase in shal expression. Remarkably, a 1300% increase was observed in shal expression in the shaker14 mutant, which is a point mutation resulting in a non-functional channel. In the shaker14 experiment, the mutant shaker transcript continues to be expressed at 80% wild type levels. Thus, the degree to which shal expression is increased correlates with the severity of altered channel function rather than the loss of shaker message. This suggests that altered channel function or altered neural activity could be the trigger for the compensatory response. These data also raise an interesting question. If the expression of one ion channel, such as shal, is specifically coupled to the expression of another channel, such as shaker, how could this be achieved by a general monitor of neural activity (Bergquist, 2010)?Several studies have now documented that prolonged inhibition of an ion channel, or genetic ablation of an ion channel, can lead to increased expression of a different ion channel with overlapping function, again suggesting coupling between specific pairs of ion channels. For example, loss of NaV1.6 causes increased expression of NaV1.1 in purkinje cells and increased expression of NaV1.2 in retinal ganglion cells. Similarly, loss of A-type potassium currents in KV4.2 (the vertebrate shal homolog) knockout animals causes a compensatory increase in both IK and ISS that preserves action potential shape and neuronal firing properties. In these examples, the compensatory changes in sodium or potassium channel expression seem to homeostatically maintain appropriate neuronal firing properties. These studies support the hypothesis that ion channels are free variables that can be adjusted by a homeostatic monitor of neural activity and that specific pairs of ion channels may be homeostatically coupled (Bergquist, 2010).
An alternate form of regulation has been suggested by work in lobster stomatogastric neurons where there is evidence for an activity-independent mechanism that couples shal and Ih expression. In this system, overexpression of shal leads to increased Ih current (channel expression was not tested). However, overexpression of a pore-blocked shal also leads to increased Ih current. Thus, altered neural function does not appear to be the trigger for a compensatory change in Ih current. Rather, the cell could monitor the level shal message or protein and regulate Ih current accordingly. This mechanism would allow for specific coupling of ion channel pairs, but appears to be different from the phenomenon identified in Drosophila motoneurons (Bergquist, 2010).
One interesting possibility is that the developmental programs that initially specify the active properties of a given neuron could, later, control ion channel expression in a homeostatic manner. Modeling studies suggest that there are large numbers of physiologically plausible combinations of ion channels that could give rise to a cell with a specific firing property. However, if the expression of pairs or combinations of ion channels are somehow coupled, then the parameter space for defining the firing properties of a given cell type would be dramatically simplified. It is interesting, therefore, to speculate that the apparent homeostatic compensation for loss of a given ion channel could represent the re-use of an earlier developmental program that initially served to balance the expression of specific pairs or combinations of ion channels during cell fate specification. It will be important to determine whether there are any general rules by which one might predict how a cell will respond to the altered expression of a specific ion channel or whether all such relationships will be defined in a cell-type specific manner (Bergquist, 2010).
The regulation of A-type currents in Drosophila motoneurons occludes trans-synaptic, homeostatic modulation of neurotransmitter release. The consequence is that the postsynaptic muscle target is unable to restore normal synaptic drive from the motoneuron terminal and remains hypo-excitable. Specifically, EPSP amplitudes are significantly smaller in the shal; GluRIIA double mutant animals compared to either shal or GluRIIA alone. Thus, at the neuromuscular junction, the regulation of motoneuron intrinsic excitability supercedes the homeostatic control of motor unit function (Bergquist, 2010).
The homeostatic modulation of synaptic transmission can be induced in seconds to minutes. By contrast, the compensatory control of ion channel expression clearly involves gene transcription and is likely to be induced more slowly. One question is whether, given enough time, the mechanisms of synaptic homeostasis can adjust to the change in ion channel expression observed in the shal mutant background. This does not appear to be the case. The GluRIIA mutation causes a persistent change in postsynaptic receptor function leading to a persistent homeostatic increase in presynaptic release that is present throughout the four days of larval development. Synaptic homeostasis is still blocked in the GluRIIA; shal double mutant and a statistically similar increase shaker transcription is observed (Bergquist, 2010).
It is worth emphasizing that the homeostatic modulation of presynaptic release appears to have been executed, unaltered in the shal mutant background because acute application of 4-AP reveals normal homeostatic compensation in the shal mutant. These data argue against the possibility that independent homeostatic signaling systems are somehow coordinated at the level of the motor unit, or perhaps neural circuit. Thus, even though an initial homeostatic action is restorative, any change in the balance of ion conductances that control the action potential could dramatically alter how a cell responds to a future perturbation. It has been speculated in systems ranging from crustacean central neurons to the vertebrate cortex, that normal cell-to-cell differences in ionic conductances recorded from an identified cell type might reflect the activity of homeostatic signaling systems. The question remains whether these different cells respond similarly to future homeostatic pressures (Bergquist, 2010).
The output of a neural circuit results from an interaction between the intrinsic properties of neurons in the circuit and the features of the synaptic connections between them. The plasticity of intrinsic properties has been primarily attributed to modification of ion channel function and/or number. This study has found a mechanism for intrinsic plasticity in rhythmically active Drosophila neurons that was not based on changes in ion conductance. Larval motor neurons had a long-lasting, sodium-dependent afterhyperpolarization (AHP) following bursts of action potentials that was mediated by the electrogenic activity of Na(+)/K(+) ATPase. This AHP persisted for multiple seconds following volleys of action potentials and was able to function as a pattern-insensitive integrator of spike number that was independent of external calcium. This current also interacted with endogenous Shal K(+) conductances to modulate spike timing for multiple seconds following rhythmic activity, providing a cellular memory of network activity on a behaviorally relevant timescale (Pulver, 2010).
One of the most important tasks of a neuron is to keep track of its own activity. This is of obvious importance for neurons that are involved in memory processes, but it is also true for many other types of neurons that need to operate within a particular activity or input-output range. Many types of plasticity mechanisms have been described that allow cells to adjust synaptic weights and intrinsic properties to reflect their activity history and maintain optimal functionality. This study has demonstrated a new form of short-term cellular memory in Drosophila larval motor neurons that is mediated by spike-dependent activation of Na+/K+-ATPase. An AHP mediated by electrogenic activity of the Na+/K+ pump is proportional to the number of proceeding spikes, even when the pattern of activity is varied. This AHP effectively acts as a spike counter at behaviorally relevant spike rates. Furthermore, this study found that this AHP can release endogenous Ishal channels from inactivation during rhythmic firing, and that this modification persists for multiple seconds in the absence of rhythmic input, providing a memory trace of the rhythmically active state (Pulver, 2010).
Na+/K+ pumps are commonly portrayed as the necessary but unglamorous workhorses of neuronal membranes. By continually moving Na+ ions out and K+ ions into cells, Na+/K+ pumps generate an electrochemical gradient across the cellular membrane; this slow activity is crucial for generating the resting membrane potential in all neurons and setting basal excitability. Na+/K+ pumps can have other functions, however. Long lasting, Na+/K+ pump-mediated AHPs have been observed in a variety of neuronal types. In Drosophila they have been shown to be engaged by pharmacological manipulation of sodium channel inactivation kinetics. In various vertebrate preparations, pump-mediated AHPs regulate rhythmic bursting by suppressing excitability. Pump-mediated AHPs have also been shown to underlie changes in the efficacy of neuromodulatory synaptic input in leech sensory neurons. This same pump current has been shown to be intimately involved in sensory coding in these neurons due to its ability to allow adaptive scaling of input signals. The present study is the first to show that a persistent (many seconds long), dynamic change in neuronal excitability can be attributed to Na+/K+ pump function under normal physiological conditions in rhythmically active motor neurons (Pulver, 2010).
One striking feature of the AHP reported in this study is that it reflects overall previous spiking activity but remains relatively insensitive to the pattern of activity in which spikes are presented. This is not a feature of the long lasting AHPs which are mediated by ionic conductances. Spike counting has been shown to underlie memory formation in other systems. In weakly electric fish, a long-lasting shift in intrinsic excitability is responsible for a pulse integrating mechanism that is immune to frequency-dependent fluctuations. This process is critical to a form of long lasting sensorimotor adaptation in electric organ discharges (Pulver, 2010).
The role of spike counting in the mature larval locomotor circuit is less clear, but the ability of AHPs to act as spike integrators or 'spike counters' through a range of activity patterns has interesting implications for computational neuroscientists interested in homeostatic plasticity. In previous work, models of how neurons keep track of their own activity have been focused on sensors of intracellular Ca2+. Intracellular Ca2+ levels, however, are not always well correlated with spiking activity in neurons. Furthermore, Ca2+-sensing mechanisms that operate on time scales over 1 sec are sometimes difficult to justify in a model, given the fast time constant of Ca2+ decay after spiking (typically ~0.5 sec). Intracellular Na+ concentrations, by contrast, are more directly linked to spiking since they directly reflect the actions of voltage-gated Na+ channels. This work suggests that activity can modify intracellular Na+ levels over multi-second time scales. Such accumulation is likely to be most significant in small neurons or geometrically constrained subcellular compartments. These results suggest that activity sensors tuned to intracellular Na+ could be potentially useful as seconds - long time scale activity integrators in computational models of homeostatic plasticity. This has special relevance to rhythmic networks since many of these circuits operate with cycle periods of this magnitude (Pulver, 2010).
An additional interesting property of the hyperpolarization produced by the larval pump is that it can release endogenous Ishal channels from inactivation and thereby modify how a cell responds to the next depolarizing input. Previous studies in the larval motor circuit concluded that Ishal currents are largely inactivated at rest and do not affect spike timing in MN30-Ib cells. This conclusion, however, was based on measurements from silent cells. This is not the usual state of MN30-Ib cells; in a behaving animal, MN30-Ibs are rhythmically active. The importance of considering a network's endogenous activity in studies of synaptic plasticity has been demonstrated in other systems. The example shown in this study highlights the importance of considering the endogenous activity of a network when measuring intrinsic properties in neurons as well. The ability of the AHP to alter the intrinsic properties of motor neurons embedded in the firing locomotor circuit marks them as having been recently active and alters the timing of motor outputs (Pulver, 2010).
The extent to which activity-dependent intrinsic properties can lead to forms of cellular memory has been widely studied=. However, putting these phenomena in a functional context is often difficult. Genetic inhibition of Na+/K+ ATPase activity in motor neurons abolishes AHPs. This genetic manipulation clearly has an impact on network output: it slows forward peristalsis by reducing central pattern generator cycle period. One possibility is that normal AHPs facilitate proper segment-to-segment coordination by restricting the time-frame of rhythmic activity within a segment during a peristaltic wave. When this restriction is removed (as in the case when dnATPase is expressed in motor neurons), activity is 'slurred' over a longer time frame in each segment, potentially leading to longer overall peristaltic wave durations. Tight control of activity bursts has been shown to be an important factor in regulating cycle period in other segmentally coupled oscillating networks. At this time, there is not enough information about synaptic connectivity within the ventral ganglion to test this and other hypotheses using computational techniques, however, future work could address this question as circuit information becomes available (Pulver, 2010).
It is important to note that in addition to abolishing AHPs, expression of dnATPase (ATPaseα) also affects motor neuron response to current injection. As a result, some of the behavior effects seen could be caused by hyperexcitation. Unfortunately, currently available genetic tools do not allow manipulation of AHP amplitude and response to current injection independently. However, the dnATPase manipulation does not significantly affect resting membrane potential; in addition, the behavioral effects of acutely depolarizing motor neurons with the heat-activated ion channel dTRPA1 (i.e. full stop, no peristalsis) are different from those reported in this study. These observations suggest that the pump-mediated affects on CPG output observed in this study are not merely the result of massive motor neuron depolarization. Overall, the results suggest that the seconds-long time scale of AHPs could act to keep motor neuron properties primed for rhythmic action and provide a complement to the longer term plasticity processes that engage translational and transcriptional mechanisms 43 to tune intrinsic properties in this circuit (Pulver, 2010).
Ionic currents underlie the firing patterns, excitability, and synaptic integration of neurons. Despite complete sequence information in multiple species, knowledge about ion channel function in central neurons remains incomplete. This study analyzes the potassium currents of an identified Drosophila flight motoneuron, MN5, in situ. MN5 exhibits four different potassium currents, two fast-activating transient ones and two sustained ones, one of each is calcium activated. Pharmacological and genetic manipulations unravel the specific contributions of Shaker and Shal to the calcium independent transient A-type potassium currents. alpha-dendrotoxin (Shaker specific) and phrixotoxin-2 (Shal specific) block different portions of the transient calcium independent A-type potassium current. Following targeted expression of a Shaker dominant negative transgene in MN5, the remaining A-type potassium current is alpha-dendrotoxin insensitive. In Shal RNAi knock down the remaining A-type potassium current is phrixotoxin-2 insensitive. Additionally, barium blocks calcium-activated potassium currents but also a large portion of phrixotoxin-2-sensitive A-type currents. Targeted knock down of Shaker or Shal channels each cause identical reduction in total potassium current amplitude as acute application of alpha-dendrotoxin or phrixotoxin-2, respectively. This shows that the knock downs do not cause upregulation of potassium channels underlying other A-type channels during development. Immunocytochemistry and targeted expression of modified GFP-tagged Shaker channels with intact targeting sequence in MN5 indicate predominant axonal localization. These data can now be used to investigate the roles of Shaker and Shal for motoneuron intrinsic properties, synaptic integration, and spiking output during behavior by targeted genetic manipulations (Ryglewski, 2009).
Shal K+ (K(v)4) channels across species carry the major A-type K+ current present in neurons. Shal currents are activated by small EPSPs and modulate post-synaptic potentials, backpropagation of action potentials, and induction of LTP. Fast inactivation of Shal channels regulates the impact of this post-synaptic modulation. This study introduced SKIP3, as the first protein interactor of Drosophila Shal K+ channels. The SKIP gene encodes three isoforms with multiple protein-protein interaction domains. SKIP3 is nervous system specific and co-localizes with Shal channels in neuronal cell bodies, and in puncta along processes. Using a genetic deficiency of SKIP, this study shows that the proportion of neurons displaying a very fast inactivation, consistent with Shal channels exclusively in a 'fast' gating mode, is increased in the absence of SKIP3. As a scaffold-like protein, SKIP3 is likely to lead to the identification of a novel regulatory complex that modulates Shal channel inactivation (Diao, 2009).
Voltage-gated potassium channels related to the Shal gene of Drosophila (Kv4 channels) mediate a subthreshold-activating current (I(SA)) that controls dendritic excitation and the backpropagation of action potentials in neurons. Kv4 channels also exhibit a prominent low voltage-induced closed-state inactivation, but the underlying molecular mechanism is poorly understood. This study has examined a structural model in which dynamic coupling between the voltage sensors and the cytoplasmic gate underlies inactivation in Kv4.2 channels. An alanine-scanning mutagenesis was performed in the S4-S5 linker, the initial part of S5, and the distal part of S6, and the mutants were functionally characterized under two-electrode voltage clamp in Xenopus oocytes. In a large fraction of the mutants (>80%) normal channel function was preserved, but the mutations influenced the likelihood of the channel to enter the closed-inactivated state. Depending on the site of mutation, low-voltage inactivation kinetics were slowed or accelerated, and the voltage dependence of steady-state inactivation was shifted positive or negative. Still, in some mutants these inactivation parameters remained unaffected. Double mutant cycle analysis based on kinetic and steady-state parameters of low-voltage inactivation revealed that residues known to be critical for voltage-dependent gate opening, including Glu 323 and Val 404, are also critical for Kv4.2 closed-state inactivation. Selective redox modulation of corresponding double-cysteine mutants supported the idea that these residues are involved in a dynamic coupling, which mediates both transient activation and closed-state inactivation in Kv4.2 channels (Diao, 2009).
Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodeling. The translational repressor Pumilio (Pum) is a regulator of sodium current [I(Na)] and excitability in Drosophila motoneurons. This study shows that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel Paralytic (Para). A putative binding site for Pum was identified in the 3' end of the para open reading frame (ORF). Characterization of the mechanism of action of Pum, using whole-cell patch clamp and real-time reverse transcription-PCR, reveals that the full-length protein is required for translational repression of para mRNA. Additionally, the cofactor Nanos is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation of I(Na) in motoneurons requires both Nanos and Brat, whereas regulation in other neuronal types seemingly requires only Nanos but not Brat. Pum is able to reduce the level of nanos mRNA and as such a potential negative-feedback mechanism has been identified that protects neurons from overactivity of Pum. Finally, coupling was shown between I(Na) (para) and I(K) (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as a direct target of Pum represents the first ion channel to be translationally regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3'-untranslated region of target transcripts (Muraro, 2008).
Neuronal activity is regulated by homeostatic mechanisms that serve to maintain membrane excitability within pre-defined limits. This is achieved, at least in part, by continual adjustment of both ligand- and voltage-gated ionic conductances to maintain stable action potential firing rates in response to changing synaptic excitation. Such regulation is predicted to be particularly predominant when neural circuit synaptic activity is changing rapidly, for example during both neuronal circuit development and in the formation of memory. However, although now well established, the molecular pathways that underlie homeostatic regulation remain largely unknown (Muraro, 2008).
Previous studies indicate that activity-dependent regulation of voltage-gated sodium channels is central to the control of membrane excitability in both mammalian and invertebrate neurons. Studies in Drosophila have shown that increased synaptic excitation of motoneurons is countered by a decrease in sodium current (INa) and membrane excitability in these cells. Similar, but opposite, changes in INa and excitability are observed in mutants which display decreased synaptic excitability. These changes require the known translational repressor Pumilio (Pum), which has been shown previously to be both necessary and sufficient for activity-dependent changes of INa in Drosophila motoneurons (Mee, 2004). The model predicts that prolonged change in exposure to synaptic excitation is countered by a reciprocal Pum-dependent regulation in translation of para mRNA and membrane excitability (Muraro, 2008).
The role of Pum is well described from studies of early Drosophila embryogenesis. Specification of the abdomen requires Pum-dependent repression of translation of hunchback (hb) mRNA. The first step begins with the recognition and binding of Pum to the Nanos Response Element (NRE)-motif located in the 3' untranslated region (UTR) of hb mRNA. Once bound, Pum then recruits the co-factors Nanos (Nos) and Brain Tumor (Brat) to form a repressor complex that results in the translational repression of hb mRNA. The mechanism of repression involves both deadenylation and poly(A)-independent silencing (Chagnovich, 2001). In addition to its characterised roles in repression of hb, Pum has also been shown to bind, and repress translation of, mRNAs encoding the eukaryotic Initiation factor 4E (eIF4E) and Cyclin B (CycB) . Indeed, these few mRNAs may represent just the tip of the iceberg as the actual list of targets is likely to be extensive based on a recent demonstration that Pum associates with more than 1,000 different mRNAs in the ovaries of adult flies. Pum proteins are evolutionarily conserved from yeast to mammals and, moreover, Pum expression is activity-dependent in mammalian neurons in culture (Muraro, 2008).
This paper reports that Pum is able to directly bind paralytic (para) mRNA (encoding the Drosophila voltage-gated Na+ channel). The mechanism of para translational repression shows similarities and differences to Pum-dependent repression of hb mRNA. Unlike repression of hb, full length Pum is necessary for para repression. As for most other Pum-dependent repressed transcripts described to date, para repression requires the presence of the co-factor Nos. However, the requirement for the co-factor Brat is neuronal type-specific. Pum was shown to be sufficient to down-regulate nos mRNA levels in the CNS, a property that may serve to protect neurons from the effects of over-activity of this translational repressor (Muraro, 2008).
Identification of the molecular components that underlie homeostasis of membrane excitability in neurons remains a key challenge. This study shows that the translational repressor Pum binds para mRNA, which encodes the Drosophila voltage-gated Na+ channel. This observation provides a mechanistic understanding for the previously documented ability of Pum to regulate INa and membrane excitability in Drosophila motoneurons (Mee, 2004). Thus, alteration in activity of Pum, in response to changing exposure to synaptic excitation, enables neurons to continually reset membrane excitability through the translational control of a voltage-gated Na+ channel (Muraro, 2008).
Previous studies report several mRNAs subject to direct Pum regulation including hb, bicoid (bcd), CycB, eIF4E, and possibly the transcript destabilization factor smaug (smg). The majority of these identified transcripts concentrate the roles of Pum to the establishment of the embryonic anterior-posterior axis (hb and bcd) and germ-line function/oogenesis (CycB). However, in the last few years, new findings have expanded the role of Pum to encompass predicted roles in memory formation, neuron dendrite morphology, and glutamate receptor expression in muscle. Indeed, the role of Pum is likely to be very much more widespread given that Pum pull-down assays followed by microarray analysis of bound mRNAs have now identified a plethora of possible additional targets of translational regulation (Gerber, 2006). The ~1000 or so genes identified are implicated to be involved in various cellular functions, suggesting that Pum-dependent translational repression might be a mechanism used in different stages of development and in diverse tissue function. To date, para is the first confirmed Pum target encoding a voltage-gated ion channel (Muraro, 2008).
Pum-binding motifs have been identified in the 3'-UTRs of many mRNAs known to bind to this protein. Analysis of 113 such genes expressed in adult Drosophila ovaries has identified a consensus 8 nt binding motif [UGUAHAUA]. This sequence contains the UGUA tetranucleotide that is a defining characteristic of the NRE-like motif described in the 3'-UTR of hb mRNA. Such an 8 nt motif has been identified within the ORF of para at the 3' end of the transcript. The biochemical binding data support the notion that this motif is indeed sufficient to bind Pum and as such represents the first such site to be localized to an ORF of any transcript. However, to translationally repress para mRNA, the data also show a requirement for regions of Pum in addition to the RBD. Interestingly, this kind of requirement has also been shown for another Pum target, eIF4E. The translational silencing of mRNAs is a complex mechanism on which only little information is available. It could involve deadenylation and degradation of the mRNA and/or the circularization of the mRNA and the recruitment of factors that would preclude translation. The fact that different Pum targets may require only the RBD (hb) or the full-length protein (eIF4E and para) suggests that Pum-mediated translational repression may follow complex target mRNA-specific mechanisms, most probably involving the interaction of other domains of Pum with additional, so far unknown, factors. In this regard, it is interesting to note that the N terminus of Pum has regions of low complexity including prion-like domains rich in Q/R. These domains may provide a platform for other proteins that influence the fate of Pum targets (Muraro, 2008).
The putative Pum binding motif lies within an exon that is common to all para splice variants identified (at least in the embryo) but is possibly subject to editing by adenosine deamination. Thus, in an analysis of splicing of para, a number of individual cDNA clones were sequenced and one splice variant was recovered that shows A-to-I editing in this motif. Together with a differential requirement for specific cofactors, editing of this motif might serve to influence how para is affected by Pum and, as such, further increase diversity in level of expression of INa in differing neurons or disease states (Muraro, 2008).
The known mechanism of action of Pum-dependent translational repression is absolutely dependent on additional cofactors. The most studied example, that of hb mRNA during early embryogenesis, requires the presence of both Nanos and Brat. However, the requirement for these two cofactors is seemingly transcript dependent. Thus, Pum-mediated repression of CycB mRNA requires Nanos but not Brat. However, Pum-dependent repression of bcd is apparently Nanos independent, because levels of Nanos in the anterior of the early embryo are undetectable. Although it was clearly shown that Pum-dependent repression of para mRNA in the Drosophila CNS requires Nanos, the requirement for Brat is less clear and seems to be neuronal cell type specific. A requirement for a different combination of cofactors for Pum-dependent translational regulation of a single gene transcript has not been reported previously, but clearly might represent an additional level of regulation. Such differential regulation might be required to spatially restrict the effect of Pum to certain cell types within the CNS. Voltage-gated Na+ currents are responsible for the initiation and propagation of the action potential and determine, together with other voltage-gated ion conductances, the membrane excitability of a neuron. Despite para being the sole voltage-gated sodium channel gene in Drosophila [compared with at least nine different genes in mammals, neuronal subpopulations nevertheless exhibit distinctive INa characteristics. To achieve this, para is known to undergo extensive alternative splicing and, additionally, RNA editing. It is highly likely that both alternative splicing and RNA editing generate mRNAs that encode channels with differing electrophysiological properties. It is also conceivable that these mechanisms might yield para transcripts that contain differing arrangements of Pum/Nanos binding sites, which may, or may not, recruit Brat. Indeed, it has been proposed that variations of the NRE consensus sequence may result in Pum-NRE-Nanos complexes with different topographies, resulting in altered recruitment abilities for additional cofactors such as Brat. Additional work is necessary to clarify where, in para mRNA, the binding sites for the Pum/Nanos complex are localized and how the recruitment of Brat is facilitated in only some neurons. In the hb repression complex, Brat has been shown to interact with the cap-binding protein d4EHP. Therefore, additional cofactors might be necessary for Pum-dependent para repression in the Brat-independent neuronal cell subtypes (Muraro, 2008).
In contrast to translational repression of hb, the data show that Nanos is unlikely to be a limiting factor of Pum-dependent repression of para translation. Consistent with this finding is the observation that overexpression of pum is sufficient to downregulate (and probably translationally repress) nanos mRNA. However, the opposite is not true; overexpression of nanos does not affect levels of pum mRNA. These data suggest that Pum is at least a principal orchestrating factor (if not the prime factor) in regulation of para translation. Moreover, the demonstration that overexpression of pum is sufficient to greatly downregulate nanos mRNA (relative to para mRNA), together with a requirement of Nanos for Pum-dependent para mRNA repression, implicates the existence of a protective negative-feedback mechanism that prevents overrepression of para mRNA. In the absence of such feedback, it is conceivable that excessive overrepression of para mRNA might lead to neurons falling silent as their membrane excitability drops below a critical threshold. Were this to happen, then signaling in the affected neuronal circuit would be severely compromised (Muraro, 2008).
Overexpression of full-length Pum in aCC/RP2 motoneurons not only causes a decrease in INa but also a significant decrease in IKfast. Additionally, pan-neuronal overexpression of Pum causes a significant decrease in Shal mRNA, a gene encoding a potassium channel known to contribute to IKfast. This result was surprising given that Shal was not identified as a Pum target from microarray analysis. That this mechanism might, therefore, be indirect is corroborated by the finding that IKfast and Shal mRNA remain at wild-type levels when Pum is overexpressed in a para-null background. It is, perhaps, counterintuitive that a reduction in INa, to achieve a reduction in membrane excitability, should be accompanied by a similar decrease in outward IKfast. However, changes in ionic conductances should not be considered in isolation and such a relationship might serve to maintain action potential kinetics within physiological constraints. Covariation of INa and IK as a mechanism for changing neuronal excitability has been described in these motoneurons previously. Moreover, there is precedent for coupling between transcripts: injection of Shal mRNA into lobster PD (pyloric dilator) neurons results in an expected increase in IA but also an unexpected linearly correlated increase in Ih, an effect that acts to preserve membrane excitability. Injection of a mutated, nonfunctional, Shal mRNA is also sufficient to increase Ih indicative that this coregulation is activity independent (MacLean, 2003). It remains to be shown whether genetic manipulation of para mRNA levels in Drosophila motoneurons will similarly evoke compensatory changes in Shal expression (Muraro, 2008).
In a previous study, it was shown that blockade of synaptic release, through pan-neuronal expression of tetanus toxin light chain, is sufficient to evoke a compensatory increase in membrane excitability in aCC/RP2 that was accompanied by increases in INa, IKfast, and also IKslow (Baines, 2001). In contrast, the current study showed that overexpression of pum is sufficient to decrease INa and IKfast but does not significantly affect IKslow (although there is a small nonsignificant reduction in this current). Clearly, the complete absence of synaptic input is a more severe change that likely elicits a greater compensatory change in these neurons than when Pum is overexpressed. However, whether removal of synaptic excitation also invokes additional compensatory mechanisms that act preferentially on IKslow remains to be determined. What is consistent, however, is that change in synaptic excitation of these motoneurons is countered by Pum-dependent regulation of both para mRNA translation and magnitude of INa (Muraro, 2008).
A key question remains as to what the mechanism is that transduces changes in synaptic excitation to altered Pum activity. Perhaps the most parsimonious mechanism will be one linked to influx of extracellular Ca2+. Indeed, experimental evidence supports a role for Ca2+, because blocking its entry can preclude changes in neuronal excitability observed as a result of activity manipulation. In addition, changes of gene expression resulting from activity-mediated Ca2+ entry have been described both in vitro and in vivo after plasticity changes such as long-term potentiation. Whether Ca2+ influx influences translation and/or transcription of Pum remains to be shown. Stimulation of mammalian neurons in culture with glutamate, after a preconditioning period of forced quiescence, results in an increase of Pum2 protein levels after just 10 min. The rapidity of this response suggests that it is mediated by a posttranscriptional mechanism. This study examined the role of Pum on Ca2+ channel activity. Neither IBa(Ca) nor levels of the voltage-gated calcium channel coded by Dmca1A (cacophony, Calcium channel α1 subunit, type A) are affected in aCC/RP2 motoneurons in which pum [full length (FL)] is overexpressed. The fact that Pum does not affect Ca2+channel activity directly could reinforce the idea of its serving as a primary sensor of activity changes (Muraro, 2008).
In summary, this study has shown that Pum is able to bind to para mRNA, an effect that is sufficient to regulate both INa and membrane excitability in Drosophila motoneurons. This mechanism requires the cofactor Nanos but does not obligatorily require Brat. Given that mammals express two Pum genes, Pum1 and Pum2, it will be of importance to determine whether this protein is also able to regulate sodium channel translation in the mammalian CNS (Muraro, 2008).
Different K+ currents participate in generating neuronal firing patterns. The Drosophila embryonic 'giant' neuron culture system has facilitated current- and voltage-clamp recordings to correlate distinct excitability patterns with the underlying K+ currents and to delineate the mutational effects of identified K+ channels. Mutations of Sh and Shab K+ channels remove part of inactivating IA and sustained IK, respectively, and the remaining IA and IK reveal the properties of their counterparts, e.g., Shal and Shaw channels. Neuronal subsets displaying the delayed, tonic, adaptive, and damping spike patterns are characterized by different profiles of K+ current voltage dependence and kinetics and by differential mutational effects. Shab channels regulate membrane repolarization and repetitive firing over hundreds of milliseconds, and Shab neurons show a gradual decline in repolarization during current injection and their spike activities become limited to high-frequency, damping firing. In contrast, Sh channels acted on events within tens of milliseconds, and Sh mutations broadened spikes and reduced firing rates without eliminating any categories of firing patterns. However, removing both Sh and Shal IA by 4-aminopyridine (4-AP) converts the delayed to damping firing pattern, demonstrating their actions in regulating spike initiation. Specific blockade of Shab IK by quinidine mimic the Shab phenotypes and convert tonic firing to a damping pattern. These conversions suggest a hierarchy of complexity in K+ current interactions underlying different firing patterns. Different lineage-defined neuronal subsets, identifiable by employing the GAL4-UAS system, display different profiles of spike properties and K+ current compositions, providing opportunities for mutational analysis in functionally specialized neurons (Peng, 2007).
Characteristic firing patterns are found among different types of neurons that subserve specific functions in the nervous system. A variety of inward Na+ and Ca2+ currents and outward K+ currents take part in shaping neuronal action potentials and firing patterns. In particular, molecular studies have revealed a much greater diversity of K+ channel subtypes than that of Na+ and Ca2+ channels. Regulation of such molecular diversity facilitates the fine tuning of neuronal excitability and thus enriches spike patterning (Peng, 2007).
K+ currents have been classified according to their gating, kinetic, and pharmacological properties. In a variety of excitable cells, voltage-activated outward K+ currents are composed of transient, inactivating IA and sustained, noninactivating IK. Drosophila Shaker (Sh) mutants, initially isolated on the basis of their abnormal shaking behavior, have made possible the cloning of the first K+ channel gene and subsequent identification of three additional Drosophila K+ channel genes of the Sh family, Shab, Shaw, and Shal, and their vertebrate counterparts Kv 1, 2, 3, and 4. In heterologous expression systems, Sh and Shal channels mediate IA-like, fast-inactivating currents, whereas both Shaw and Shab regulate IK-like, slowly inactivating currents. Drosophila point mutations of Sh and Shab have demonstrated the in vivo roles of IA and IK channels at different levels, from cellular physiology to behavior, and can provide information about the regulation of neuronal excitability. Rich repertories of neuronal spike patterns have been described in several Drosophila semi-intact preparations. Nevertheless, it is difficult to delineate the spike patterns generated by synaptic interactions from those reflecting intrinsic neuronal membrane excitability in these preparations. Dissociated cell culture systems provide isolated conditions without cell-cell contacts and controlled ionic environment to eliminate contributions from synaptic interactions (Peng, 2007).
The 'giant' neuron culture system of Drosophila derived from cytokinesis-arrested embryonic neuroblasts displays differentiated morphological and molecular characteristics of different neuronal lineages. These enlarged cells facilitate Ca2+ imaging and electrophysiological recordings of Na+ and Ca2+ action potentials of different firing patterns. To explore the biophysical characteristics and functional roles of Sh and Shab channels, current- and voltage-clamp recordings were performed on the same cells in this culture system. The remaining IA in Sh and IK in Shab null mutants could provide opportunities to distinguish properties of Sh versus non-Sh IA channels (encoded by Shal and possibly other unidentified genes) as well as Shab versus non-Shab IK channels (encoded by Shaw and other candidate genes). The current results demonstrate the profound effect of Shab mutations on spike firing patterns and the distinctions between Sh channels and their counterpart, such as Shal channels. Advantage was taken of the GAL4-UAS system to demonstrate different profiles of K+ current kinetics and firing properties in cell-lineage defined neuronal subsets and their specific alterations by Sh and Shab mutations (Peng, 2007).
This study demonstrates that the Drosophila 'giant' neuron culture system can provide a bridge between heterologous expression systems and in vivo preparations to facilitate the study of how firing patterns are generated by interactions of molecularly identified channel subtypes and controlled by genes of interest. The current-clamp study has shown a diversity of firing patterns in WT cultures. Voltage-clamp recordings on the same cells further revealed the relationship of firing patterns and the compositions of underlying K+ currents. In addition, manipulating K+ channel compositions by employing mutations and pharmacological agents provided independent lines of evidence for the distinct contributions of each K+ current component to the control of membrane excitability (Peng, 2007).
In Drosophila muscles, it has been demonstrated that IA is eliminated by Sh mutations and IK is affected by Shab mutations. Mutations of ether a go-go (eag), another gene encoding a K+ channel subunit, can also reduce IA and IK in muscles. Several studies propose a different K+ channel expression pattern in neurons: IA channels are encoded by both Sh and Shal, but major component of IK is produced by Shab channels (Tsunoda, 1995). Consistently, in 'giant' neuron cultures, Sh and Shab null mutations only reduce, but did not eliminate IA or IK, indicating the presence of a substantial non-Sh component for IA and a minor non-Shab component for IK in neurons (Peng, 2007).
The results provide a first description of the striking phenotype of Shab neurons. During sustained current injection, Shab neurons rely on other noninactivating K+ currents for action potential repolarization as the transient IA becomes progressively inactivated. The resultant abnormal damping spike pattern and unusual regenerative activities contrast the roles of Shab and non-Shab channels (including Shaw). In most Shab neurons, spiking or nonspiking, a novel 'repolarization decline' phenotype is observed that reflects a failure in maintaining a steady level of membrane repolarization. Furthermore, the spiking activity in Shab cultures was restricted to the damping firing pattern and was coupled with T3 current kinetics. Consistently, quinidine-treatments, which specifically remove Shab currents in WT neurons, closely mimic the 'repolarization decline' phenotype and converted spiking patterns into damping firing (Peng, 2007).
The mutational effects demonstrate that Shab IK is more important for signal processing at time scales of hundreds of milliseconds. Shab mutant neurons are capable of generating normal action potentials in response to brief stimuli, but prolonged current injection causes abnormal high-frequency, run-away spikes that degenerate into dwindling oscillations. WT neurons expressing both Shab and non-Shab sustained currents fire full-blown action potentials ~25 Hz on average. In contrast, T3 Shab neurons (IS/IP >0.5) that retain substantial non-Shab currents, including Shaw, generate high-frequency firing (up to 60 Hz) either immediately on depolarization or with a gradual development. Interestingly, it has been shown that pharmacological blockade or mutations of Shaw-like Kv3 channels disable the high-frequency firing (often up to 1 kHz) found in certain neuronal types in the hippocampus, basal ganglia, and auditory nuclei. Because the exact dynamics of membrane repolarization can determine the timing of recovery of inward Na+ and Ca2+ currents from inactivation for resetting another cycle of spike activity, the balancing act between Shab and non-Shab IK currents can enrich spike frequency control during repetitive firing (Peng, 2007).
An interesting parallel of the striking Shab phenotype during repetitive firing is observed at the larval neuromuscular junction (Ueda, 2006). With single nerve stimuli, mutant Shab synaptic transmission appears normal. During high-frequency nerve stimulation, an explosive neuromuscular transmission (up to 10-fold gain, termed the 'big bang' phenomenon) could suddenly occur when cumulative inactivation of IA reaches a critical level. The phenomenon can also be induced in WT by repetitive stimulation following quinidine treatment and by single nerve stimuli in quinidine-treated Sh preparations (Ueda, 2006). The generation of a plateau membrane potential in the motor axon terminals, which are enriched with both Na+ and Ca2+ channels, has been proposed to account for this phenomenon when both IA and IK are weakened by mutations, drugs, or activity-dependent inactivation. These observations are reminiscent of the 'repolarization decline' in Shab neurons and quinidine-treated WT neurons during prolonged current injection. Taken together, an intricate interaction between slowly inactivating IK and fast inactivating IA is important during the dynamic process of repetitive firing for maintaining the cycles of membrane excitation and repolarization (Peng, 2007).
Notably, simple removal of Shab current by acute quinidine treatment on WT cultures converts firing patterns to damping rather than nonspiking activities. However, in Shab mutant cultures, a drastic increase of nonspiking neurons was observed, contrary to the expectation of increased excitability caused by reduced IK. A clue to this unexpected finding is provided by the observation that in some nonspiking Shab cells, regenerative oscillations could still be initiated when the transient IA was suppressed by 4-AP or a depolarizing prepulse. Voltage-clamp measurements yielded direct evidence for an increase in the inactivating IA in Shab cultures. The peak total current (IP), which represents the sum of peak IA and IK, remained undiminished despite the fact that the sustained current component (IS) was significantly decreased in mutant Shab neurons. In contrast, pharmacological removal of Shab currents reduced both IS (~35%) and IP (~30%). These observations resemble the homeostatic regulation of ion channel previously reported in other preparations. When neuronal spike activity is manipulated, a homeostatic regulation can be initiated to adjust the relative abundance of ion channels and other proteins. Over-expression of Shal IA in lobster neurons triggers a compensatory increase of hyperpolarization-activated inward Ih. Therefore a compensatory upregulation of the transient K+ current component in mutant Shab cultures could account for the lower percentage of spiking cells. Overexpression of transient IA could prevent spike initiation in the standard current-clamp protocols employed in this study. Additional types of compensatory mechanisms, such as decreased expression of inward Na+ or Ca2+ currents, might also occur in Shab neurons. However, preliminary results indicate that Ca2+ current density in Shab mutant cultures remains unaltered compared with that in WT cells, although potential modification of Na+ currents require further investigation (Peng, 2007).
In contrast to Shab channels, Sh channels play a role in regulating rapid events within a millisecond time scale. Broadened action potentials were observed in mutant Sh neurons with delayed and tonic firing patterns. This demonstrates a role of action potential repolarization for Sh channels, consistent with greatly prolonged action potentials documented in the cervical giant fiber of Sh mutants (Peng, 2007).
A well-established function of transient K+ currents is pertinent to the control of spike initiation time during excitatory inputs. Mutational and pharmacological analyses confirm the important but overlapping roles of Sh and Shal IA channels in controlling spike initiation. Neurons exhibiting delayed firing persisted in Sh cultures, supporting the idea that within only a small subset of neurons, IA channels are exclusively or predominantly encoded by Sh. Furthermore, no disproportional reduction in any of the categories of firing patterns was observed in Sh cultures, indicating that Sh and Shal IA may serve redundant roles in suppressing transient depolarization, and thus the action of Shal IA alone could retain the dynamic manifestation of the delayed and other firing patterns (Peng, 2007).
Delayed or tonic firing in WT and Sh cultures could be converted into damping patterns after 4-AP treatment, suggesting that with total elimination of transient IA, spike repolarization deteriorates during repetitive firing. Nevertheless, such damping firing patterns were distinct from those observed in Shab or quinidine-treated WT neurons, in that the high-frequency oscillations typical of Shab neurons were never observed. In summary, a clear rank of firing rate was observed in the damping patterns of the three genotypes: Shab > WT > Sh (Peng, 2007).
The slower decay of transient IA in mutant Sh cultures is in agreement with previous reports on pupal and larval cultures, indicating that the remaining Shal channels inactivate more slowly than Sh channels. This is also consistent with the conclusion based on Sh and Shal channel expression experiments in the frog oocyte. The results also suggest that Shal channels follow a slower kinetics of recovery from inactivation, something that awaits confirmation in other preparations. However, steady-state inactivation measurements of IA in Sh cultures suggest that Shal channels inactivate at voltages more positive than that for Sh channels, contrary to the preceding reports. The reason for the discrepancy is unknown, although expression patterns of the Sh and Shal splice variants as well as their potential association with auxiliary channel subunits could differ among the embryonic, larval and pupal cultures used in these studies. Splice variants of the Sh products are known to mediate K+ currents of varying degrees of inactivation when expressed heterologously in Xenopus oocytes. Similarly, subtypes of mammalian Kv1 channels also display different inactivation properties (Peng, 2007).
In some cases, 4-AP did not completely eliminate the transient component in T3 cells in WT and Sh cultures. Such 4-AP insensitive transient component may reflect low sensitivity to 4-AP in certain splicing isoforms of Sh or Shal subunits. Alternatively, inactivating isoforms of Shaw may exist, since a mammalian homologue of Shaw, Kv3.4, mediates inactivating K+ currents (Peng, 2007).
Although the tonic firing pattern is insensitive to 4-AP in WT neurons, 4-AP blockade of the remaining Shal currents in Sh neurons converts the tonic firing pattern to adaptive or damping in two out of three cases. Apparently, the currents contributing to the tonic firing pattern have been reconfigured in some Sh neurons relative to WT neurons. In addition, the population of nonspiking neurons is also increased in Sh cultures, contrary to the expectation that removal of Sh currents leads to hyperexcitability. Again, the possibility of downregulation of inward Na+ and Ca2+ currents in Sh neurons must be considered, similar to the case for Shab mutant neurons. Furthermore, upregulation of Shal or other transient currents in Sh mutant neurons is possible. However, over-expression of non-Sh transient currents would be rather limited because a significant reduction of IP was still evident in Sh cultures. Taken together, these observations suggest diverse and cell-specific compensatory mechanisms still await further exploration in the heterogeneous population of the nervous system (Peng, 2007).
Both Sh and Shab mutations alter the abundance of the individual kinetic categories of total voltage-activated K+ currents among cultured neurons. This redistribution conceivably leads to a population conversion of neuronal types in mutant cultures. For example, part of the more populated T1-like neurons in Shab cultures reflects a conversion from T2 and T3 neurons on removal of Shab IK as indicated by the unusually fast decay kinetics in some Shab T1 cells that resemble the decay time course of WT T2 and T3 neurons. Similarly, the overpopulated T3 cells in Sh cultures might be converted from T1 and T2 neurons, reflecting enhanced representation of Shal currents. The assumption of population conversion on removal of Sh and Shal channels is corroborated by the results of drug treatments in both voltage- and current-clamp experiments that reveal differential expression of K+ channels required for generation of different firing patterns. Notably, neurons with delayed firing patterns were converted by 4-AP to damping patterns but were not affected by quinidine, suggesting an abundance of transient IA coupled with a deficiency of Shab IK in this cell category. In contrast, quinidine converted tonic to damping firing patterns, indicating a strong dependence on Shab IK during tonic firing. These observations suggest that the damping firing pattern requires actions of fewer K+ channel subtypes. In other words, an apparent order of complexity exists for K+ current interactions that underlie different firing patterns, suggesting an interesting scheme, in which differential expression or modulation of K+ channels may generate a diversity of neuronal firing patterns to fulfill the specific tasks and functional plasticity of neuronal circuits (Peng, 2007).
Several Drosophila Gal4 lines have been used to drive targeted expression of UAS-GFP to identify specific neuronal subsets in larval or pupal culture systems. Using this approach, it was demonstrated that in embryonic 'giant' neuron cultures, neurons of different cell lineages displayed characteristic excitability properties and K+ current kinetics. This study observed a substantial inactivating K+ current component in 201Y(+) cells in embryonic 'giant' neuron cultures, similar to that reported for cultured neurons dissociated from larval mushroom body (Peng, 2007).
It should be noted that channel distribution among different neuronal compartments in dissociated neuron cultures might not exactly match that of native neurons with their interacting partners in vivo and thus may modify the firing pattern of particular neuronal subpopulations. Moreover, in cell division-arrested embryonic neurons, the channel expression profile may reflect a combination of expression patterns of the neuronal descendants. It is known that the progeny of one insect neuroblast can develop different excitability properties (Peng, 2007).
Despite these caveats, electrophysiological and Ca2+ imaging studies on the same preparation have indicated a disparity between soma and neurites in K+ channel distribution similar to the pattern found in neurons of other in vivo preparations. In addition, the present physiological results of different K+ currents are directly comparable to the measurements from acutely dissociated CNS neurons of Drosophila larvae, pupae and adults in terms of kinetic categories, current compositions and mutational effects. Therefore the 'giant' neuron culture system can serve the purpose to link data of different levels that obtained from heterologous expression systems and in vivo preparations. In this system, results from electrophysiological analyses can be integrated with Ca2+ imaging to reveal functional specialization in different neuronal lineages. Combining the baseline information obtained in neuronal cultures with in vivo studies will help elucidate the cellular mechanisms that enable the neuronal circuits of interest to perform particular functional tasks in Drosophila (Peng, 2007).
Shaker, a voltage-dependent K+ channel, is enriched in the mushroom bodies (MBs), the locus of olfactory learning in Drosophila. Mutations in the shaker locus are known to alter excitability, neurotransmitter release, synaptic plasticity, and olfactory learning. However, a direct link of Shaker channels to MB intrinsic neuron (MBN) physiology has not been documented. This study found that transcripts for shab, shaw, shaker, and shal, among which only Shaker and Shal have been reported to code for A-type currents, are present in the MBs. The electrophysiological data showed that the absence of functional Shaker channels modifies the distribution of half-inactivation voltages (V(i1/2)) in the MBNs, indicating a segregation of Shaker channels to only a subset (approximately 28%) of their somata. In harmony with this notion, this study found that approximately one-fifth of MBNs lacking functional Shaker channels displayed dramatically slowed-down outward current inactivation times and reduced peak-current amplitudes. Furthermore, whereas all MBNs were sensitive to 4-aminopyridine, a nonspecific A-type current blocker, a subset of neurons (approximately 24%) displayed little sensitivity to a Shal-specific toxin. This subset of neurons displaying toxin-insensitive outward currents had more depolarized V(i1/2) values attributable to Shaker channels. These findings provide the first direct evidence that altered Shaker channel function disrupts MBN physiology in Drosophila. Surprisingly, the experimental data also indicate that Shaker channels segregate to a minor fraction of MB neuronal somata (20%-30%) and that Shal channels contribute the somatic A-type current in the majority of MBNs (Gasque, 2005).
This study has used dye fills and electrophysiological recordings to identify and characterize a cluster of motor neurons in the third instar larval ventral ganglion. This cluster of neurons is similar in position to the well-studied embryonic RP neurons. Dye fills of larval dorsomedial neurons demonstrate that individual neurons within the cluster can be reproducibly identified by observing their muscle targets and bouton morphology. The terminal targets of these five neurons are body wall muscles 6/7, 1, 14, and 30 and the intersegmental nerve (ISN) terminal muscles (1, 2, 3, 4, 9, 10, 19, 20). All cells except the ISN neuron, which has a type Is ending, display type Ib boutons. Two of these neurons appear to be identical to the embryonic RP3 and aCC cells, which define the most proximal and distal innervations within a hemisegment. The targets of the other neurons in the larval dorsomedial cluster do not correspond to embryonic targets of the neurons in the RP cluster, suggesting rewiring of this circuit during early larval stages. Electrophysiological studies of the five neurons in current clamp revealed that type Is neurons have a longer delay in the appearance of the first spike compared with type Ib neurons. Genetic, biophysical, and pharmacological studies in current and voltage clamp show this delay is controlled by the kinetics and voltage sensitivity of inactivation of a current whose properties suggest that it may be the Shal I(A) current. The combination of genetic identification and whole cell recording allows direct exploration of the cellular substrates of neural and locomotor behavior in an intact system (Choi, 2004).
The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the cAMP-dependent protein kinase A (PKA) cascades have been implicated in neural mechanisms underlying learning and memory as supported by mutational analyses of the two enzymes in Drosophila. While there is mounting evidence for their roles in synaptic plasticity, less attention has been directed toward their regulation of neuronal membrane excitability and spike information coding. This study reports genetic and pharmacological analyses of the roles of PKA and CaMKII in the firing patterns and underlying K(+) currents in cultured Drosophila central neurons. Genetic perturbation of the catalytic subunit of PKA (DC0) did not alter the action potential duration but disrupted the frequency coding of spike-train responses to constant current injection in a subpopulation of neurons. In contrast, selective inhibition of CaMKII by the expression of an inhibitory peptide in ala transformants prolonged the spike duration but did not affect the spike frequency coding. Enhanced membrane excitability, indicated by spontaneous bursts of spikes, was observed in CaMKII-inhibited but not in PKA-diminished neurons. In wild-type neurons, the spike train firing patterns were highly reproducible under consistent stimulus conditions. However, disruption of either of these kinase pathways led to variable firing patterns in response to identical current stimuli delivered at a low frequency. Such variability in spike duration and frequency coding may impose problems for precision in signal processing in these protein kinase learning mutants. Pharmacological analyses of mutations that affect specific K(+) channel subunits demonstrated distinct effects of PKA and CaMKII in modulation of the kinetics and amplitude of different K(+) currents. The results suggest that PKA modulates Shaker A-type currents, whereas CaMKII modulates Shal-A type currents plus delayed rectifier Shab currents. Thus differential regulation of K(+) channels may influence the signal handling capability of neurons. This study provides support for the notion that, in addition to synaptic mechanisms, modulations in spike activity patterns may represent an important mechanism for learning and memory that should be explored more fully (Yao, 2001).
Perceptual decisions require the accumulation of sensory information to a response criterion. Most accounts of how the brain performs this process of temporal integration have focused on evolving patterns of spiking activity. This study reports that subthreshold changes in membrane voltage can represent accumulating evidence before a choice. alphabeta core Kenyon cells (alphabetac KCs) in the mushroom bodies of fruit flies integrate odor-evoked synaptic inputs to action potential threshold at timescales matching the speed of olfactory discrimination. The forkhead box P transcription factor (FoxP) sets neuronal integration and behavioral decision times by controlling the abundance of the voltage-gated potassium channel Shal (KV4) in alphabetac KC dendrites. alphabetac KCs thus tailor, through a particular constellation of biophysical properties, the generic process of synaptic integration to the demands of sequential sampling (Groschner, 2018).
A de novo mutation in the KCND2 gene, which encodes the Kv4.2 K(+) channel, was identified in twin boys with intractable, infant-onset epilepsy and autism. Kv4.2 channels undergo closed-state inactivation (CSI), a mechanism by which channels inactivate without opening during subthreshold depolarizations. CSI dynamically modulates neuronal excitability and action potential back propagation in response to excitatory synaptic input, controlling Ca(2+) influx into dendrites and regulating spike timing-dependent plasticity. This study shows that the V404M mutation specifically affects the mechanism of CSI, enhancing the inactivation of channels that have not opened while dramatically impairing the inactivation of channels that have opened. The mutation gives rise to these opposing effects by increasing the stability of the inactivated state and in parallel, profoundly slowing the closure of open channels, which according to the data, is required for CSI. The larger volume of methionine compared with valine is a major factor underlying altered inactivation gating. The results suggest that V404M increases the strength of the physical interaction between the pore gate and the voltage sensor regardless of whether the gate is open or closed. Furthermore, in contrast to previous proposals, the current data strongly suggest that physical coupling between the voltage sensor and the pore gate is maintained in the inactivated state. The state-dependent effects of V404M on CSI are expected to disturb the regulation of neuronal excitability and the induction of spike timing-dependent plasticity. These results strongly support a role for altered CSI gating in the etiology of epilepsy and autism in the affected twins (Lin, 2018).
The K(+) channel pore-forming subunit Kv4.3 is expressed in a subset of nonpeptidergic nociceptors within the dorsal root ganglion (DRG), and knockdown of Kv4.3 selectively induces mechanical hypersensitivity, a major symptom of neuropathic pain. K(+) channel modulatory subunits KChIP1, KChIP2, and DPP10 are coexpressed in Kv4.3(+) DRG neurons, but whether they participate in Kv4.3-mediated pain control is unknown. This study shows the existence of a Kv4.3/KChIP1/KChIP2/DPP10 complex (abbreviated as the Kv4 complex) in the endoplasmic reticulum and cell surface of DRG neurons. After intrathecal injection of a gene-specific antisense oligodeoxynucleotide to knock down the expression of each component in the Kv4 complex, mechanical hypersensitivity develops in the hindlimbs of rats in parallel with a reduction in all components in the lumbar DRGs. Electrophysiological data further indicate that the excitability of nonpeptidergic nociceptors is enhanced. The expression of all Kv4 complex components in DRG neurons is downregulated following spinal nerve ligation (SNL). To rescue Kv4 complex downregulation, cDNA constructs encoding Kv4.3, KChIP1, and DPP10 were transfected into the injured DRGs (defined as DRGs with injured spinal nerves) of living SNL rats. SNL-evoked mechanical hypersensitivity was attenuated, accompanied by a partial recovery of Kv4.3, KChIP1, and DPP10 surface levels in the injured DRGs. By showing an interdependent regulation among components in the Kv4 complex, this study demonstrates that K(+) channel modulatory subunits KChIP1, KChIP2, and DPP10 participate in Kv4.3-mediated mechanical pain control. Thus, these modulatory subunits could be potential drug targets for neuropathic pain (Kuo, 2017).
Voltage-gated K(+) (Kv) channel activation depends on interactions between voltage sensors and an intracellular activation gate that controls access to a central pore cavity. It was hypothesized that this gate is additionally responsible for closed-state inactivation (CSI) in Kv4.x channels. These Kv channels undergo CSI by a mechanism that is still poorly understood. To test the hypothesis, the state of the Kv4.1 channel intracellular gate was deduced by exploiting the trap-door paradigm of pore blockade by internally applied quaternary ammonium (QA) ions exhibiting slow blocking kinetics and high-affinity for a blocking site. Inactivation gating seemingly traps benzyl-tributylammonium (bTBuA) when it enters the central pore cavity in the open state. However, bTBuA fails to block inactivated Kv4.1 channels, suggesting gated access involving an internal gate. In contrast, bTBuA blockade of a Shaker Kv channel that undergoes open-state P/C-type inactivation exhibits fast onset and recovery inconsistent with bTBuA trapping. Furthermore, the inactivated Shaker Kv channel is readily blocked by bTBuA. It is concluded that Kv4.1 closed-state inactivation modulates pore blockade by QA ions in a manner that depends on the state of the internal activation gate (Fineberg, 2016).
Shal (Kv4) alpha-subunits are the most conserved among the family of voltage-gated potassium channels. Previous work has shown that the Shal potassium channel subfamily underlies the predominant fast transient outward current in Drosophila neurons and the fast transient outward current in mouse heart muscle. This study shows that Shal channels also play a role as the predominant transient outward current in Caenorhabditis elegans muscle. Green fluorescent protein promoter experiments also revealed SHL-1 expression in a subset of neurons as well as in C. elegans body wall muscle and in male-specific diagonal muscles. The shl-1 (ok1168) null mutant removed all fast transient outward current from muscle cells. SHL-1 currents strongly resembled Shal currents in other species except that they were active in a more depolarized voltage range. It was also determined that the remaining delayed-rectifier current in cultured myocytes was carried by the Shaker ortholog SHK-1. In shl-1 (ok1168) mutants there was a significant compensatory increase in the SHK-1 current. Male shl-1 (ok1168) animals exhibited reduced mating efficiency resulting from an apparent difficulty in locating the hermaphrodite vulva. SHL-1 channels are apparently important in fine-tuning complex behaviors, such as mating, that play a crucial role in the survival and propagation of the species (Fawcett, 2006).
Search PubMed for articles about Drosophila Shal
Bergquist, S., Dickman, D. K. and Davis, G. W. (2010). A hierarchy of cell intrinsic and target-derived homeostatic signaling. Neuron 66(2): 220-234. PubMed ID: 20434999
Choi, J. C., Park, D. and Griffith, L. C. (2004). Electrophysiological and morphological characterization of identified motor neurons in the Drosophila third instar larva central nervous system. J Neurophysiol 91(5): 2353-2365. PubMed ID: 14695352
Diao, F., Waro, G. and Tsunoda, S. (2009). Fast inactivation of Shal (K(v)4) K+ channels is regulated by the novel interactor SKIP3 in Drosophila neurons. Mol Cell Neurosci 42(1): 33-44. PubMed ID: 19463952
Diao, F., Chaufty, J., Waro, G. and Tsunoda, S. (2010). SIDL interacts with the dendritic targeting motif of Shal (K(v)4) K+ channels in Drosophila. Mol Cell Neurosci 45(1): 75-83. PubMed ID: 20550966
Fawcett, G. L., Santi, C. M., Butler, A., Harris, T., Covarrubias, M. and Salkoff, L. (2006). Mutant analysis of the Shal (Kv4) voltage-gated fast transient K+ channel in Caenorhabditis elegans. J Biol Chem 281(41): 30725-30735. PubMed ID: 16899454
Feng, G., Pang, J., Yi, X., Song, Q., Zhang, J., Li, C., He, G. and Ping, Y. (2018a). Down-regulation of KV4 channel in Drosophila mushroom body neurons contributes to Abeta42-induced courtship memory deficits. Neuroscience 370: 236-245. PubMed ID: 28627422
Feng, G., Zhang, J., Li, M., Shao, L., Yang, L., Song, Q. and Ping, Y. (2018b). Control of sleep onset by Shal/Kv4 channels in Drosophila circadian neurons. J Neurosci. 38(42):9059-9071. PubMed ID: 30185460
Fineberg, J. D., Szanto, T. G., Panyi, G. and Covarrubias, M. (2016). Closed-state inactivation involving an internal gate in Kv4.1 channels modulates pore blockade by intracellular quaternary ammonium ions. Sci Rep 6: 31131. PubMed ID: 27502553
Gasque, G., Labarca, P., Reynaud, E. and Darszon, A. (2005). Shal and shaker differential contribution to the K+ currents in the Drosophila mushroom body neurons. J Neurosci 25(9): 2348-2358. PubMed ID: 15745961
Groschner, L. N., Chan Wah Hak, L., Bogacz, R., DasGupta, S. and Miesenbock, G. (2018). Dendritic Integration of Sensory Evidence in Perceptual Decision-Making. Cell 173(4): 894-905 e813. PubMed ID: 29706545
Jegla, T., et al. (2016). Bilaterian giant Ankyrins have a common evolutionary origin and play a conserved role in patterning the axon initial segment. PLoS Genet 12(12): e1006457. PubMed ID: 27911898
Kulik, Y., Jones, R., Moughamian, A. J., Whippen, J. and Davis, G. W. (2019). Dual separable feedback systems govern firing rate homeostasis. Elife 8. PubMed ID: 30973325
Kuo, Y. L., Cheng, J. K., Hou, W. H., Chang, Y. C., Du, P. H., Jian, J. J., Rau, R. H., Yang, J. H., Lien, C. C. and Tsaur, M. L. (2017). K(+) Channel Modulatory Subunits KChIP and DPP Participate in Kv4-Mediated Mechanical Pain Control. J Neurosci 37(16): 4391-4404. PubMed ID: 28330877
Lin, M. A., Cannon, S. C. and Papazian, D. M. (2018). Kv4.2 autism and epilepsy mutation enhances inactivation of closed channels but impairs access to inactivated state after opening. Proc Natl Acad Sci U S A 115(15): E3559-E3568. PubMed ID: 29581270
Muraro, N. I., et al. (2008). Pumilio binds para mRNA and requires Nanos and Brat to regulate sodium current in Drosophila motoneurons. J. Neurosci. 28(9): 2099-109. PubMed Citation: 18305244
Parrish, J. Z., Kim, C. C., Tang, L., Bergquist, S., Wang, T., Derisi, J. L., Jan, L. Y., Jan, Y. N. and Davis, G. W. (2014). Kruppel mediates the selective rebalancing of ion channel expression. Neuron 82: 537-544. PubMed ID: 24811378
Peng, I. F. and Wu, C. F. (2007). Differential contributions of Shaker and Shab K+ currents to neuronal firing patterns in Drosophila. J. Neurophysiol. 97(1): 780-94. PubMed ID: 17079336
Pimentel, D., Donlea, J. M., Talbot, C. B., Song, S. M., Thurston, A. J. F. and Miesenbock, G. (2016). Operation of a homeostatic sleep switch. Nature 536(7616): 333-337. PubMed ID: 27487216
Ping, Y., Waro, G., Licursi, A., Smith, S., Vo-Ba, D. A. and Tsunoda, S. (2011a). Shal/K(v)4 channels are required for maintaining excitability during repetitive firing and normal locomotion in Drosophila. PLoS One 6(1): e16043. PubMed ID: 21264215
Ping, Y. and Tsunoda, S. (2011b). Inactivity-induced increase in nAChRs upregulates shal K(+) channels to stabilize synaptic potentials. Nat Neurosci 15:90-97. PubMed ID: 22081160
Pulver, S. R. and Griffith, L. C. (2010). Spike integration and cellular memory in a rhythmic network from Na+/K+ pump current dynamics. Nat Neurosci 13(1): 53-59. PubMed ID: 19966842
Ryglewski, S. and Duch, C. (2009). Shaker and Shal mediate transient calcium-independent potassium current in a Drosophila flight motoneuron. J Neurophysiol 102(6): 3673-3688. PubMed ID: 19828724
Schaefer, J. E., Worrell, J. W. and Levine, R. B. (2010). Role of intrinsic properties in Drosophila motoneuron recruitment during fictive crawling. J Neurophysiol 104(3): 1257-1266. PubMed ID: 20573969
Smith, P., Buhl, E., Tsaneva-Atanasova, K. and Hodge, J. J. L. (2019). Shaw and Shal voltage-gated potassium channels mediate circadian changes in Drosophila clock neuron excitability. J Physiol 597(23): 5707-5722. PubMed ID: 31612994
Srinivasan, S., Lance, K. and Levine, R. B. (2012). Segmental differences in firing properties and potassium currents in Drosophila larval motoneurons. J Neurophysiol 107(5): 1356-1365. PubMed ID: 22157123
Tsunoda, S. and Salkoff, L. (1995). The major delayed rectifier in both Drosophila neurons and muscle is encoded by Shab. J Neurosci 15(7 Pt 2): 5209-5221. PubMed ID: 7623146
Ueda, A. and Wu, C. F. (2006). Distinct frequency-dependent regulation of nerve terminal excitability and synaptic transmission by IA and IK potassium channels revealed by Drosophila Shaker and Shab mutations. J Neurosci 26(23): 6238-6248. PubMed ID: 16763031
Wolfram, V., Southall, T. D., Gunay, C., Prinz, A. A., Brand, A. H. and Baines, R. A. (2014). The transcription factors islet and lim3 combinatorially regulate ion channel gene expression. J Neurosci 34: 2538-2543. PubMed ID: 24523544
Yao, W. D. and Wu, C. F. (2001). Distinct roles of CaMKII and PKA in regulation of firing patterns and K(+) currents in Drosophila neurons. J Neurophysiol 85(4): 1384-1394. PubMed ID: 11287463
date revised: 2 December 2019
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