InteractiveFly: GeneBrief

paralytic: Biological Overview | References

BIOLOGICAL OVERVIEW

The voltage-gated sodium channel (Na_v) plays a key role in regulation of neuronal excitability. Aberrant regulation of Na_v expression and/or function can result in an imbalance in neuronal activity which can progress to epilepsy. Regulation of Na_v activity is achieved by coordination of a multitude of mechanisms including RNA alternative splicing and translational repression. Understanding of these regulatory mechanisms is complicated by extensive genetic redundancy: the mammalian genome encodes ten Na_vs. By contrast, the genome of the fruitfly, Drosophila melanogaster, contains just one Na_v homologue, encoded by paralytic (DmNa_v). Analysis of splicing in DmNa_v shows variants exhibit distinct gating properties including varying magnitudes of persistent sodium current (INaP) (see Schematic of the predicted topology of the voltage-gated sodium channel showing approximate locations of Drosophila spliced exons). Splicing by Pasilla, an identified RNA splicing factor, alters INaP magnitude as part of an activity-dependent mechanism. Enhanced INaP promotes membrane hyperexcitability that is associated with seizure-like behaviour in Drosophila. Nova-2, a mammalian Pasilla homologue, has also been linked to splicing of Na_vs and, moreover, mouse gene knockouts display seizure-like behaviour. Expression level of Na_vs is also regulated through a mechanism of translational repression in both flies and mammals. The translational repressor Pumilio (Pum) can bind to Na_v transcripts and repress the normal process of translation, thus regulating sodium current (INa) density in neurons. Pum2-deficient mice exhibit spontaneous EEG abnormalities. Taken together, aberrant regulation of Na_v function and/or expression is often epileptogenic. As such, a better understanding of regulation of membrane excitability through RNA alternative splicing and translational repression of Na_vs should provide new leads to treat epilepsy (Lin, 2014).

Shudderer (Shu) is an X-linked dominant mutation in Drosophila melanogaster identified more than 40 years ago. A previous study showed that Shu caused spontaneous tremors and defects in reactive climbing behavior, and that these phenotypes were significantly suppressed when mutants were fed food containing lithium, a mood stabilizer used in the treatment of bipolar disorder. This unique observation suggested that the Shu mutation affects genes involved in lithium-responsive neurobiological processes. The present study identified Shu as a novel mutant allele of the voltage-gated sodium (Na_v) channel gene paralytic (para). Given that hypomorphic para alleles and RNA interference-mediated para knockdown reduced the severity of Shu phenotypes, Shu was classified as a para hypermorphic allele. It was demonstrated that lithium could improve the behavioral abnormalities displayed by other Na_v mutants, including a fly model of the human generalized epilepsy with febrile seizures plus. Electrophysiological analysis of Shu showed that lithium treatment did not acutely suppress Na_v channel activity, indicating that the rescue effect of lithium resulted from chronic physiological adjustments to this drug. Microarray analysis revealed that lithium significantly alters the expression of various genes in Shu, including those involved in innate immune responses, amino acid metabolism, and oxidation-reduction processes, raising the interesting possibility that lithium-induced modulation of these biological pathways may contribute to such adjustments. Overall, these findings demonstrate that Na_v channel mutants in Drosophila are valuable genetic tools for elucidating the effects of lithium on the nervous system in the context of neurophysiology and behavior (Kaas, 2016).

Since the initial discovery in 1949 that lithium possesses mood-stabilizing, the alkali metal has remained one of the most widely used medications for bipolar disorder (BPD). Over the years, various hypotheses have been proposed to explain the physiological effects of lithium. These stem mainly from lithium's ability to inhibit, either directly or indirectly, particular enzymes such as glycogen synthase kinase 3β (GSK-3β), inositol monophosphatase (IMPase), and inositol 1-polyphosphate phosphatase (IPPase). By inhibiting these enzymes, lithium is thought to alter the signaling cascades in which they participate, ultimately influencing an array of physiological. However, the exact mechanisms by which lithium modifies neural function and improves pathophysiological behaviors are still not fully understood (Kaas, 2016).

Studies using animal models suggest that the effects of lithium on the nervous system have common features across a range of diverse animal species. In particular, the fruit fly Drosophila melanogaster displays a neurobiological response to lithium resembling that observed in mammals. For instance, chronic treatment with lithium lengthens the free-running period of the circadian rhythm in both mice and fruit flies. Likewise, lithium can protect against polyglutamine-induced neurotoxicity in Drosophila, as is observed in rodent models of Huntington's disease. Furthermore, lithium treatment can rescue the behavioral deficits exhibited by the Drosophila fragile X mental retardation 1 (dfmr1) mutant, as was observed in human fragile X patients and rodent models of the disease. These findings indicate that studies in Drosophila can provide valuable insights into the basic mechanisms of lithium action on neural function and behavior (Kaas, 2016).

Shudderer (Shu) was originally isolated more than 40 years ago, through an ethyl methanesulfonate mutational screen in Drosophila, as an X chromosome-linked dominant mutation that causes sporadic jerks and defects in reactive climbing behavior (Williamson, 1982). Interestingly, Williamson reported that these behavioral phenotypes were significantly suppressed when adult Shu mutants were fed a diet supplemented with lithium salts. This finding implied that the Shu mutation affects genes related to lithium-responsive neurobiological processes. No research on Shu has followed since the original report in 1982, and this mutation remains to be characterized at both the molecular and functional levels. The present study has identified Shu as a hypermorphic mutation in the Drosophila voltage-gated sodium (Na_v) channel gene paralytic (para) and found that lithium also mitigates behavioral phenotypes of some other para mutants. A combination of molecular, pharmacological, electrophysiological, and behavioral analyses from this study suggests that lithium's suppressive effect on Shu phenotypes is not due to acute actions of the drug but rather through long-term physiological adjustments to lithium treatment. These results connecting lithium with Na_v channel dysfunction demonstrate that Shu and other Na_v channel mutants in Drosophila are valuable genetic tools for investigating the effects of lithium on the nervous system in the context of neurophysiology and behavior. (Kaas, 2016).

This study has identified Shu, a Drosophila mutation causing severe behavioral defects, as a novel hypermorphic allele of the Na_v channel gene para. The notion that the Shu mutation is hypermorphic is based on Shu's morphological, electrophysiological, and behavioral phenotypes as well as the observation that Shu phenotypes were substantially suppressed when para function was reduced using para hypomorphs or para-RNAi. These results suggest that the Shu Na_v channels cause an increase in sodium currents, and that this defect is compensated by reduced para function, leading to a more balanced neuronal output (Kaas, 2016).

It was possible to at least partly recapitulate Shu phenotypes by driving expression of Shu Na_v channels in wild-type flies. However, the effect of the Shu transgene was mild when the motor neuron-positive driver C164-Gal4 was used, and there was little effect with a pan-neuronal driver elav-Gal4. This result somewhat resembles that of a previous attempt to reproduce the para^bss1 mutant phenotype with pan-neuronal expression of the para^bss1 transgene (Parker, 2011), where only a small subset (1.6%) of wild-type flies expressing the para^bss1 transgene displayed the bang-sensitive phenotype. Although the reasons underlying the weak effect of these mutant transgenes remain unknown, a lack of alternative splicing may contribute. Previous studies revealed that 27-29 distinct para splice variants exist, and that they are functionally diverse (Olson, 2008; Lin, 2009). In the current study and that of Parker (2011), the cDNA of the Na_v 1-1 para isoform, which is presumably the most common splice variant in adult flies, was utilized to construct the Shu and para^bss1 transgenes. To completely mimic the effects of these dominant para mutations, the transgenes may need to be expressed, in specific cell types, as certain splice variants (Kaas, 2016).

Importantly, as originally reported by Williamson (1982), this study observed suppression of the seizure-like phenotypes of Shu when the mutants were fed lithium-containing food. This study also demonstrated that GF motor outputs of Shu mutants exhibit hyperexcitable, seizure-like discharges and that lithium partially rescues these phenotypes as well. The results show that lithium can improve physiological as well as behavioral defects originally caused by aberrant sodium channel function. Consistently, lithium had a rescue effect on the heat-induced seizure phenotype of para^GEFS+ and the bang-sensitive phenotype of para^bss1. Based on their electrophysiological phenotypes, both para^GEFS+ and para^bss1 are considered to be para gain-of-function alleles (Parker, 2011; Sun, 2012). In contrast, lithium does not seem to improve the phenotypes of two para loss-of-function alleles, para^DS and para^ts1. This apparent correlation between types of Na_v channel mutations (i.e., gain- and loss-of-function) and the effects of lithium suggest that lithium may preferentially improve defects caused by aberrantly activated Na_v channels (Kaas, 2016).

Interestingly, many antiepileptic drugs targeting Na_v channels, such as lamotrigine, are effective for BPD. This therapeutic regimen for BPD suggests that its pathophysiology may be influenced by abnormal Na_v channel function. Further evidence for the connection between BPD and Na_v channels comes from genome-wide association studies, which have implicated ankyrin-G (ANK3) as a potential risk factor for BPD. ANK3 encodes an adaptor protein that regulates Na_v channel assembly. Likewise, a gene expression study in the postmortem brains of BPD patients found that the α- and β-subunits of the voltage-gated type I sodium channel genes were upregulated. Overall, these data provide support for the notion that the dysregulation of Na_v channel function may play a role in the etiology of BPD, and that some of the most effective pharmacological therapies for this disorder may counteract the effect of aberrant Na_v channel activity (Kaas, 2016).

Lithium is commonly thought to elicit its mood-stabilizing effect by inhibiting GSK-3β, IMPase, or IPPase. However, the current experiments did not yield results that directly connect lithium's rescue effect with these enzymes. For instance, pharmacological inhibition of GSK-3β using AR-A014418, which was shown to reduce tau-induced pathology by inhibiting GSK-3β, did not suppress behavioral phenotypes of Shu. The median values of percentage of time spent inside were 42.8 and 47.9% for control flies, respectively. Likewise, feeding Shu mutants with L-690,330, which is known to be ∼1000-fold more potent than lithium in inhibiting IMPase, did not lead to phenotypic suppression either. The median values of percentage of time spent inside were 43.4 and 39.4% for control flies, respectively. These negative data with the enzyme inhibitors suggest that lithium may act independently of GSK-3β and inositol signaling in exerting its rescue effect on Shu mutants. (Kaas, 2016).

Early voltage-clamp studies demonstrated that the permeability of Na_v channels to Li⁺ is nearly identical to their permeability to Na⁺ itself. In bovine adrenal chromaffin cells, Li⁺ was shown to inhibit radioactive Na⁺ influx through veratridine-activated sodium channels, independently of GSK-3β. Such observations raised the possibility that lithium may suppress activity of Shu Na_v channels to confer a rescue effect. However, direct application of lithium did not cause acute suppression of hyperexcitability of motor neurons displayed by Shu larvae. Moreover, total replacement of Na⁺ with Li⁺ did not alter the hyperexcitability phenotype in the larval motor axon. The results are consistent with the previous observation that Li⁺, like ouabain, can enhance neuromuscular excitability in wild-type larvae via inhibition of Na⁺/K⁺ ATPase, in contrast to the suppression effect of chronic lithium feeding to Shu mutants. Furthermore, a recent report demonstrates interesting effects of long-term lithium treatment of induced pluripotent stem cells derived from lithium-responsive BPD patients. The differentiated neurons from such patients display hyperexcitability that can be suppressed after 1-week incubation in lithium-containing culture medium. A reduction in neuronal firing is coupled with a decrease in Na⁺ and K⁺ currents, consistent with a long-term physiological readjustment of neuronal excitability mechanisms (Kaas, 2016).

Microarray and RT-PCR analyses revealed that expression of innate immune response genes is enhanced in the head of Shu mutants and that lithium tends to normalize their expression levels. The correlation between immune gene expression and severity of Shu phenotypes raises the possibility that the innate immune system is involved in manifestation of Shu phenotypes and their modulation by lithium. Changes in immune gene expression were also observed in two other seizure-prone para knock-in mutants and are associated with genetic suppression of seizure-like phenotypes of Drosophila easily shocked (eas) mutants. In addition, experimental and clinical studies have provided accumulating evidence for a strong relationship between an activated innate immune system and the pathophysiology of psychiatric and neurological disorders, including BPD and human epilepsies, as well as in rodent epilepsy models. Recent studies using mice also demonstrated that lithium can attenuate innate immune responses in a GSK-3β-independent manner both in vitro and in vivo (Kaas, 2016).

A combination of microarray and bioinformatics analyses revealed additional genes and biological pathways significantly affected by lithium treatment in Shu. Particularly notable are those related to amino acid metabolism and oxidation-reduction processes. Interestingly, alterations of brain amino acid metabolism are implicated in the antiepileptic effect of the ketogenic diet, whereas redox reactions, particularly those involved in oxidative stress, are suggested to play critical roles in initiation and progression of epilepsy. Future experiments are required to determine whether these biological pathways as well as the innate immune system have functional significance in lithium-dependent improvement of Shu phenotypes caused by abnormal Na_v channel activity (Kaas, 2016).

Despite several decades of scientific investigation, the exact etiology and the mechanisms of drug action for many psychiatric disorders, such as BPD, remain largely unknown. Attempts to better understand the pathophysiology of these illnesses using animal models, including Drosophila, continue to be a challenge but also prove highly promising. Taken together, the current results suggest that Shu and other Drosophila sodium channel mutants have the potential to be useful and experimentally amenable tools for elucidating the actions of lithium in the nervous system, and that they may ultimately contribute to an understanding of lithium-responsive disorders in humans (Kaas, 2016).

Drosophila voltage-gated sodium channels are only expressed in active neurons and are localized to distal axonal initial segment-like domains

In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the axonal initial segment in vertebrates. An intronic Minos-Mediated Integration Cassette was used to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila, para. Despite being the only NaV channel in the fly, this study shows that only 23 ± 1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. A single-cell transcriptomic atlas of the whole third instar larval brain was generated to identify para expressing neurons; it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23&177; 1% of larval neurons may be capable of firing NaV-dependent APs. Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which is named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates (Ravenscroft, 2020).

Action potentials (APs) are generated by the sequential opening of voltage-gated sodium (NaV) and potassium channels (KV) in the axons of neurons. Mammalian CNS neurons are typically multipolar; and APs initiate at the dense concentration of NaV channels in the axonal initial segment (AIS) close to the soma, and propagate along the axon via the nodes of Ranvier. In addition to AP initiation, the AIS forms a barrier between the soma and the axon, preventing the free diffusion of organelles, proteins, and lipids between the two compartments. Invertebrate neurons are typically unipolar with the dendrites impinging on the axon distal to the cell body. Whether an AIS is present in these neurons, and where it is located along the axon, is unresolved (Ravenscroft, 2020).

In order to determine the site of AP initiation, and if and where the AIS is in invertebrate neurons, the location was examined of the sole NaV channel gene in Drosophila melanogaster, paralytic (para). Unlike mammals, which have multiple NaV encoding genes (SCN1-5A, 8-11A), the genome of D. melanogaster encodes only two genes predicted to encode NaV proteins para and Na channel protein 60E (NaCP60E). para is the putative NaV channel as NaCP60E null animals are viable with no loss of inward sodium currents detected in neurons using patch clamp. In contrast, para null animals die as first instar larvae with no detectable inward sodium current in neurons using patch clamp. Despite having one NaV gene, compared with nine in mammals, it is possible that a similar degree of channel protein diversity is achieved via alternate splicing. para has 60 predicted isoforms, some of which have different developmental expression. Very little is known about the expression pattern or subcellular localization of Para. ISH studies determined that para is expressed in the nervous system from embryos to adults. Whether para is expressed in all or just some cells in the nervous system, and where it is subcellularly localized, remains to be established (Ravenscroft, 2020).

To determine the expression pattern and protein localization of NaV channels in Drosophila neurons, previously established tools were used to develop two novel fly models: a model where the endogenous Para is tagged with GFP to determine Para subcellular localization and another with para replaced with GAL4 to determine para gene expression. Surprisingly, it was found that para is present in a small fraction of CNS neurons in embryos and third instar larvae, while it is broadly expressed in neurons in the adult CNS. A single-cell transcriptomic atlas was generated of the whole third instar larval brain to identify that para correlates with RNAs of active zone proteins and mature neuron markers; hence, para expression is restricted to active, differentiated neurons in larvae. Neurons that coexpress para and active zone protein RNAs are abundant in the adult CNS but only represent 23 ± 1% of neurons in third instar larvae. In neurons where para is expressed, Para protein is enriched at an AIS-like region in axons distal to where the dendritic tree connects to the axons in a distal axonal segment (DAS). Para localized far from the soma is functionally verified electrophysiologically. In longer neurons, Para is expressed throughout the axon, likely to maintain AP propagation to the synapses (Ravenscroft, 2020).

The site of AP initiation in invertebrate neurons is unclear. In vertebrates, APs are generated at the AIS. The major functional hallmark of vertebrate AIS is clustering of NaV channels. Therefore, the gene expression and subcellular localization of the sole NaV channel in Drosophila, (para) was examined. Despite being the only NaV channel in flies, this study found that para is only expressed in 23 ± 1% of neurons in the third instar larval CNS. In contrast, in the adult, para expression is far broader. By generating a single-cell transcriptomic atlas of the third instar larval brain, it was determined that para expression correlates with the expression of ARGs, genes expressed in active zones, and markers of mature neurons. This implies that cells expressing para, while proportionally small, likely represent the active population of neurons in the third instar larval CNS. This correlation occurs also in the adult CNS, indicating that the number of active neurons is likely much higher in the adult than in the third instar larvae. In the neurons expressing para, it was found that Para distributed into axonal segments that are far removed from the soma, distal to where dendrites integrate into the axon. This structure shares some features of the mammalian AIS, and whole-cell patch-clamp electrophysiological recordings are consistent with the interpretation that Nav-dependent APs initiate there; however, because of its distal location to the cell soma, this region was named the DAS (Ravenscroft, 2020).

The AIS identifies the origin of AP propagation through the high density of NaV channels. The clustering of NaV channels at the DAS suggests that this is the site of AP initiation in fly neurons. Identification that TTX-sensitive, inward sodium currents that occur after long delays in response to somatic depolarization are also suggestive of a distal site of AP initiation. In vertebrate neurons, TTX-sensitive inward sodium currents are measured immediately after somatic depolarization as the AIS is so close to the soma. Single-cell electrophysiological recordings performed on fly neurons in the CNS are a challenge because of their small size. Recordings performed in the neurons of larger invertebrates, Aplysia, crab, and leeches also exhibit delayed activation and poor space clamp, indicating that APs are initiated at a site distal relative to the cell body. This suggests that AP initiation at the DAS is conserved across invertebrate species. Because of their small size, patch-clamp recordings are made on the soma. Therefore, the soma receives back-propagating APs from the DAS, and passive fluctuations in membrane potential are readily measured in the soma as well in Drosophila CNS neurons. While APs appear to initiate from the DAS, the sources and ionic mechanisms of passive membrane potentials are not clear; however, fluctuations in membrane potential clearly influence the pattern of AP firing in Drosophila neurons. For example, large fluctuations in membrane potential drive burst firing in lLNv, whereas stable membrane potential is associated with regular tonic AP firing. Further, transition between burst and tonic firing AP firing pattern is CaV-dependent as it is modulated by cobalt block of CaV channels. Surprisingly, either TTX or cobalt abolishes lLNv APs altogether, suggesting complex interactions between Para and CaVs in Drosophila neurons. Therefore, the electrical activity recorded at the soma may not be representative of the neuron's actual firing activity, as in some cases the DAS is far away from the soma and these cell bodies may not undergo any depolarization. This emphasizes the need to establish voltage-sensing reporters to simultaneously enable accurate reporting of APs and calcium events at high spatiotemporal resolution. While genetically encoded calcium indicators are often used for a proxy for inferring APs, intracellular calcium sources are highly diverse (CaVs, TRP channels, nonselective cation channels, multiple intracellular stores, etc.) (Ravenscroft, 2020).

As an alternative to electrophysiological recordings, computational models of compartmentalized fly neurons have been used to model their electrophysiological properties. Models of different neurons in the fly CNS predict that the site of AP propagation occurs, not proximal to the cell body as in mammals, but distally in the axon after the last dendrite innervates the axon. The mapping of the DAS and the ability to visualize it in any fly neuron using Para-GFSTF endorse the accuracy of these models and enable the generation of models with greater precision to better predict and analyze neuron activity and dynamics (Ravenscroft, 2020).

Some studies have indicated that an AIS-like region may be present in some Drosophila neurons, based on GAL4-mediated overexpression of Drosophila homologs of vertebrate AIS proteins, such as Ank1 and Shal in MB neurons. The overexpressed proteins were shown to cluster in axons downstream of the dendritic tree, in the MB of the third instar larval and the adult CNS. Using a similar approach, it has been shown that proximal axons of ddaE neurons in the PNS also may contain AIS-like regions. However, the overexpression of proteins may lead to their restriction in cellular compartments as it has been shown that the mobility of transmembrane molecules is restricted to areas before and after dendritic branches, suggesting that there are barriers in axons that restrict protein localization. The DAS reported in this study is of comparable size and location to the AIS-like region previously reported in adult MB neurons. AnkG is key for NaV channel clustering at the vertebrate AIS. The similarity in the reported AIS-like localization of Ank1 localization and Nav enrichment at the DAS as defined in this study indicate that the role of ankyrins in NaV clustering may be conserved in invertebrates, despite differences in ankyrin structure across species. Interestingly, this study found that the distance between the soma and the length of the DAS is correlated, with a longer DAS present in neurons where the DAS is more distal. This is in contrast to what is observed in vertebrates where the more distal the AIS, the shorter the length of the AIS (Ravenscroft, 2020).

Low levels of Para distribution were observed along long axons, suggesting that AP propagation is maintained by Para across the length of the axon. APs are all-or-nothing signals that rapidly propagate along axons; however, if no NaV channel is present, the signal should decay rapidly. It is argued that the low levels of Para distributed along the length of long axons enable the APs to reach the synapse without signal depletion. This is seen clearly in neurons, such as motor neurons in larval and adult CNS, and neurons in the adult brain that cross from one hemisphere to the other. Neurons with smaller axons may also have Para continually distributed along axons at undetectable levels or may not require Para at all. However, given the expression data discussed below, the latter is unlikely (Ravenscroft, 2020).

The sparse number of CNS neurons in the third instar larvae that express para is an unanticipated observation. Only 23 ± 1% of elav-positive neurons in the larval CNS express para, and historical gene expression tracing shows that those neurons that are para-negative never express para. These data are also in agreement with the single-cell sequencing data, which document a highly restricted expression pattern for para in the third instar larval CNS. Neurons expressing para also express genes that produce neurotransmitters as well as proteins that have been shown to be upregulated in many Drosophila models of enhanced neuronal activity, both in the third instar larval and adult brain single-cell RNA sequencing datasets. This provides evidence that this restricted cell population is likely capable of firing APs, that para expression is a potential marker for active AP firing neurons, and that only 23 ± 1% of cells in the third instar larvae fit this criterion. Whether the para-negative cells still have electrical activity in lieu of NaV channels, either through passive signaling or using CaV, is unknown. para expression has been demonstrated to positively correlate with neural progenitor proliferation in third instar larvae. The single-cell transcriptomic atlas of the whole third instar larval CNS generated in this study identifies neuronal progenitors at immature, intermediate, and mature maturation steps, and para expression was not detected in any neuroblasts lineage at any stage of maturation (Ravenscroft, 2020).

This study has identified the DAS, a compartment of Drosophila axons analogous to the AIS in vertebrates. The characterization of NaV channels throughout development in D. melanogaster is reported, uncovering the presence of the DAS in most axons after the dendritic tree, where AP is likely initiated, and continual low-level Para distribution in long axons to maintain AP. Additionally, comprehensive single-cell RNA sequencing of the third instar larval CNS was performed. This single-cell atlas in conjunction with para-T2A-GAL4 revealed that only 23 ± 1% of the cells in the third instar larval brain express para and other genes expressed in AP firing neurons. These data are consistent with the notion that most neurons are developing in third instar larvae, especially in the brain lobes. In contrast, para expression is broad in the adult CNS. The models generated herein should allow the mapping of DAS in any neuron and provide a GAL4 driver to target differentiated, AP firing neurons (Ravenscroft, 2020).

Molecular chaperone Calnexin regulates the function of Drosophila sodium channel Paralytic

Neuronal activity mediated by voltage-gated channels provides the basis for higher-order behavioral tasks that orchestrate life. Chaperone-mediated regulation, one of the major means to control protein quality and function, is an essential route for controlling channel activity. This study presents evidence that Drosophila ER chaperone Calnexin colocalizes and interacts with the alpha subunit of sodium channel Paralytic. Co-immunoprecipitation analysis indicates that Calnexin interacts with Paralytic protein variants that contain glycosylation sites Asn313, 325, 343, 1463, and 1482. Downregulation of Calnexin expression results in a decrease in Paralytic protein levels, whereas overexpression of the Calnexin C-terminal calcium-binding domain triggers an increase reversely. Genetic analysis using adult climbing, seizure-induced paralysis, and neuromuscular junction indicates that lack of Calnexin expression enhances Paralytic-mediated locomotor deficits, suppresses Paralytic-mediated ghost bouton formation, and regulates minature excitatory junction potentials (mEJP) frequency and latency time. Taken together, these findings demonstrate a need for chaperone-mediated regulation on channel activity during locomotor control, providing the molecular basis for channlopathies such as epilepsy (Xiao, 2017).

Parallel Visual Conditional protein tagging methods reveal highly specific subcellular distribution of ion channels in motion-sensing neurons

Neurotransmitter receptors and ion channels shape the biophysical properties of neurons, from the sign of the response mediated by neurotransmitter receptors to the dynamics shaped by voltage-gated ion channels. Therefore, knowing the localizations and types of receptors and channels present in neurons is fundamental to understanding of neural computation. This study developed two approaches to visualize the subcellular localization of specific proteins in Drosophila: the flippase-dependent expression of GFP-tagged receptor subunits in single neurons and 'FlpTag', a versatile new tool for the conditional labelling of endogenous proteins. Using these methods, the subcellular distribution of the receptors GluClα, Rdl, and Dα7 and the ion channels Para and Ih in motion-sensing T4/T5 neurons of the Drosophila visual system was investigated. A strictly segregated subcellular distribution of these proteins a sequential spatial arrangement of glutamate, acetylcholine, and GABA receptors was discovered along the dendrite that matched the previously reported EM-reconstructed synapse distributions (Fendl, 2020).

How neural circuits implement certain computations in order to process sensory information is a central question in systems neuroscience. In the visual system of Drosophila, much progress has been made in this direction: numerous studies examined the response properties of different cell-types in the fly brain and electron microscopy studies revealed the neuronal wiring between them. However, one element crucial to understanding is still missing; these are the neurotransmitter receptors used by cells at the postsynaptic site. This knowledge is essential since neurotransmitters and corresponding receptors define the sign and the time-course of a connection, that is whether a synapse is inhibitory or excitatory and whether the signal transduction is fast or slow. The same neurotransmitter can act on different receptors with widely differing effects for the postsynaptic neuron. Glutamate for instance is mainly excitatory, however, in invertebrates it can also have inhibitory effects when it acts on a glutamate-gated chloride channel, known as GluClα. Recently, it has also been shown that acetylcholine, usually excitatory, might also be inhibitory in Drosophila, if it binds to the muscarinic mAChR-A receptor. Hence, knowledge inferring the type of transmitter receptor at a synapse is essential for understanding of the way neural circuits process information (Fendl, 2020).

Moreover, voltage-gated ion channels shape synaptic transmission and the integration of synaptic inputs by defining the membrane properties of every neural cell type. The voltage-gated calcium channel cacophony, for instance, mediates influx of calcium ions that drives synaptic vesicle fusion at presynaptic sites. Voltage-gated sodium channels like Paralytic (Para) are important for the cell's excitability and the generation of sodium-dependent action potentials. The voltage-gated channel Ih influences the integration and kinetics of excitatory postsynaptic potentials. However, only little is known about how these channels are distributed in neurons and how this shapes the neural response properties (Fendl, 2020).

One of the most extensively studied neural circuits in Drosophila is the motion vision pathway in the optic lobe and the underlying computation for direction-selectivity. The optic lobe comprises four neuropils: lamina, medulla, lobula, and lobula plate. As in the vertebrate retina, the fly optic lobe processes information in parallel ON and OFF pathways. Along the visual processing chain, T4/T5 neurons are the first neurons that respond to visual motion in a direction selective way. T4 dendrites reside in layer 10 of the medulla and compute the direction of moving bright edges (ON-pathway). T5 dendrites arborize in layer 1 of the lobula and compute the direction of moving dark edges (OFF-pathway). The four subtypes of T4/T5 neurons (a, b, c, d), project axon terminals to one of the four layers in the lobula plate, each responding only to movement in one of the four cardinal directions, their preferred direction (Fendl, 2020).

How do T4/T5 neurons become direction-selective? Both T4 and T5 dendrites span around eight columns collecting signals from several presynaptic input neurons, each of which samples information from visual space in a retinotopic manner. The functional response properties of the presynaptic partners of T4/T5 have been described in great detail along with their neurotransmitter phenotypes. T4 dendrites receive glutamatergic, GABAergic and cholinergic input, whereas T5 dendrites receive GABAergic and cholinergic input only. These input synapses are arranged in a specific spatial order along T4/T5 dendrites (Fendl, 2020).

Which receptors receive this repertoire of different neurotransmitters at the level of T4/T5 dendrites? Recently, several RNA-sequencing studies described the gene expression pattern of nearly all cell-types in the optic lobe of the fruit fly including T4/T5 neurons. T4/T5 neurons were found to express numerous receptor subunits of different transmitter classes and voltage-gated ion channels at various expression strengths. However, RNA-sequencing studies do not unambiguously answer the above question for two reasons: mRNA and protein levels are regulated in complex ways via post-transcriptional, translational, and protein degradation mechanisms making it difficult to assign protein levels to RNA levels. Secondly, standard RNA-sequencing techniques cannot provide spatial information about receptor localizations, hence, they are not sufficient to conclude which transmitter receptors receive which input signal. Both shortcomings could in principle be overcome by antibody staining since immunohistochemical techniques detect neurotransmitter receptors at the protein level and preserve spatial information. However, high-quality antibodies are not available for every protein of interest and may have variable affinity due to epitope recognition. Furthermore, labeling ion channels via antibodies and ascribing expression of a given channel to a cell-type in dense neuronal tissue remains challenging. The disadvantages of the above techniques highlight the need for new strategies for labeling neurotransmitter receptors in cell types of interest (Fendl, 2020).

This study employed existing and generated new genetic methods to label and visualize ion channels in Drosophila. For endogenous, cell-type-specific labeling of proteins, a generalizable method called FlpTag was developed that expresses a GFP-tag conditionally. Using these tools, the subcellular distribution was determined of the glutamate receptor subunit GluClα, the acetylcholine receptor subunit Dα7, and the GABA receptor subunit Rdl in motion-sensing T4/T5 neurons. These receptor subunits were differentially localized between dendrites and axon terminals. Along the dendrites of individual T4/T5 cells, the receptor subunits GluClα, Rdl, and Dα7 reveal a distinct distribution profile that can be assigned to specific input neurons forming synapses in this area. Furthermore, it was demonstrated the generalizability of the FlpTag approach by generating lines for the metabotropic GABA receptor subunit Gaba-b-r1 and the voltage-gated ion channels para and Ih. The strategies described in this study can be applied to other cells as well as other proteins to reveal the full inventory and spatial distribution of the various ion channels within individual neurons (Fendl, 2020).

Neurotransmitter receptors are essential neuronal elements that define the sign and temporal dynamics of synaptic connections. For understanding of complex neural circuits, it is indispensable to examine which transmitter receptor types are used by the participating neurons and to which compartment they localize. This study developed FlpTag, a generalizable method for endogenous, cell-type-specific labeling of proteins. Alongside several GFP-tagged UAS-lines, the newly developed FlpTag lines were developed to explore the distribution of receptor subunits GluClα, Rdl, Dα7, Gaba-b-r1 and voltage-gated ion channels Para and Ih in motion-sensing T4/T5 neurons of the visual system of Drosophila. These ion channels were found to be localized to either the dendrite, the axonal fiber or the axon terminal. Even at the level of individual dendrites, GluClα, Rdl and Dα7 were differentially distributed precisely matching the locations where T4 and T5 neurons sample signals from their glutamatergic, cholinergic, or GABAergic input neurons, respectively (Fendl, 2020).

Working with Drosophila as model organism bears some unrivaled advantages when it comes to genetic tools. The MiMIC and FlyFos libraries, for instance, are large-scale approaches of enormous value for the fly community as they provide GFP-tagged protein lines for thousands of Drosophila genes including several neurotransmitter receptors and voltage-gated ion channels. Recently, Kondo expanded these existing libraries with T2A-Gal4 insertions in 75 neurotransmitter receptor genes that can also be exchanged by the fluorescent protein tag Venus (Kondo, 2020). While all these approaches tag genes at their endogenous locus, none of them are conditional, for example they cannot be applied in a cell-type-specific manner. Hence, ascribing the expression of the pan-neuronally tagged proteins to cell-types of interest are challenging in dense neuronal tissue (Fendl, 2020).

To overcome these difficulties, two conditional strategies were used for the investigation of membrane protein localizations in the cell types of interest, T4 and T5 neurons. First, GFP-tagged UAS-lines were developed for GluClα and Rdl, and an existing UAS-Dα7::GFP line was tested. As stated above, aberrant localization of overexpressed proteins can occur, however, this is not always the case. Overexpression of UAS-GluClα::GFP shows a similar receptor localization pattern as both MiMIC and FlpTag endogenous lines, thus, validating the use of UAS-GluClα::GFP for studying receptor distribution. Additionally, previous studies reported that the UAS-Dα7::GFP line showed proper localization of the acetylcholine receptor to endogenous synapses when compared to antibody stainings or endogenous Bruchpilot (Brp) puncta. This study confirmed confirmed this finding and further showed that Dα7::GFP presumably localizes only to cholinergic synapses. Overexpressing Dα7::GFP in a medulla neuron that is devoid of endogenous Dα7 demonstrated that Dα7::GFP localized to apparent cholinergic synapses. Hence, the UAS-Dα7::GFP line can be used to study the distribution of cholinergic synapses, but not the exact composition of cholinergic receptor subunits. A recent study showed that quantitatively the levels of the postsynaptic density protein PSD95 change when overexpressed, but qualitatively the localization is not altered. Altogether, this suggests that tagged overexpression lines can be used for studying protein localizations, but they have to be controlled carefully and drawn conclusions might be different for every line (Fendl, 2020).

Ideally, a tool for protein tagging should be both endogenous and conditional. This can be achieved by introducing an FRT-flanked STOP cassette upstream of the gene of interest which was engineered with an epitope tag or fluorescent protein. Only upon cell-type specific expression of Flp, the tagged protein will be expressed in a cell-type specific manner. This genetic strategy was utilized by two independent studies to label the presynaptic protein Brp, the histamine channel Ort and the vesicular acetylcholine transporter VAChT. Recently, a new approach based on the split-GFP system was utilized for endogenous, conditional labeling of proteins in two independent studies. However, all these aforementioned approaches are not readily generalizable and easily applicable to any gene of interest (Fendl, 2020).

The FlpTag strategy presented in this study overcomes these caveats by allowing for endogenous, conditional tagging of proteins and by offering a generalizable toolbox for targeting many genes of interest. Similar to the conditional knock-out tools FlpStop and FlipFlop, FlpTag utilizes a FLEx switch to conditionally control expression of a reporter gene, in this case GFP. Likewise, FlpTag also easily integrates using the readily available intronic MiMIC insertions. This study attempted to generate FlpTag lines for six genes, GluClα, Rdl, Dα7, Gaba-b-r1, para and Ih. Four out of these six lines yielded conditional GFP-tagged protein lines (GluClα, Gaba-b-r1, para, Ih). The FlpTag cassette was injected in MI02620 for Rdl and MI12545 for Dα7, but no GFP expression was detected across the brain. The MiMiC insertion sites used for Rdl and Dα7 seem to be in a suboptimal location for tagging the protein (Fendl, 2020).

As of now, there are MiMIC insertions in coding introns for more than 2800 genes available, which covers approximately 24% of neuronal genes. Additionally, the attP insertion sites generated in the study by Kondo provide possible landing sites for the FlpTag cassette for 75 neurotransmitter receptor genes (Kondo, 2020). Transmembrane proteins such as neurotransmitter receptors form complex 3D structures making fluorescent tagging especially difficult. Neither the MiMIC insertion sites, nor the target sites of the Kondo study at the C-terminus of several transmitter receptor genes, ensure a working GFP-tagged protein line. For genes of interest lacking a suitable MiMIC insertion site a homology directed repair (HDR) cassette was generated that utilizes CRISPR/Cas9-mediated gene editing to integrate the FlpTag cassette in any desired gene locus. The plasmid consists of the FlpTag cassette flanked by multiple cloning sites for the insertion of homology arms (HA). Through HDR the FlpTag cassette can be knocked-in into any desired locus. Taken together, the FlpTag cassette is a generalizable tool that can be integrated in any available attP-site in genes of interest or inserted by CRISPR-HDR into genes lacking attP landing sites. This allows for the investigation of the endogenous spatial distributions of proteins, as well as the correct temporal dynamics of protein expression (Fendl, 2020).

Further, the FlyFos project demonstrated that most fly lines with an extra copy of GFP-tagged protein-coding genes worked normally and GFP-tagged proteins could be imaged in living fly embryos and pupae. In principle, live-imaging of the GFP-tagged lines that were created could be performed during different developmental stages of the fruit fly. In general, the tools generated in this study can be used as specific postsynaptic markers, visualizing glutamatergic, GABAergic, and cholinergic synapses with standard confocal light microscopy. This extends the existing toolbox of Drosophila postsynaptic markers for studying the localization and development of various types of synapses (Fendl, 2020).

T4/T5 neurons combine spatiotemporal input from their presynaptic partners, leading to selective responses to one of the four cardinal directions. Numerous studies investigated the mechanisms underlying direction-selective responses in T4/T5 neurons, yet the computation is still not fully understood. At an algorithmic level, a three-arm detector model is sufficient to describe how direction-selective responses in T4/T5 neurons arise. This model relies on the comparison of signals originating from three neighboring points in space via a delay-and-compare mechanism. The central arm provides fast excitation to the neuron. While one flanking arm amplifies the central signal for stimuli moving along the preferred direction, the other inhibits the central signal for stimuli moving along the null direction of the neuron. Exploring the neurotransmitter receptors and their distribution on T4/T5 dendrites allows defining the sign as well as the temporal dynamics of some of the input synapses to T4/T5 (Fendl, 2020).

According to the algorithmic model, an excitatory, amplifying input signal on the distal side of T4/T5 dendrites was expected. This study found that T4 cells receive an inhibitory, glutamatergic input from Mi9 via GluClα, which, at first sight, seems to contradict expectation. However, since Mi9 has an OFF-center receptive field, this glutamatergic synapse will invert the polarity from Mi9-OFF to T4-ON. Theoretically, in darkness, Mi9 inhibits T4 via glutamate and GluClα, and this inhibition is released upon an ON-edge moving into its receptive field. The concomitant closure of chloride channels and subsequent increased input resistance in T4 cells results in an amplification of a subsequent excitatory input signal from Mi1 and Tm3. As shown by a recent modeling study, this biophysical mechanism can indeed account for preferred direction enhancement in T4 cells (Borst, 2018). Some studies failed to detect preferred direction enhancement in T4/T5 neurons and they proposed that the enhanced signal in PD seen in GCaMP recordings could be a result from a non-linear calcium-to-voltage transformation. If this was really the case, the role of Mi9 and GluClα must be reconsidered and future functional experiments will shed light onto this topic (Fendl, 2020).

Nevertheless, Strother (2017) showed that the RNAi- knock-down of GluClα in T4/T5 neurons leads to enhanced turning responses on the ball set-up for faster speeds of repeating ON and OFF edges (Strother, 2017). Although this observation cannot answer the question about preferred direction enhancement in T4 cells, it indicates that both T4 and T5 receive inhibitory input and that removal of such create enhanced turning responses at the behavioral level. In line with these observations, the glutamate receptor GluClα was also found in T4/T5 axon terminals. A possible functional role of these inhibitory receptors in the axon terminals could be a cross-inhibition of T4/T5 cells with opposite preferred directions via lobula plate intrinsic neurons (LPis). Glutamatergic LPi neurons are known to receive a cholinergic, excitatory signal from T4/T5 neurons within one layer and to inhibit lobula plate tangential cells, the downstream postsynaptic partners of T4/T5 neurons, via GluClα in the adjacent oppositely tuned layer. This mechanism induces a motion opponent response in lobula plate tangential cells and increases their flow-field selectivity. In addition, LPi neurons could also inhibit T4/T5 neurons presynaptically at their axon terminals via GluClα in order to further sharpen the flow-field selectivity of lobula plate tangential cells. Taken together, exploring the subcellular distribution of GluClα in T4/T5 neurons highlights its differential functional roles in different parts of these cell types (Fendl, 2020).

Secondly, the Dα7 signal in the center of T4/T5 dendrites discovered in this study, corresponds to ionotropic, cholinergic input from Mi1 and Tm3 for T4, and Tm1, Tm2 and Tm4 for T5. These signals correspond to the central, fast, excitatory arm of the motion detector model. As T4 and T5 express a variety of different ACh receptor subunits, the exact subunit composition and underlying biophysics of every cholinergic synapse on T4/T5 dendrites still awaits further investigations (Fendl, 2020).

Third, inhibition via GABA plays an essential role in creating direction-selective responses in both T4 and T5 neurons by providing null direction suppression. Computer simulations showed that direction selectivity decreases in T4/T5 motion detector models without this inhibitory input on the null side of the dendrite. This study shows that T4 and T5 neurons possess the inhibitory GABA receptor subunit Rdl mainly on the proximal base on the null side of their dendrites, providing the synaptic basis for null direction suppression. The metabotropic GABA receptor subunit Gaba-b-r1 was not detected in T4/T5 neurons using the newly generated FlpTag Gaba-b-r1 line. Finally, all of the receptor subunits GluClα, Rdl and Dα7 investigated in this study are ionotropic, fast receptors, which presumably do not add a temporal delay at the synaptic level. In the detector model described above, the two outer arms provide a slow and sustained signal, and such properties are already intrinsic properties of these input neurons. However, it cannot be excluded that slow, metabotropic receptor subunits for acetylcholine or GABA (e.g. Gaba-br2) which are also present in T4/T5 and could induce additional delays at the synaptic level (Fendl, 2020).

Furthermore, the subcellular distribution was investigated of the voltage-gated ion channels Para and Ih in T4/T5 neurons. Para, a voltage-gated sodium channel, was found to be distributed along the axonal fibers of both T4 and T5 neurons. As Para is important for the generation of sodium-dependent action potentials, it will be interesting for future functional studies to investigate, if T4/T5 really fire action potentials and how this shapes their direction-selective response. Further, Ih, a voltage-gated ion channel permeable for several types of ions, was detected in T4/T5 dendrites using the FlpTag strategy. Ih channels are activated at negative potentials below -50 mV and as they are permeable to sodium and potassium ions, they can cause a depolarization of the cell after hyperpolarization. Loss-of-function studies will unravel the functional role of the Ih channel for direction-selective responses in T4/T5 neurons (Fendl, 2020).

Since the ability to combine synaptic inputs from different neurotransmitters at different spatial sites is common to all neurons, the approaches described in this study represent an important future perspective for other circuits. The tools can be used to study the ion channels GluClα, Rdl, Dα7, Gaba-b-r1, para and Ih in any given Drosophila cell-type and circuit. Furthermore, the FlpTag tool box can be used to target many genes of interest and thereby foster molecular questions across fields (Fendl, 2020).

The techniques described in this study can be transferred to other model organisms as well, to study the distribution of different transmitter receptors. For instance, in the mouse retina - similar to motion-sensing T4/T5 neurons in the fruit fly - so-called On-Off direction-selective ganglion cells receive asymmetric inhibitory GABAergic inputs from presynaptic starburst amacrine cells during null-direction motion. A previous study investigated the spatial distribution of GABA receptors of these direction-selective ganglion cells using super-resolution imaging and antibody staining. Additionally, starburst amacrine cells also release ACh onto ganglion cells which contributes to the direction-selective responses of ganglion cells. Thus, mapping the distribution of ACh receptors on direction-selective ganglion cells will be the next important step to further investigate cholinergic transmission in this network (Fendl, 2020).

Overall, this study has demonstrated the importance of exploring the distributions of neurotransmitter receptors and ion channels for systems neuroscience. The distinct distributions in T4/T5 neurons discovered in this study and the resulting functional consequences expand knowledge of the molecular basis of motion vision. Although powerful, recent RNAseq studies lacked information about spatial distributions of transmitter receptors which can change the whole logic of wiring patterns and underlying synaptic signs. Future studies can use this knowledge to target these receptors and directly probe their role in functional experiments or incorporate the gained insights into model simulations. However, this study is only highlighting some examples of important neural circuit components: expanding the approaches described in this study to other transmitter receptors and ion channels, as well as gap junction proteins will reveal the full inventory and the spatial distributions of these decisive determinants of neural function within an individual neuron (Fendl, 2020).

Calcium imaging of neuronal activity in Drosophila can identify anticonvulsive compounds

This study uses neuronal expression of GCaMP, a potent calcium reporter, to image neuronal activity. Expression in motoneurons in the isolated CNS of third instar larvae shows waves of calcium-activity that pass between segments of the ventral nerve cord. Time between calcium peaks, in the same neurons, between adjacent segments usually shows a temporal separation of greater than 200 ms. Exposure to proconvulsants (picrotoxin or 4-aminopyridine) reduces separation to below 200 ms showing increased synchrony of activity across adjacent segments. Increased synchrony, characteristic of epilepsy, is similarly observed in genetic seizure mutants: bangsenseless¹ and paralytic^K1270T . Exposure of bss1 to clinically-used antiepileptic drugs (phenytoin or gabapentin) significantly reduces synchrony. The measure of synchronicity was used to evaluate the effectiveness of known and novel anticonvulsive compounds (antipain, isethionate, etopiside rapamycin and dipyramidole) to reduce seizure-like CNS activity. It was shown that such compounds also reduce the Drosophila voltage-gated persistent Na+ current (INaP) in an identified motoneuron (aCC). These combined assays provide a rapid and reliable method to screen unknown compounds for potential to function as anticonvulsants (Streit, 2016).

Distinct modulating effects of TipE-homologs 2-4 on Drosophila sodium channel splice variants

The Drosophila melanogaster TipE protein is thought to be an insect sodium channel auxiliary subunit functionally analogous to the β subunits of mammalian sodium channels. Besides TipE, four TipE-homologous proteins (TEH1-TEH4) have been identified. It has been reported that TipE and TEH1 have both common and distinct effects on the gating properties of splice variants of the Drosophila sodium channel, DmNa_v (Para). However, limited information is available on the effects of TEH2, TEH3 and TEH4 on the function of DmNa_v channel variants. This study found that TEH2 increased the amplitude of peak current, but did not alter the gating properties of three examined DmNa_v splice variants expressed in Xenopus oocytes. In contrast, TEH4 had no effect on peak current, yet altered the gating properties of all three channel variants. Furthermore, TEH4 enhanced persistent current and slowed sodium current decay. The effects of TEH3 on DmNa_v variants are similar to those of TEH4, but the data were collected from a small portion of oocytes because co-expression of TEH3 with DmNa_v variants generated a large leak current in the majority of oocytes examined. In addition, TEH3 and TEH4 enhanced the expression of endogenous currents in oocytes. Taken together, these results reveal distinct roles of TEH proteins in modulating the function of sodium channels and suggest that TEH proteins might provide an important layer of regulation of membrane excitability in vivo. These results also raise an intriguing possibility of TEH3/TEH4 as auxiliary subunits of other voltage-gated ion channels besides sodium channels (Wang, 2015).

Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials in almost all excitable cells. Mammalian sodium channels are composed of a pore-forming α subunit and one or more β subunits. The sodium channel α subunit is composed of four homologous domains (I–IV), each containing six transmembrane segments (S1–S6). The S1–S4 segments serve as a voltage-sensing module. The S5 and S6 segments and the reentrant loops connecting them form the inner pore. In response to membrane depolarization, the S4 segments moves outward which initiates the channel activation process (Catterall, 2012). Within a few milliseconds, sodium channels close or inactivate. This fast inactivation is mediated by an inactivation particle formed by residues in the linker connecting domains III and IV which physically occludes the inner pore, preventing sodium ions from flowing into the cell (Catterall, 2012). Under prolonged depolarization, sodium channels enter into another inactivation state called the slow inactivated state. Unlike fast inactivation where recovery takes tens of milliseconds, recovery from slow inactivation requires seconds to minutes of membrane repolarization to return to a resting state. The processes of sodium channel activation, fast inactivation and slow inactivation play critical roles in regulating membrane excitability (Wang, 2015).

In mammals, there are at least nine sodium channel isoforms with different gating properties and expression patterns in various cell types, tissues, and developmental stages. There are four genes encoding sodium channel β subunits (β1–β4). The β subunits are small transmembrane proteins that possess an extracellular immunoglobulin (Ig) domain, a single transmembrane segment, and a short intracellular C-terminal domain. They modulate gating properties and expression of sodium channels. In addition to their roles in channel modulation, β subunits also function as cell adhesion molecules interacting with both cytoskeleton and extracellular matrix, regulating cell migration and cellular aggregation (Wang, 2015).

In contrast, most insects species have only one sodium channel gene that encodes the α subunit equivalent of mammalian sodium channels, such as para, also known as DmNa_v, in Drosophila melanogaster. Instead, insects take advantage of alternative splicing and RNA editing to generate a large collection of sodium channel variants, such as DmNa_v in D. melanogaster, which exhibit diverse gating properties. Drosophila lacks any orthologs of mammalian β subunits. However, there is a small transmembrane protein, TipE, which is critical for sodium channel expression and neuronal activities, and functions as an auxiliary subunit for insect sodium channels (Feng, 1995; Warmke, 1997). TipE⁻ mutant flies exhibit a temperature-sensitive paralytic phenotype, similar to sodium channel mutants, and [H³]saxitoxin binding studies of insect embryonic neurons indicate that tipE⁻ mutant flies have reduced sodium channel density. Consistently, whole-cell patch recording indicates that sodium current is decreased about 40%–60% in dissociated embryonic neurons of tipE⁻ mutant flies. TipE also increases peak current of sodium channels transiently expressed in Xenopus oocytes (Wang, 2015).

In addition to TipE, there are four TipE-homologous genes (TEH1–4) in D. melanogaster and three to four orthologs in other insect species. TEH1 is exclusively expressed in the nervous system, while transcripts of TipE and TEH2–4 are detected in both neuronal and non-neuronal tissues (Derst, 2006). All four TEH proteins differentially modified one DmNa_v variant expressed in Xenopus oocytes. A recent study compared the modulation of three different DmNa_v splice variants by TipE or TEH1 and revealed that TEH1 extensively modified the functional properties of all three variants, whereas TipE only modified the gating of one of the variants (Wang, 2013; Wang, 2015 and references therein).

Whereas TEH2–4 have been shown to modulate the expression and some gating properties of a DmNa_v variant, the effects of TEH2, TEH3, or TEH4 on the gating properties of different DmNa_v splice variants remain unknown. This study examined the modulating effects of TEH2–4 on gating properties of three variants: DmNa_v9-1, DmNa_v22, and DmNa_v26 expressed in Xenopus oocytes. The data revealed strikingly distinct effects of TEH2, TEH3 and TEH4 on the expression and function of these variants. In addition to modulating sodium channel gating properties, TEH3 and TEH4 enhanced an outward current from an endogenous channel in Xenopus oocytes. These results suggest that TEH proteins provide an important layer of regulation of membrane excitability in vivo. The results also raise an intriguing possibility of TEH3 and TEH4 as auxiliary subunits of other voltage-gated ion channels besides sodium channels (Wang, 2015).

Differential effects of TipE and a TipE-homologous protein on modulation of gating properties of sodium channels from Drosophila melanogaster

β subunits of mammalian sodium channels play important roles in modulating the expression and gating of mammalian sodium channels. However, there are no orthologs of β subunits in insects. Instead, an unrelated protein, TipE in Drosophila melanogaster and its orthologs in other insects, is thought to be a sodium channel auxiliary subunit. In addition, there are four TipE-homologous genes (TEH1-4) in D. melanogaster and three to four orthologs in other insect species. TipE and TEH1-3 have been shown to enhance the peak current of various insect sodium channels expressed in Xenopus oocytes. However, limited information is available on how these proteins modulate the gating of sodium channels, particularly sodium channel variants generated by alternative splicing and RNA editing. This study, compared the effects of TEH1 and TipE on the function of three Drosophila sodium channel splice variants, DmNav9-1, DmNav22, and DmNav26, in Xenopus oocytes. Both TipE and TEH1 enhanced the amplitude of sodium current and accelerated current decay of all three sodium channels tested. Strikingly, TEH1 caused hyperpolarizing shifts in the voltage-dependence of activation, fast inactivation and slow inactivation of all three variants. In contrast, TipE did not alter these gating properties except for a hyperpolarizing shift in the voltage-dependence of fast inactivation of DmNav26. Further analysis of the gating kinetics of DmNav9-1 revealed that TEH1 accelerated the entry of sodium channels into the fast inactivated state and slowed the recovery from both fast- and slow-inactivated states, thereby, enhancing both fast and slow inactivation. These results highlight the differential effects of TipE and TEH1 on the gating of insect sodium channels and suggest that TEH1 may play a broader role than TipE in regulating sodium channel function and neuronal excitability in vivo (Wang, 2013).

Seizure suppression through manipulating splicing of a voltage-gated sodium channel

Seizure can result from increased voltage-gated persistent sodium current expression. Although many clinically-approved antiepileptic drugs target voltage-gated persistent sodium current, none exclusively repress this current without also adversely affecting the transient voltage-gated sodium current. Achieving a more selective block has significant potential for the treatment of epilepsy. Recent studies show that voltage-gated persistent sodium current amplitude is regulated by alternative splicing offering the possibility of a novel route for seizure control. This study identified 291 splicing regulators that, on knockdown, alter splicing of the Drosophila voltage-gated sodium channel (Paralytic) to favour inclusion of exon K, rather than the mutually exclusive exon L. This change is associated with both a significant reduction in voltage-gated persistent sodium current, without change to transient voltage-gated sodium current, and to rescue of seizure in this model insect. RNA interference mediated knock-down, in two different seizure mutants, shows that 95 of these regulators are sufficient to significantly reduce seizure duration. Moreover, most suppress seizure activity in both mutants, indicative that they are part of well conserved pathways and likely, therefore, to be optimal candidates to take forward to mammalian studies. Proof-of-principle is provided for such studies by showing that inhibition of a selection of regulators, using small molecule inhibitors, is similarly effective to reduce seizure. Splicing of the Drosophila sodium channel shows many similarities to its mammalian counterparts, including altering the amplitude of voltage-gated persistent sodium current. This study provides the impetus to investigate whether manipulation of splicing of mammalian voltage-gated sodium channels may be exploitable to provide effective seizure control (Lin, 2015).

Knock-in model of Dravet syndrome reveals a constitutive and conditional reduction in sodium current

Hundreds of mutations in the SCN1A sodium channel gene confer a wide spectrum of epileptic disorders, requiring efficient model systems to study cellular mechanisms and identify potential therapeutic targets. \Drosophila knock-in flies carrying the K1270T SCN1A mutation known to cause a form of genetic epilepsy with febrile seizures plus (GEFS+) exhibit a heat-induced increase in sodium current activity and seizure phenotype. To determine whether different SCN1A mutations cause distinct phenotypes in Drosophila as they do in humans, this study focuses on a knock-in line carrying a mutation that causes a more severe seizure disorder termed Dravet syndrome (DS). Introduction of the DS SCN1A mutation (S1231R) into the Drosophila sodium channel gene para results in flies that exhibit spontaneous and heat-induced seizures with distinct characteristics and lower onset temperature than the GEFS+ flies. Electrophysiological studies of GABAergic interneurons in the brains of adult DS flies reveal, for the first time in an in vivo model system, that a missense DS mutation causes a constitutive and conditional reduction in sodium current activity and repetitive firing. In addition, feeding with the serotonin precursor 5-HTP suppresses heat-induced seizures in DS but not GEFS+ flies. The distinct alterations of sodium currents in DS and GEFS+ GABAergic interneurons demonstrate that both loss- and gain-of-function alterations in sodium currents are capable of causing reduced repetitive firing and seizure phenotypes. The mutation-specific effects of 5-HTP on heat-induced seizures suggest the serotonin pathway as a potential therapeutic target for DS (Schutte, 2014).

Activity-dependent alternative splicing increases persistent sodium current and promotes seizure

Activity of voltage-gated Na channels (Na_v) is modified by alternative splicing. However, whether altered splicing of human Na_vs contributes to epilepsy remains to be conclusively shown. This study shows that altered splicing of the Drosophila Na_v (paralytic, DmNa_v) contributes to seizure-like behavior in identified seizure mutants. Attention was focused on a pair of mutually exclusive alternate exons (termed K and L), which form part of the voltage sensor (S4) in domain III of the expressed channel. The presence of exon L results in a large, non-inactivating, persistent I_Nap (a persistent sodium current). Many forms of human epilepsy are associated with an increase in this current. In wild-type (WT) Drosophila larvae, ~70–80% of DmNa_v transcripts contain exon L, and the remainder contain exon K. Splicing of DmNa_v to include exon L is increased to ∼100% in both the slamdance and easily-shocked seizure mutants. This change to splicing is prevented by reducing synaptic activity levels through exposure to the antiepileptic phenytoin or the inhibitory transmitter GABA. Conversely, enhancing synaptic activity in WT, by feeding of picrotoxin is sufficient to increase I_Nap and promote seizure through increased inclusion of exon L to 100%. The underlying activity-dependent mechanism requires the presence of Pasilla, an RNA-binding protein. Finally, computational modeling was used to show that increasing I_Nap is sufficient to potentiate membrane excitability consistent with a seizure phenotype. Thus, increased synaptic excitation favors inclusion of exon L, which, in turn, further increases neuronal excitability. Thus, at least in Drosophila, this self-reinforcing cycle may promote the incidence of seizure (Lin, 2012).

Alternative splicing involves the substitution, removal, and/or inclusion of exonic sequences within a pre-mRNA to produce transcripts encoding related protein isoforms. Estimates indicate that ∼95% of human genes are alternatively spliced. How splicing influences function of voltage-gated Na channel (Na_v) transcripts, and whether such changes promote seizure, is complicated by the genetic redundancy present in the mammalian genome. Recent reports suggest, however, that Na_vs show altered splicing in mesial temporal lobe epilepsy and that a single nucleotide polymorphism is sufficient to influence splicing of exon 5N in Na_v1.1, an effect that is associated with altered sensitivity to established antiepileptic drugs and possibly increased risk of febrile seizures (Lin, 2012).

In contrast to mammals, the genome of the fruitfly Drosophila melanogaster contains only one Na_v channel homolog: encoded by paralytic. This, coupled with the high degree of structural and functional homology, makes DmNa_v an ideal model with which to study the role of alternative splicing of this ion channel family. Previous work described the complete pattern of alternative splicing of DmNa_v isolated from Drosophila embryonic CNS (Lin, 2009). In particular, a pair of mutually exclusive, membrane-spanning exons (termed K and L) were identified that markedly affect the magnitude of the persistent current (I_Nap) that arises from incomplete inactivation of the channel. The magnitude of I_Nap ranges from 4.1 to 9.5% of peak transient current (I_Nat) in transcripts containing exon L. In contrast, inclusion of exon K reduces this to 1.5-2.4%. Although relatively small compared with I_Nat, the effect I_Nap has on membrane excitability can be substantial. Indeed, a number of mutations in Na_v channels, seemingly causative of human epilepsy, specifically increase I_Nap. Intriguingly, it has been recently reported that the seizure phenotype characteristic of the larval Drosophila slamdance (sda) mutant is also associated with an increased I_Nap in central motoneurons. In contrast, the magnitude of I_Nat was not affected (Marley, 2011). How loss of the sda gene, which encodes the fly homolog of mammalian aminopeptidase N (APN), results in heightened seizures remains unknown. In mammals, APN is widely expressed and catalyzes the removal of basic and neutral amino acids from the N terminals of peptides. Intriguingly, the related insulin-regulated amino peptidase has been implicated to contribute to seizure, primarily through an as yet undefined interaction with angiotensin IV. However, the precise mechanistic details also remain unknown (Lin, 2012).

This study shows that the choice to splice either exons K or L is perturbed in the sda mutant to favor exclusive inclusion of L. This change is rescued by pretreatment of sda larvae with either the antiepileptic phenytoin or the inhibitory transmitter GABA and recapitulated in wild type (WT) by exposure to picrotoxin (PTx), a known proconvulsive. These observations are indicative that the underlying splicing mechanism is activity dependent. Increased inclusion of exon L requires the presence of Pasilla (Ps), a known RNA-binding protein that has been previously shown to regulate splicing of exons K and L (Park, 2004; Lin, 2009). Finally, a computational approach was used to show that increasing I_Nap is sufficient to increase membrane excitability consistent with the sda epileptic phenotype. Together, these results indicate that increased synaptic activity influences the decision to splice in exon L, which, in turn, may promote seizure (Lin, 2012).

This study shows that a key splicing decision in DmNa_v is influenced by the level of synaptic excitation present in the CNS. Thus, increasing synaptic excitation, through either genetic (e.g., sda) or pharmacological (e.g., PTx) means, is sufficient to favor inclusion of exon L at the expense of the mutually exclusive exon K. Splicing of these exons dramatically influences the magnitude of I_Nap carried by the expressed channel. Increased inclusion of L results in a larger I_Nap, which, in turn, is predicted to further increase action potential firing. These observations provide experimental support for the premise that self-reinforcing cycles of activity contribute to the emergence of epilepsy in susceptible individuals. Moreover, early intervention to break these cycles may offer the exciting prospect of preventing certain types of epilepsy from developing (Lin, 2012).

Although a linkage between synaptic activity and splicing of ion channels has been reported previously, whether this mode of regulation represents the norm remains to be determined. For example, splicing of exon 20 of the NMDA receptor 1 (NR1) is activity dependent. Increased activity promotes splicing to favor the C2 variant, whereas activity blockade results in a predominance of the C2' variant. The C2' variant accelerates NR1 trafficking from the ER to the synapse. Activity, or more specifically the lack of it, decreases the inclusion of the stress axis-regulated exon (STREX) in the mammalian BK K⁺ channel (also known as Slowpoke). Based on homologous expression studies, this change is predicted to decrease action potential firing in neurons expressing this variant. The demonstration that activity influences a splice decision in an Na_v channel is the first such report for this channel type. Importantly, it also extends the number of known examples of such regulation. Recently a structure-function analysis was conducted for the more common splice variants of DmNa_v identified in embryonic CNS (Lin, 2009). Of particular relevance for this study are the mutually exclusive exons K and L. These exons, which differ in 16 of 41 aa, are membrane spanning and are located in homology domain III/S4, which forms part of the voltage sensor. These exons influence the magnitude of I_Nap in expressed channels. When L is present, I_Nap ranges from 4.1% to 9.5% of the peak transient current, and this value drops to 1.5-2.4% when K is included (Lin, 2012).

The molecular origin of I_Nap is still unclear but is believed to result from incomplete inactivation of the channel. Significantly, many channelopathies identified from human epilepsy sufferers show amino acid substitutions in Na_v channels that, when expressed, produce channels with larger than normal I_Nap. This current component is also increased in motoneurons of seizure-sensitive Drosophila mutants, including the sda mutant used in this study (Marley, 2011). Most human Na_v channels exhibit >50% identity to exon K/L, including a region of 11 residues that are identical. Although this region (termed exon 18 in mammalian Na_vs) is also subject to splicing, the outcome differs: a full-length channel containing exon 18 (termed 18A), a truncated channel containing an alternate exon 18 that encodes a stop codon (18N), or a channel that lacks either exon 18A/N (Δ18). Intriguingly, splicing of this region in Na_v is developmentally regulated, keeping open the possibility that aberrant splicing during embryogenesis could lead to altered patterns of activity that may predispose susceptible individuals to epilepsy (Lin, 2012).

An increase in I_Nap has been shown to be associated with, and even causative of, epilepsy in a number of studies. One such example is provided by pilocarpine induction of status epilepticus in rat. Such treatment promotes the appearance of spontaneous recurrent seizures after 1-4 weeks, yet recordings from CA1 pyramidal cells show an associated 1.5-fold increase in I_Nap and a switch to burst firing within 1 week. Similar observations have been reported in other neuron types, for example, entorhinal cortex layer 5, indicative that acute increases in synaptic excitation may be sufficient to increase this current component in all temporal lobe structures. The underlying mechanism(s) is unknown but has been speculated to include transcriptional and/or posttranslational modifications of Na_vs. The results highlight that changes to splicing may also contribute to this phenomenon. This study shows that the known RNA-binding protein Ps is required for the activity-dependent increase in inclusion of exon L in DmNa_v. Ps and its mammalian homologs Nova-1 and Nova-2 are predicted to bind to [T/C]CA[T/C] motifs in pre-mRNAs; multiple copies of these motifs are located in the downstream introns of both exons K and L in DmNa_v. A comparative analysis has recently concluded that the RNA regulatory map between Ps and Nova-1/2 is highly conserved between fly and mammals and that putative pre-mRNA targets of Nova-1/2 include Na_v1.1 and Na_v1.5 (Licatalosi, 2008; Brooks, 2011). Thus, it is probable that splicing of mammalian Na_vs are regulated by the Nova proteins. Significantly, transcription of at least Nova-2 is activity dependent and is downregulated after treatment with pilocarpine. Whether activity also regulates expression of ps is unknown (Lin, 2012).

The human brain has been estimated to contain ~100 billion neurons, each of which receives an average of 10,000 synapses. Because stable circuit function requires matching of presynaptic and postsynaptic activity, it is perhaps surprising, given this level of complexity, that epilepsy is not more prevalent. That it is not is attributable to regulatory mechanisms that continually monitor and, when required, modify both synaptic connectivity and levels of presynaptic and postsynaptic activity. These mechanisms are likely to be particularly important during neurogenesis when both neurons and neural circuits first form. Thus, from the outset, neurons are required not only to make appropriate connections but also to express suitable mixtures of ion channels to enable them to become functional members of individual networks. Once in a network, these same neurons must then continually monitor the level of excitatory and inhibitory synaptic drive to which they are exposed and adapt accordingly. It has been hypothesized that the establishment of an excitation/inhibition balance in adult cortical neurons, critical for circuit stability, may arise from developmental coregulation of developing glutamatergic and GABAergic synapses (Lin, 2012).

Significantly, perturbation of electrical activity in the early developing CNS is sufficient to evoke homeostatic changes in numbers of glutamatergic and GABAergic neurons. Thus, it is possible that any alterations to activity patterns early in the development of the CNS may manifest as changes to the excitation/inhibition balance in mature neural networks. Such altered networks may be prone to seizure-like activity. The early CNS of sda larvae shows increased synaptic activity (Marley, 2011), which may be a consequence of the altered splicing of DmNa_v that is shown in this study. Increased inclusion of exon L is likely to promote self-reinforcing cycles of activity that may disturb the excitation/inhibition balance of the developing CNS, possibly resulting in a seizure-like phenotype in more mature larvae. Consistent with this hypothesis is the observation that feeding phenytoin to WT larvae is sufficient to promote a seizure phenotype that is associated with a significant increase in I_Nap in the aCC (and RP2) motoneuron. Analysis of splicing of DmNa_v isolated from whole CNS from WT fed phenytoin also shows complete saturation of exon L. Thus, it seems likely that perturbation of neural activity in WT, mediated by phenytoin, is also sufficient to induce a change in DmNa_v splicing (Lin, 2012).

The results also show that both splicing of K/L and seizure-like activity can be manipulated in the postembryonic larval CNS. Thus, feeding of PTx to WT, or GABA to sda, is sufficient to either promote or reduce inclusion of exon L and the associated increase in seizure-like activity. This observation is important because it suggests that the consequence of embryonic patterns of neural activity can be overwritten at a later stage. However, whether the effects of these manipulations persist long after exposure to the causative agent has ceased has yet to be determined. Persistence of effect was, however, observed in a previous study, which showed that exposure to a subthreshold amount of phenytoin during embryogenesis is sufficient to prevent the appearance of seizure-like activity in subsequent sda larvae (Marley, 2011). Analysis of DmNa_v splicing in aCC neurons isolated from such 'treated' sda larvae shows an expected reduction in inclusion of exon L. The most parsimonious conclusion is that the presence of phenytoin, during embryogenesis, effectively capped hyperexcitability and, by doing so, uncoupled the positive feedback that is predicted to lead to circuit instability. Clearly, this possibility demands additional investigation (Lin, 2012).

In summary, this study has shown that increased synaptic activity is sufficient to alter splicing of DmNa_v to promote inclusion of alternate exon L. This splicing increases the magnitude of I_Nap of the expressed channel protein isoforms, which, in turn, promotes membrane excitability. This cycle of events may offer a possible mechanistic explanation of the long appreciated phenomenon that 'seizures beget seizures.' For example, in the kindling model, multiple successive small electrical stimuli that are initially without obvious effect eventually lead to full-blown seizure-like behavior (Lin, 2012).

A knock-in model of human epilepsy in Drosophila reveals a novel cellular mechanism associated with heat-induced seizure

Over 40 missense mutations in the human SCN1A sodium channel gene are linked to an epilepsy syndrome termed genetic epilepsy with febrile seizures plus (GEFS+). Inheritance of GEFS+ is dominant, but the underlying cellular mechanisms remain poorly understood. This study reports that knock-in of a GEFS+ SCN1A mutation (K1270T) into the Drosophila sodium channel gene, para, causes a semidominant temperature-induced seizure phenotype. Electrophysiological studies of GABAergic interneurons in the brains of adult GEFS+ flies reveal a novel cellular mechanism underlying heat-induced seizures: the deactivation threshold for persistent sodium currents reversibly shifts to a more negative voltage when the temperature is elevated. This leads to sustained depolarizations in GABAergic neurons and reduced inhibitory activity in the central nervous system. Furthermore, the data indicate a natural temperature-dependent shift in sodium current deactivation (exacerbated by mutation) may contribute to febrile seizures in GEFS+ and perhaps normal individuals (Sun, 2012).

Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures

This study reports the identification of bang senseless (bss), a Drosophila melanogaster mutant exhibiting seizure-like behaviors, as an allele of the paralytic (para) voltage-gated Na(+) (Na_v) channel gene. Mutants are more prone to seizure episodes than normal flies because of a lowered seizure threshold. The bss phenotypes are due to a missense mutation in a segment previously implicated in inactivation, termed the 'paddle motif' of the Na_v fourth homology domain. Heterologous expression of cDNAs containing the bss¹ lesion, followed by electrophysiology, shows that mutant channels display altered voltage dependence of inactivation compared to wild type. The phenotypes of bss are the most severe of the bang-sensitive mutants in Drosophila and can be ameliorated, but not suppressed, by treatment with anti-epileptic drugs. As such, bss-associated seizures resemble those of pharmacologically resistant epilepsies caused by mutation of the human Na_v SCN1A, such as severe myoclonic epilepsy in infants or intractable childhood epilepsy with generalized tonic-clonic seizures (Parker, 2011).

Alternative splicing in the voltage-gated sodium channel DmNa_v regulates activation, inactivation, and persistent current

Diversity in neuronal signaling is a product not only of differential gene expression, but also of alternative splicing. However, although recognized, the precise contribution of alternative splicing in ion channel transcripts to channel kinetics remains poorly understood. Invertebrates, with their smaller genomes, offer attractive models to examine the contribution of splicing to neuronal function. This study reports the sequencing and biophysical characterization of alternative splice variants of the sole voltage-gated Na+ gene (DmNa_v, paralytic), in late-stage embryos of Drosophila melanogaster. Twenty-seven unique splice variants were identified, based on the presence of 15 alternative exons. Heterologous expression, in Xenopus oocytes, shows that alternative exons j, e, and f primarily influence activation kinetics: when present, exon f confers a hyperpolarizing shift in half-activation voltage (V1/2), whereas j and e result in a depolarizing shift. The presence of exon h is sufficient to produce a depolarizing shift in the V1/2 of steady-state inactivation. The magnitude of the persistent Na+ current, but not the fast-inactivating current, in both oocytes and Drosophila motoneurons in vivo is directly influenced by the presence of either one of a pair of mutually exclusive, membrane-spanning exons, termed k and L. Transcripts containing k have significantly smaller persistent currents compared with those containing L. Finally, it waa shown that transcripts lacking all cytoplasmic alternatively spliced exons still produce functional channels, indicating that splicing may influence channel kinetics not only through change to protein structure, but also by allowing differential modification (i.e., phosphorylation, binding of cofactors, etc.). These results provide a functional basis for understanding how alternative splicing of a voltage-gated Na+ channel results in diversity in neuronal signaling (Lin, 2009).

Pumilio binds para mRNA and requires Nanos and Brat to regulate sodium current in Drosophila motoneurons

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 I_Na 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 I_Na 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 I_Na 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 I_Na but also a significant decrease in I_Kfast. Additionally, pan-neuronal overexpression of Pum causes a significant decrease in Shal mRNA, a gene encoding a potassium channel known to contribute to I_Kfast. 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 I_Kfast 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 I_Na, to achieve a reduction in membrane excitability, should be accompanied by a similar decrease in outward I_Kfast. 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 I_Na and I_K 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 I_A but also an unexpected linearly correlated increase in I_h, an effect that acts to preserve membrane excitability. Injection of a mutated, nonfunctional, Shal mRNA is also sufficient to increase I_h 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 I_Na, I_Kfast, and also I_Kslow (Baines, 2001). In contrast, the current study showed that overexpression of pum is sufficient to decrease I_Na and I_Kfast but does not significantly affect I_Kslow (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 I_Kslow 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 I_Na (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 I_Ba(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 I_Na 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).

Molecular and functional characterization of voltage-gated sodium channel variants from Drosophila melanogaster

Extensive alternative splicing and RNA editing have been documented for the transcript of DmNa_v (formerly para), the sole sodium channel gene in Drosophila melanogaster. However, the functional consequences of these post-transcriptional modifications are not well understood. This study isolated 64 full-length DmNa_v cDNA clones from D. melanogaster adults. Based on the usage of 11 alternative exons, 64 clones could be grouped into 29 splice types. When expressed in Xenopus oocytes, 33 DmNa_v variants generated sodium currents large enough for functional characterization. Among these variants, DmNa_v5-1 and DmNa_v7-1 channels activated at the most hyperpolarizing potentials, whereas DmNa_v1-6 and DmNa_v19 channels activated at the most depolarizing membrane potentials. An A-to-I editing event was identified in DmNa_v5-1 that is responsible for its uniquely low-voltage-dependent activation. The wide range of voltage dependence of gating properties exhibited by DmNa_v variants represents a rich resource for future studies to determine the role of DmNa_v in regulating sodium channel gating, pharmacology, and neuronal excitability in insects (Olson, 2008).

Regulation of neuronal excitability through Pumilio-dependent control of a sodium channel gene

Dynamic changes in synaptic connectivity and strength, which occur during both embryonic development and learning, have the tendency to destabilize neural circuits. To overcome this, neurons have developed a diversity of homeostatic mechanisms to maintain firing within physiologically defined limits. This study show that activity-dependent control of mRNA for a specific voltage-gated Na+ channel [encoded by paralytic (para)] contributes to the regulation of membrane excitability in Drosophila motoneurons. Quantification of para mRNA, by real-time reverse-transcription PCR, shows that levels are significantly decreased in CNSs in which synaptic excitation is elevated, whereas, conversely, they are significantly increased when synaptic vesicle release is blocked. Quantification of mRNA encoding the translational repressor pumilio (pum) reveals a reciprocal regulation to that seen for para. Pumilio is sufficient to influence para mRNA. Thus, para mRNA is significantly elevated in a loss-of-function allele of pum (pum^bemused), whereas expression of a full-length pum transgene is sufficient to reduce para mRNA. In the absence of pum, increased synaptic excitation fails to reduce para mRNA, showing that Pum is also necessary for activity-dependent regulation of para mRNA. Analysis of voltage-gated Na+ current (I_Na) mediated by para in two identified motoneurons (termed aCC and RP2) reveals that removal of pum is sufficient to increase one of two separable I_Na components (persistent I_Na), whereas overexpression of a pum transgene is sufficient to suppress both components (transient and persistent). Through use of anemone toxin (ATX II) it was shown that alteration in persistent I_Na is sufficient to regulate membrane excitability in these two motoneurons (Mee, 2004).

Search PubMed for articles about Drosophila Paralytic

Baines, R. A., Uhler, J. P., Thompson, A., Sweeney, S. T. and Bate, M. (2001). Altered electrical properties in Drosophila neurons developing without synaptic transmission. J Neurosci 21(5): 1523-1531. PubMed ID: 11222642

Borst, A. (2018). A biophysical mechanism for preferred direction enhancement in fly motion vision. PLoS Comput Biol 14(6): e1006240. PubMed ID: 29897917

Brooks, A. N., Yang, L., Duff, M. O., Hansen, K. D., Park, J. W., Dudoit, S., Brenner, S. E. and Graveley, B. R. (2011). Conservation of an RNA regulatory map between Drosophila and mammals. Genome Res 21(2): 193-202. PubMed ID: 20921232

Catterall, W. A. (2012). Voltage-gated sodium channels at 60: structure, function and pathophysiology. J Physiol 590(11): 2577-2589. PubMed ID: 22473783

Chagnovich, D. and Lehmann, R. (2001). Poly(A)-independent regulation of maternal hunchback translation in the Drosophila embryo. Proc Natl Acad Sci U S A 98(20): 11359-11364. PubMed ID: 11562474

Derst, C., Walther, C., Veh, R. W., Wicher, D. and Heinemann, S. H. (2006). Four novel sequences in Drosophila melanogaster homologous to the auxiliary Para sodium channel subunit TipE. Biochem Biophys Res Commun 339(3): 939-948. PubMed ID: 16325765

Fendl, S., Vieira, R. M. and Borst, A. (2020). Parallel Visual Conditional protein tagging methods reveal highly specific subcellular distribution of ion channels in motion-sensing neurons. Elife 9. PubMed ID: 33079061

Feng, G., Deak, P., Chopra, M. and Hall, L. M. (1995). Cloning and functional analysis of TipE, a novel membrane protein that enhances Drosophila para sodium channel function. Cell 82(6): 1001-1011. PubMed ID: 7553842

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date revised: 1 January 2024

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