cacophony


REGULATION

Functional Characterization of Cacophony

The neuropeptide CAP2b stimulates fluid transport obligatorily via calcium entry, nitric oxide, and cGMP in Drosophila melanogaster Malpighian (renal) tubules. The Drosophila L-type calcium channel alpha1-subunit genes Dmca1D and Dmca1A (nbA/cacophony) are both expressed in tubules. CAP2b-stimulated fluid transport and cytosolic calcium concentration ([Ca2+]i) increases are inhibited by the L-type calcium channel blockers verapamil and nifedipine. cGMP-stimulated fluid transport is verapamil and nifedipine sensitive. Furthermore, cGMP induces a slow [Ca2+]i increase in tubule principal cells via verapamil- and nifedipine-sensitive calcium entry; RT-PCR shows that tubules express Drosophila Cyclic nucleotide-gated channel (Cng). Additionally, thapsigargin-induced [Ca2+]i increase is verapamil sensitive. Phenylalkylamines bind with differing affinities to the basolateral and apical surfaces of principal cells in the main segment; however, dihydropyridine binds apically in the tubule initial segment. Immunocytochemical evidence suggests localization of alpha1-subunits to both basolateral and apical surfaces of principal cells in the tubule main segment. Roles for L-type calcium channels and cGMP-mediated calcium influx in both calcium signaling and fluid transport mechanisms in Drosophila are suggested (MacPherson, 2002).

mRNA editing of Cacophony

Messenger RNA editing of transcripts encoding voltage-sensitive ion channels has not been extensively analyzed -- least of all in Drosophila, for which several channel-encoding genes are known. Previous sequence studies of D. melanogaster's cacophony gene, which encodes an alpha 1 calcium-channel subunit called Dmca1A, suggested that several nucleotides within the ORF of the primary transcript are edited such that 'A-to-G' substitutions occur (these two nucleotides being the adenine that is found at the relevant sites in the sense strand of genomic DNA or the primary transcript, compared to the substitution of guanine that is detected at the level of cDNA analysis). Such A-to-G changes are the same kind of post-transcriptional variations originally discovered (in a neurobiological context) for a ligand-sensitive channel in vertebrates. RNA was extracted from adult flies and it has been revealed, by RT-PCR and restriction-enzyme analyses, that transcript heterogeneity exists in vivo for three distinct edited sites within the cac-encoded RNA. Each such nucleotide would lead to channel variability at the level of the Dmca1A polypeptide. Owing to cacophony being originally identified as a 'behavioral gene', the possible significance of Dmca1A RNA editing for influencing the relevant neuro-functional phenotypes is discussed (Smith, 1998a).

Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release

The molecular organization of presynaptic active zones during calcium influx-triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) are entirely lost, Ca2+ channels are reduced in density, evoked vesicle release is depressed, and short-term plasticity is altered. BRP-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel, 2006).

Synaptic communication is mediated by the fusion of neurotransmitter-filled vesicles with the presynaptic membrane at the active zone, a process triggered by Ca2+ influx through clusters of voltage-gated channels. The spacing between Ca2+ channels and vesicles at active zones is particularly thought to influence the dynamic properties of synaptic transmission (Kittel, 2006).

The larval Drosophila neuromuscular junction (NMJ) is frequently used as a model of glutamatergic synapses. The monoclonal antibody Nc82 specifically stains individual active zones by recognizing a coiled-coil domain protein of roughly 200 kD named Bruchpilot (Brp). Brp shows homologies to the mammalian active zone components CAST [cytoskeletal matrix associated with the active zone (CAZ)-associated structural protein], also called ERC (ELKS, Rab6-interacting protein 2, and CAST). Whereas confocal microscopy recognized diffraction limited spots, the subdiffraction resolution of stimulated emission depletion (STED) fluorescence microscopy revealed donut-shaped Brp structures at active zones. Viewed perpendicular to the plane of synapses, both single and multiple 'rings' were uncovered, of similar size to freeze-fracture-derived estimates of fly active zones. The donuts were up to 0.16 µm high, as judged by images taken parallel to the synaptic plane (Kittel, 2006).

Brp seems to demark individual active zones associated with clusters of Ca2+ channels. Transposon-mediated mutagenesis allowed isolation of a mutant chromosome (brp69) in which nearly the entire open reading frame of Brp was deleted. brp mutants [brp69/df(2R)BSC29] develop into mature larvae but do not form pupae. The Nc82 label is completely lost from the active zones of brp mutant NMJs but can be restored by re-expressing the brp cDNA in the brp mutant background with use of the neuron-specific driver lines ok6-GAL4. This also rescued larval lethality. Mutants had slightly smaller NMJs and somewhat fewer individual synapses. However, individual receptor fields, identified by the glutamate receptor subunit GluRIID, were enlarged in brp mutants. Thus, principal synapse formation occurred in brp mutants, with individual postsynaptic receptor fields increased in size but moderately decreased in number (Kittel, 2006).

In electron micrographs of brp mutant NMJs, synapses with pre- and postsynaptic membranes in close apposition were present at regular density, and consistent with the enlarged glutamate receptor fields postsynaptic densities appeared larger while otherwise normal. However, intermittent rufflings of the presynaptic membrane were noted, and brp mutants completely lacked presynaptic dense projections (T-bars). Occasionally, very little residual electron-dense material attached to the presynaptic active zone membrane was identified. After re-expressing the Brp protein in the mutant background, T-bar formation could be partially restored, although these structures were occasionally somewhat aberrant in shape. Thus, Brp assists in the ultrastructural assembly of the active zone and is essential for T-bar formation (Kittel, 2006).

In brp mutant larvae a drastic decrease was noted in evoked excitatory junctional current (eEJC) amplitudes by using two-electrode voltage clamp recordings of postsynaptic currents at low stimulation frequencies. This drop in current amplitude could be partially rescued through brp re-expression within the presynaptic motoneurons by using either elav-GAL4 or ok6-GAL4. In contrast, the amplitude of miniature excitatory junctional currents (mEJCs) in response to single, spontaneous vesicle fusion events was increased over control levels. This is consistent with the enlarged individual glutamate receptor fields of brp mutants and excludes a lack of postsynaptic sensitivity as the cause of the reduced eEJC amplitudes (Kittel, 2006).

It follows that the number of vesicles released per presynaptic action potential (AP) (quantal content) was severely compromised at brp mutant NMJs and could not be attributed solely to the moderate decrease in synapse number. The ultrastructural defects of brp mutant synapses may interfere with the proper targeting of vesicles to the active zone membrane and thereby impair exocytosis. The number of vesicles directly docked to active zone membranes was slightly decreased in brp mutants. However, the amplitude distribution and sustained frequency of mEJCs showed that brp mutant synapses did not appear to suffer from extrasynaptic release, as would be caused by a misalignment of vesicle fusion sites with postsynaptic receptors. Consistent with the appropriate deposition of exo- and endocytotic proteins, an apparently normal distribution of Syntaxin, Dap160, and Dynamin was observed at brp mutant synapses (Kittel, 2006).

The exact amplitude and time course of AP-triggered Ca2+ influx in the nerve terminal governs the amplitude and time course of vesicle. Nerve-evoked responses of brp mutants were delayed when compared with controls, whereas in contrast mEJC rise times were unchanged. Thus, evoked vesicle fusion events were less synchronized with the invasion of the presynaptic terminal by an AP. Spatiotemporal changes in Ca2+ influx have a profound effect on short-term plasticity. Whereas at 10 Hz controls exhibited substantial short-term depression of eEJC amplitudes, brp mutants showed strong initial facilitation before stabilizing at a slightly lower but frequency-dependent steady-state current. As judged by the initial facilitation at 10 Hz, neither a reduction in the number of releasable vesicles nor available release sites could fully account for the low quantal content of brp mutants at moderate stimulation frequencies. Furthermore, the altered short-term plasticity of brp mutant synapses suggested a change in the highly Ca2+-dependent vesicle release probability. Paired-pulse protocols were applied to the NMJ. Closely spaced stimuli lead to a buildup of residual Ca2+ in the vicinity of presynaptic Ca2+ channels, enhancing the probability of a vesicle within this local Ca2+ domain to undergo fusion after the next pulse. The absence of marked facilitation at control synapses could be explained by a depletion of release-ready vesicles. At brp mutant NMJs, however, the prominent facilitation at short interpulse intervals showed that the enhancement of release probability strongly outweighed the depletion of releasable vesicles. Thus, initial vesicle release probability was low, and release at brp synapses particularly benefited from the accumulation of intracellular Ca2+ (Kittel, 2006).

Vesicle fusion is highly sensitive to the spacing between Ca2+ channels and vesicles at release sites. It has been calculated that doubling this distance from 25 to 50 nm decreases the release probability threefold, and the larger this distance, the more effective the slow synthetic Ca2+ buffer EGTA should become in suppressing release. Indeed, after bath application of membrane permeable EGTA-AM, the reduction of evoked vesicle release was more pronounced at brp mutant than at control NMJs (Kittel, 2006).

The Ca2+-channel subunit Cacophony governs release at Drosophila NMJs. By using a fully functional, GFP (green fluorescent protein)-labeled variant (CacGFP), Ca2+ channels were visualized in vivo. Consistently, Ca2+ channel expression was severely reduced over the entire NMJ and at synapses lacking Brp (Kittel, 2006).

Thus, it is concluded that brp mutants suffer from a diminished vesicle release probability due to a decrease in the density of presynaptic Ca2+ channel clusters. It is conceivable that Brp tightly surrounds but is not part of the T-bar structure, contained within the unlabeled center of donuts. Brp may establish a matrix, required for both T-bar assembly as well as the appropriate localization of active zone components including Ca2+ channels, possibly by mediating their integration into a restricted number of active zone slots. Related mechanisms might underlie functional impairments of mammalian central synapses lacking active zone components and natural physiological differences between synapse types. Electron microscopy has identified regular arrangements at active zones of mammalian CNS synapses ('particle web') and frog NMJs ('ribs'), where these structures have also been proposed to organize Ca2+ channel clustering. At calyx of Held synapses (an axosomatic synapse in the auditory brainstem), both a fast and a slow component of exocytosis have been described. The fast component recovers slowly and is believed to owe its properties to vesicles attached to a matrix tightly associated with Ca2+ channels, whereas the slow component recovers faster and is thought to be important for sustaining vesicle release during tetanic stimulation. In agreement with this concept, the absence or impairment of such a matrix at brp synapses has a profound effect on vesicle release at low stimulation frequencies, but this effect subsides as the frequency increases. The sustained frequency of mEJCs at brp synapses could be explained if spontaneous fusion events arise from the slow release component or a pathway independent of evoked vesicle fusion (Kittel, 2006).

Synapses lacking Brp and T-bars exhibited a defective coupling of Ca2+ influx with vesicle fusion, whereas the vesicle availability did not appear rate-limiting under low frequency stimulation. The activity-induced addition of presynaptic dense bodies has been proposed to elevate vesicle release probability. This work supports this hypothesis and suggests an involvement of Brp or related factors in synaptic plasticity by promoting Ca2+ channel clustering at the active zone membrane (Kittel, 2006).

Maturation of active zone assembly by Drosophila Bruchpilot

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel-clustering defects. This study used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, BRP was identified as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains (Fouquet, 2009).

This study addressed whether BRP signals T-bar formation (without being a direct component of the T-bar) or whether the protein itself is an essential building block of this electron-dense structure. Evidence is provided that BRP is a direct T-bar component. Immuno-EM identifies the N terminus of BRP throughout the whole cross section of the T-bar, and genetic approaches show that a truncated BRP, lacking the C-terminal 30% of the protein's sequence, forms truncated T-bars. Immuno-EM and light microscopy consistently demonstrate that N- and C-terminal epitopes of BRP are segregated along an axis vertical to the AZ membrane and suggest that BRP is an elongated protein, which directly shapes the T-bar structure (Fouquet, 2009).

In brp5.45 (predicted as aa 1-866), T-bars were not detected, whereas brp1.3 (aa 1-1,389) formed T-bar-like structures, although fewer and smaller than normal. Moreover, the BRPD1-3GFP construct (1-1,226) did not rescue T-bar assembly. Thus, domains between aa 1,226 and 1,390 of BRP may also be important for the formation of T-bars. Clearly, however, the assembly scheme for T-bars is expected to be controlled at several levels (e.g., by phosphorylation) and might involve further protein components. Nonetheless, it is highly likely that the C-terminal half of BRP plays a crucial role (Fouquet, 2009).

Since BRP represents an essential component of the electron-dense T-bar subcompartment at the AZ center, it might link Ca2+ channel-dependent release sites to the synaptic vesicle cycle. Interestingly, light and electron microscopic analysis has located CAST at mammalian synapses both with and without ribbons. Overall, this study is one of the first to genetically identify a component of an electron-dense synaptic specialization and thus paves the way for further genetic analyses of this subcellular structure (Fouquet, 2009).

The N terminus of BRP is found significantly closer to the AZ membrane than the C terminus, where it covers a confined area very similar to the area defined by the CacGFP epitope. Electron tomography of frog NMJs suggested that the cytoplasmic domains of Ca2+ channels, reminiscent of pegs, are concentrated directly beneath a component of an electron-dense AZ matrix resembling ribs. In addition, freeze-fracture EM identified membrane-associated particles at flesh fly AZs, which, as judged by their dimensions, might well be Ca2+ channels. Peg-like structures were observed beneath the T-bar pedestal. Similar to fly T-bars, the frog AZ matrix extends up to 75 nm into the presynaptic cytoplasm. Based on the amount of cytoplasmic Ca2+ channel protein it has been concluded that Ca2+ channels are likely to extend into parts of the ribs. Thus, physical interactions between cytoplasmic domains of Ca2+ channels and components of ribs/T-bars might well control the formation of Ca2+ channel clusters at the AZ membrane. However, a short N-terminal fragment of BRP (aa 1-320) expressed in the brp-null background was unable to localize to AZs efficiently and consistently failed to restore Cac clustering (unpublished data) (Fouquet, 2009).

The mean Ca2+ channel density at AZs is reduced in brp-null alleles. In vitro assays indicate that the N-terminal 20% of BRP can physically interact with the intracellular C terminus of Cacaphony (Cac). Notably, it was found that the GFP epitope at the very C terminus of CacGFP was closer to the AZ membrane than the N-terminal epitope of BRP. It is conceivable that parts of the Cac C terminus extend into the pedestal region of the T-bar cytomatrix to locally interact with the BRP N terminus. This interaction might play a role in clustering Ca2+ channels beneath the T-bar pedestal (Fouquet, 2009).

Clearly, additional work will be needed to identify the contributions of discrete protein interactions in the potentially complex AZ protein interaction scheme. This study should pave the way for a genetic analysis of spatial relationships and structural linkages within the AZ organization. Moreover, the current findings should integrate in the framework of mechanisms for Ca2+ channel trafficking, clustering, and functional modulation (Fouquet, 2009).

The imaging assays allowed a temporally resolved analysis of AZ assembly in vivo. BRP is a late player in AZ assembly, arriving hours after DLiprin-α and also clearly after the postsynaptic accumulation of DGluRIIA. Accumulation of Cac was late as well, although it slightly preceded the arrival of BRP, and impaired Cac clustering at AZs lacking BRP became apparent only from a certain synapse size onwards, form at sites distant from preexisting ones and grow to reach a mature, fixed size. Thus, the late, BRP-dependent formation of the T-bar seems to be required for maintaining high Ca2+ channel levels at maturing AZs but not for initializing Ca2+ channel clustering at newly forming sites. As the dominant fraction of neuromuscular AZs is mature at a given time point, the overall impression is that of a general clustering defect in brp mutants. In reverse, it will be of interest to further differentiate the molecular mechanisms governing early Ca2+ channel clustering. Pre- to postsynaptic communication via neurexin-neuroligin interactions might well contribute to this process. A further candidate involved in early Ca2+ channel clustering is the Fuseless protein, which was recently shown to be crucial for proper Cac localization at AZs (Fouquet, 2009).

In summary, during the developmental formation of Drosophila NMJ synapses, the emergence of a presynaptic dense body, which is involved in accumulating Ca2+ channels, appears to be a central aspect of synapse maturation. This is likely to confer mature release probability to individual AZs and contribute to matching pre- and postsynaptic assembly by regulating glutamate receptor composition. Whether similar mechanisms operate during synapse formation and maturation in mammals remains an open question (Fouquet, 2009).

This study concentrated on developmental synapse formation and maturation. The question arises whether similar mechanisms to those relevant for AZ maturation might control activity-dependent plasticity as well and whether maturation-dependent changes might be reversible at the level of individual synapses. Notably, experience-dependent, bidirectional changes in the size and number of T-bars (occurring within minutes) were implied at Drosophila photoreceptor synapses by ultrastructural means. Moreover, at the crayfish NMJ, multiple complex AZs with double-dense body architecture were produced after stimulation and were associated with higher release probability. In fact, a recent study has correlated the ribbon size of inner hair cell synapses with Ca2+ microdomain amplitudes. Thus, a detailed understanding of the AZ architecture might permit a prediction of functional properties of individual AZs (Fouquet, 2009).

Mutations in a Drosophila α2δ voltage-gated calcium channel subunit reveal a crucial synaptic function

Voltage-dependent calcium channels regulate many aspects of neuronal biology, including synaptic transmission. In addition to their α1 subunit, which encodes the essential voltage gate and selective pore, calcium channels also contain auxiliary α2δ, β, and γ subunits. Despite progress in understanding the biophysical properties of calcium channels, the in vivo functions of these auxiliary subunits remain unclear. Mutations were isolated in the gene encoding an α2δ calcium channel subunit (dα2δ-3) using a forward genetic screen in Drosophila. Null mutations in this gene are embryonic lethal and can be rescued by expression in the nervous system, demonstrating that the essential function of this subunit is neuronal. The photoreceptor phenotype of dα2δ-3 mutants resembles that of the calcium channel α1 mutant cacophony (cac), suggesting shared functions. Genotypes that survive to the third-instar stage have been examined in detail. Electrophysiological recordings demonstrate that synaptic transmission is severely impaired in these mutants. Thus the α2δ calcium channel subunit is critical for calcium-dependent synaptic function. As such, this Drosophila isoform is the likely partner to the presynaptic calcium channel α1 subunit encoded by the cac locus. Consistent with this hypothesis, cacGFP fluorescence at the neuromuscular junction is reduced in dα2δ-3 mutants. This is the first characterization of an α2δ-3 mutant in any organism and indicates a necessary role for α2δ-3 in presynaptic vesicle release and calcium channel expression at active zones (Dickman, 2008).

Calcium channels have well established roles in synaptic transmission, cell excitability, intracellular signaling, and disease. Voltage-gated calcium channels have a unique responsibility for converting electrical changes across the plasma membrane into intracellular changes in calcium concentration. Molecularly, they contain a pore-forming α1 subunit that confers many of the basic properties of the ion channel, including its voltage-sensitive gating, selectivity for calcium, and pharmacological properties. However, calcium channels also contain α2δ and β subunits that can have a substantial influence on the properties of calcium channels when expressed in heterologous systems. Both α2δ and β subunits can markedly increase surface expression of the channels and can also influence the gating properties of the channel. The β subunit is entirely intracellular and is the target for several pathways that modulate calcium channel function. The α2δ subunit, in contrast, lacks an intracellular domain. This subunit consists of two polypeptides that are transcribed as a single transcript and posttranslationally cleaved into the α2 and δ chains, which remain linked by a disulfide bond (Klugbauer, 2003). The α2 portion is entirely extracellular and heavily glycosylated, whereas the δ chain also includes a C-terminal transmembrane domain. In addition, there is a γ subunit whose role is controversial and that need not assemble with the calcium channel complex. Although much has been gained about the biophysical properties of calcium channels, the roles of the auxiliary subunits in regulating calcium channels in vivo is less clear (Dickman, 2008).

Some insights into the role of these accessory subunits in vivo come from a series of spontaneously occurring mutations in mice. These include mutations in a β subunit in lethargic, an α2δ subunit in ducky (Barclay, 2001; Brodbeck, 2002) and in a spontaneous variant of C57BL/10 strain mice, as well as a γ subunit in stargazer. Interestingly, each of these mutants displays ataxia and some form of epilepsy. Moreover, the α2δ calcium channel subunit has been shown to be a target of the anti-epileptic drug gabapentin, although the role of this subunit in the disease remains unclear (Dickman, 2008).

One complication in the genetic analysis of these accessory subunits has been the presence of multiple isoforms in the genome. With regard to α2δ subunits, the number of genes in an organism's genome has remained relatively constant: there are three α2δ isoforms in worms and flies and four isoforms in mammals; these have been classed as α2δ-1,2,3, and 4. In mammals, the α2δ-1 subunit is expressed ubiquitously, whereas the α2δ-2 subunit, the subunit mutated in ducky, is expressed in the brain, kidney, heart, and testes. The α2δ-3 subunit is expressed only in brain. In ducky mice, loss of the α2δ-2 subunit decreases the amplitude of calcium currents in Purkinje cells, in which it is highly expressed, but not in all neurons. Purkinje cells also have abnormal morphologies. At neuromuscular junctions, however, ducky mutations have little effect on transmitter release. Loss of the α2δ-4 subunit causes abnormalities in the outer plexiform layer of the retina. At present, it is uncertain which α1 calcium channel assembles with which α2δ subunit in vivo. In heterologous systems, various combinations promote channel expression, but their associations may be less promiscuous in vivo. Thus, it has not been determined whether synaptic calcium channels also require an α2δ subunit and, if so, what significance that subunit would hold for the physiology of the synapse (Dickman, 2008).

In a forward genetic screen for mutations affecting synaptic transmission, mutations were isolated in the Drosophila α2δ-3 calcium channel subunit. This subunit (dα2δ-3) is essential for viability in Drosophila and shares many of the phenotypes described in mutations of the α1 calcium channel subunit, cacophony. A critical role is demonstrated for dα2δ-3 in synaptic function in both photoreceptors and motorneurons (Dickman, 2008).

α2δ-2 mutant mice show no physiological defects at synapses beyond what can be attributed to the small size of the animals. α2δ-1 has not been studied genetically, but it is expressed in both neuronal and non-neuronal tissues and therefore is likely to have a more general function. Loss of the murine α2δ-4 subunit (Cacna2d4) results in a phenotype that comes closest to that which was observed for loss of dα2δ-3: defects in the synaptic endings of photoreceptors, as revealed by electroretinograms and histology (Wycisk, 2006). The Drosophila genome also contains predicted isoforms of α2δ-1 and α2δ-2, but they do not appear to be functionally redundant with α2δ-3 as α2δ-3 null alleles are lethal and mutations in this isoform produce severe phenotypes in both photoreceptors and neuromuscular junctions. Thus, despite studies in heterologous expression systems that indicate that each α2δ isoform will promiscuously promote the surface expression of any α1 subunit, their functions in vivo are sufficiently distinct that loss of a single subunit can cause a severe phenotype (Dickman, 2008).

Similarly, studies of mammalian channels have not resolved whether each α2δ isoform is associated in vivo with a particular α1 isoform, although there does appear to be some level of selective association. This pairing may derive primarily from the expression patterns of the α2δ and α1 subunits. However, from studies on gabapentin and on ducky mice, α2δ-2 and α2δ-3 appear to be the primary subunits in brain and preferentially associate with P- and N-type calcium channels. In Drosophila, it was found that loss of 2δ-3 gives an electrophysiological phenotype similar to loss of the cac-encoded presynaptic α1 subunit in the ERG and neuromuscular junction. cac is the only Drosophila member of the Cav2 family, homologous to N-, P- and Q-type channels of mammals. Indeed, the cac channel has been established by both electrophysiological studies and cytochemical localization to be the major calcium channel in active zones for driving vesicle release. The present data indicate that the α2δ-3 subunit is its partner and necessary for its proper localization to the active zone. A similar pairing may occur in C. elegans in which unc-36, an α2δ subunit mutant, displays an identical phenotype to the α1 subunit mutant unc-2. At murine photoreceptor synapses, L-type calcium channels mediate transmitter release and therefore, in a subset of mammalian synapses, α2δ-4 may play a similar role to α2δ-3, but partnering L-type rather than N-type channels (Wycisk, 2006), although this has not been investigated with direct recordings of synaptic properties. In the present study, transgenic rescue experiments demonstrated that the only essential function of α2δ-3 in Drosophila is in the nervous system. Other isoforms are thus likely to promote calcium channel expression in other cell types including muscle cells, which express the L-type α1 subunit Dmca1D (Dickman, 2008).

How does the dα2δ-3 subunit contribute to presynaptic function? The leading hypothesis from mammalian work and studies in heterologous systems is that α2δ subunits promote robust plasma membrane expression of the α1 subunit, at least in part by stabilizing them at the plasma membrane (Bernstein, 2007). The phenotype of 2δ-3k10814 is consistent with the hypothesis that 2δ-3 is similarly required for proper synaptic expression of the cacophony α1 subunit. Although it is not possible to record directly from these synaptic boutons to determine the amplitude of calcium currents, the reduction in quantal content per active zone and the decreased amplitude of the EJP are consistent with a decrease in calcium influx at the terminals attributable to decreased channel density. Because of the fourth-order dependence of release on calcium influx, even a 33% reduction in channel density could account for the ~5-fold observed reduction in vesicles released per active zone in the 2δ-3k10814 allele. By fluorescent imaging of the cacGFP transgene, a 25-60% reduction in the level of the cac α1 subunit was observed in 2 different allelic combinations that, because they are not completely null, survive to the third-instar stage. This degree of reduction in α1 subunits at the active zone is consistent with the physiological findings and the hypothesis that the synaptic role of 2δ-3 is to promote the expression, localization, or retention of the cac α1 subunit at active zones. Similarly, the ability of cac overexpression to extend the lifespan of 2δ-3DD196/Df(2)7128 mutants suggests that the 2δ-3 phenotype arises from an insufficiency of synaptic cac channels. In the context of the P-element insertion allele 2δ-3k10814, however, in which substantial amounts of the α1 subunit likely were already present in the terminals, this overexpression was not adequate to significantly increase synaptic transmission (Dickman, 2008).

The 20% decrease in active zones observed per neuromuscular junction, suggested by the decrease in nc82-immunoreactive puncta, is likely to account for a significant component of the 37% decrease in mEJP frequency, but a portion of the decrease may also arise from a change in calcium channel density. Similar decreases in mini frequency arise when neuromuscular junctions are placed in calcium-free salines, indicating that mEJPs are at least partially dependent on the entry of extracellular calcium. One possibility is that the ambient, resting calcium concentration in the synaptic cytosol depends on the amount of calcium that enters through spontaneous openings of calcium channels even at hyperpolarized potentials. Alternatively, sporadic, spontaneous calcium channel openings at the active zones of unstimulated terminals may cause brief, local increases in cytosolic calcium and trigger a portion of the observed minis (Dickman, 2008).

An unexpected feature of the dα2δ-3 mutants was their anatomical phenotype at the neuromuscular junction. In particular, mutants had slightly more boutons per junction, although the muscles themselves were smaller, and these boutons had a lower density of active zones, as scored by detectable puncta of nc82 immunoreactivity. This led to an overall 20% decrease in active zones per muscle (Dickman, 2008).

Changes in the size and morphology of the Drosophila neuromuscular junction have been shown to occur as a result of perturbations in both presynaptic activity. However, many other perturbations of synaptic function do not have these effects, including viable mutations in synaptotagmin, syntaxin and SNAP-25. Mutations in the calcium channel α1 subunit, cac, did not cause an overgrowth of boutons in these studies, and others have reported fewer than normal boutons in cac alleles (Rieckhof, 2003; Xing, 2005). The changes in bouton number and active zone density in 2δ-3 mutants therefore merit additional study. At present, these phenotypes may be direct consequences of the loss of this subunit or may include indirect consequences, possibly compensatory, in response to changes in the activation of the terminals, calcium influx, or transmitter release (Dickman, 2008).

In summary, although much attention has been paid to the pore-forming α1 subunits, the calcium channel is a multi-subunit complex whose other subunits can also serve essential functions. The profound synaptic consequences of the loss of the 2δ-3 subunit highlights the need to understand these subunits in their normal cellular milieu in which both physiological and developmental phenotypes may emerge that could not be appreciated in heterologous systems. These in vivo phenotypes should ultimately refine understanding of the calcium channel complex in neuronal development, function, and disease (Dickman, 2008).

TRP, TRPL and cacophony channels mediate Ca(2+) influx and exocytosis in photoreceptors axons in Drosophila

In Drosophila photoreceptors Ca(2+)-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, the distribution and physiological properties were studied of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca(2+) channel Cacophony is also present there. Measurements in the lamina with the Ca(2+) fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca(2+) increments mediated by Cacophony. Additional Ca(2+) influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on Cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca(2+) also contributes to exocytosis, since this process was reduced after Ca(2+)-store depletion. Therefore, several Ca(2+) pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves Cacophony; this is complemented by internal Ca(2+) release and the activation of TRP and TRPL coupled to Ca(2+) depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons (Astorga, 2012).

Light transduction in Drosophila occurs in retinal microvillar arrangements running along the photoreceptor soma, termed rhabdomere (see Drosophila visual system and brain slices). The axon of this non-spiking neuron releases histamine in a tonic manner. It presents a T-bar ribbon synapse, a particular structure of the active zones specialized for fast and sustained multivesicular neurotransmitter release in response to graded membrane depolarizations. R1-R6 photoreceptors make multiple axo-axonic synaptic contacts with large monopolar (LI-L3) and amacrine cells in the lamina. Cell somata are located in the outermost part of this neuropile, leading to a particular situation where axonal arrays (named cartridges) are the predominant components of the lamina. The axons of centrifugal medullar neurons (C2-C3), a T-shaped centripetal neuron (T1) and a wide field tangential neuron (Tan) are also found in the lamina. In the rhabdomere, photon absorption triggers rhodopsin isomerization into an active state which, upon interaction with a Gq-protein, activates phospholipase C (PLCβ4). This enzyme, encoded by norpA, hydrolyses phosphatidylinositol biphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). This signaling cascade has been widely implicated in the activation of TRP and TRPL. Although the mechanism of channel gating remains undetermined, there is evidence that under experimental conditions, DAG, polyunsaturated fatty acids (PUFAs), PIP2 and protons are involved in opening TRP and TRPL, whereas IP3 receptor does not. Interestingly, TRP and TRPL expressed in heterologous systems are activated by Ca2+ depletion of the endoplasmic reticulum (ER). This study confirmed the presence of TRP in the lamina, where TRPL is also expressed. For the first time, evidence is provided that these channels are implicated in neurotransmitter release in the lamina, where they apparently allow Ca2+ influx via a store-operated channel (SOC) mechanism and could also be regulated by a PLC-mediated cascade. Furthermore, it was shown that the voltage-dependent Ca2+ channel Cacophony, the only fly homologue of vertebrate N-, P/Q- and R-type, is present in the lamina where it plays an important role in photoreceptor synaptic transmission, probably as a first step in a complex cascade involving both intracellular and extracellular Ca2+ signalling (Astorga, 2012).

TRP and TRPL are the targets of Drosophila phototransduction in the rhabdomere, gated by an as yet undetermined PLC-dependent mechanism independent of internal membrane systems, which are absent in the microvilli. This study provides the first evidence that both channels additionally participate in exocytosis in photoreceptor synaptic terminals, where they can be activated by depletion of Ca2+ stores. It is also demonstrated that the voltage-dependent Ca2+ channel, Cacophony, plays a critical role in exocytosis (Astorga, 2012).

This study confirmed that, in addition to the rhabdomere, TRP localizes to the lamina and the medulla. Additionally, TRPL was found in these two neuropiles, where photoreceptors synapse with secondary neurons. The lamina, where most photoreceptors make synaptic connections into well-defined structures, was studied (Astorga, 2012).

A Drosophila slice preparation suitable for immunohistochemistry and functional experiments in the lamina was studied. In addition to TRP and TRPL, PLC and PKC exhibited high expression levels, while Gq and INAD were scarce and rhodopsin was absent. The four former proteins colocalized with ectopically expressed GFP, used as photoreceptor marker, whereas Gq and INAD colocalization with GFP was low. While TRP, TRPL and PLC were not restricted to photoreceptors, the relevant conclusion is that their presence in photoreceptors axons in the lamina suggests a participation in presynaptic events (Astorga, 2012).

The prominent cacophony immunostaining in the lamina is relevant. This Ca2+ channel is involved in synaptic transmission in Drosophila neuromuscular junction, brain and retina, suggesting a role in synaptic transmission in the lamina. A role of cacophony in photoreceptor synaptic transmission is supported by the observation that inhibition of this channel by PLTX-II affected bouton labeling. Although the possibility that PLTX-II could also affect other Ca2+ channels cannot be ruled out, the role of cacophony in vesicle release was further strengthened by the substantial reduction in FM4-64 fluorescence in the thermosensitive cacophony mutant cacTS at non-permissive temperature. In agreement with this, a mutation in the 2δ-3 gene encoding a cacophony subunit abolishes the ERG 'on' transient. In contrast depolarization-induced G-CaMP Ca2+ fluorescence changes in the photoreceptors were significantly decremented by PLTX-II, providing additional evidence involving cacophony in the synaptic events (Astorga, 2012).

The observations that TRP and TRPL are also in the photoreceptors axons and are considerably Ca2+-permeable (PCa:PNa ~100:1 and ~4:1, respectively) suggested a synaptic role. Accordingly, vesicle release was drastically impaired in the double mutant. Opening a Ca2+ pathway with the ionophore induced exocytosis in this mutant, an observation that opposes to a generalized degeneration of synaptic machinery. This evidence shows that TRP and TRPL are involved in exocytosis. Only one of these channels was sufficient for sustaining exocytosis (Astorga, 2012).

FM4-64 is presumably incorporated by all lamina neurons and therefore not only photoreceptor boutons should be labeled. However, it is expected that the dramatic changes in release observed include photoreceptor terminals, which represent the most numerous synaptic contacts in the lamina. Altogether, these results support the participation of TRP, TRPL and cacophony in synaptic transmission in photoreceptor terminals (Astorga, 2012).

What is PLC doing in photoreceptors synaptic terminals? Depolarization-induced exocytosis was markedly reduced in norpA mutant, suggesting a role of PLC in neurotransmitter release. An obvious possibility is that it mediates TRP/TRPL activation. In principle, PLC may act by either DAG or IP3. PUFAs can activate the light-dependent channels when added to intact ommatidia, as well as to excised rhabdomeric membrane patches, in which DAG can do the same. Thus, it is conceivable that these lipids may also activate TRP/TRPL channels in the lamina. Nevertheless, there is no evidence that PUFAs are generated in these photoreceptors (Astorga, 2012).

How is PLC activated? In Drosophila photoreceptors, a level of PLC activity has been observed both in vitro and in vivo. This basal activity is probably a property of the PLC molecule itself, as it is not affected by mutation of Gq-protein. In addition, a positive modulation of PLC activity by micromolar Ca2+ has been reported in Drosophila head membranes. Therefore, basal PLC activity could be boosted by Ca2+ influx through cacophony (and additional Ca2+ pathways described in this study) during depolarization-induced vesicle exocytosis, representing a feed-forward mechanism in this graded synapse. Alternatively, PLC activation may be a consequence of a direct activation of Gq by depolarization, as reported in other insects. In contrast, the substantial PKC expression in the terminals suggests that it may down-regulate PLC, as in the rhabdomere (Astorga, 2012).

Calcium reservoirs appear to be involved in exocytosis, since inhibition of SERCA with Thg deeply affected vesicle release. Moreover, exposure of sercaTS to the non-permissive temperature considerably decreased bouton labeling compared to permissive temperature, and this study shows that this decrease cannot be explained exclusively by a temperature effect. These results strongly implicate ER Ca2+ release in photoreceptors exocytosis (Astorga, 2012).

The robust Ca2+ signals in the lamina after Ca2+ depletion implicated TRP/TRPL, as it was absent in trpl;trp animals. This supports the function of TRP/TRPL as SOCs in the synaptic terminals, allowing Ca2+ influx. This mechanism drives exocytosis, as indicated by the Ca2+-depletion protocol, where bouton labeling was significant. Interestingly, TRP and TRPL function as SOCs in heterologous expression systems, but not in the rhabdomere (Astorga, 2012).

Mammalian homologues of Drosophila TRP, TRPC1, 2, 4 and 6, are proposed to function as SOCs in different cell types. Moreover, TRPC1 operating as SOC regulates Ca2+ influx related to neurotransmission in rods and cones. The Drosophila genome has one gene encoding STIMh, an ER Ca2+ sensor protein that forms functional SOCs in association with TRPC1. It remains to be determined whether TRP/TRPL could form equivalent presynaptic macromolecular complexes in photoreceptors (Astorga, 2012).

This study showed that it is improbable that in the Ca2+-depletion experiments TRPL/TRP opening could be induced by a PLC-dependent mechanism mediated by phospholipase activation by a cytoplasmic Ca2+ increase due to altered reticular release/uptake balance during Thg treatment. In these experiments PLC contribution to exocytosis was possibly by-passed. In normal conditions, this enzyme may elicit Ca2+ elevation in the synaptic terminals by DAG-mediated activation of TRP/TRPL and/or by inducing Ca2+ release (Astorga, 2012).

Photoreceptors synaptic transmission must accurately follow the fast photoresponses generated in the rhabdomere. As graded synapses support rapid changes in neurotransmitter release, they should undergo fast variations in internal free Ca2+. Small and fast Ca2+ increments induce correspondent changes in release, something that would be implausible if a threshold were involved, as in non-graded synapses (Astorga, 2012).

Besides cacophony contribution to exocytosis, the presence of the ryanodine receptor (RyR) in the lamina suggests the participation of Ca2+-induced Ca2+ release (CICR), but direct evidence for this is lacking. CICR regulates exocytosis in rods allowing high rates of neurotransmitter release. A reasonable expectation is that Drosophila photoreceptors use all available Ca2+ pathways (cacophony; TRP/TRPL; the IP3 receptor, IP3R and RyR) to satisfy the synaptic demands required by their extremely fast photoresponse. It has been speculated that the IP3R might reinforce transmitter release, but no direct evidence for it has been shown. This possibility is supported by the current results implicating PLC. Moreover, the observation that Ca2+ from the ER contributes to depolarization-induced exocytosis strengthens the possibility of internal release via IP3R and/or RyR (Astorga, 2012).

Bouton labeling experiments were conducted under prolonged depolarization, implying that vesicle exocytosis was at steady-state. Thg experiments under such conditions show that released Ca2+ plays an essential role in neurotransmission. In tonic synapses, this mechanism may be crucial to sustain synaptic transmission for extensive periods of time (Astorga, 2012).

The following model is proposed for the synaptic events at the axon terminals (see Model for photoreceptor synaptic events in the lamina): the receptor potential activates cacophony in the axon, allowing its propagation towards the axonal terminal, where Ca2+ enters through cacophony inducing vesicle release, perhaps enhanced by CICR. Additionally, PLC activated by an unknown mechanism which may be Ca2+ itself or depolarization, generates IP3, triggering Ca2+ release through IP3Rs. ER Ca2+ depletion in turn opens TRP/TRPL by a SOC mechanism, incrementing the Ca2+ supply. These channels may also be opened by lipid and pH changes resulting of PLC activity. This multi-source transient Ca2+ increment guarantees efficient, rapid and sustained neurotransmitter release. After depolarization, resting Ca2+ levels would be restored by extrusion by the Na+/Ca2+ exchanger and uptake by the ER (Astorga, 2012).

It is important to integrate the data into a plausible working model that could be helpful for designing further experiments. Although the model accounts for the data, it is by no means the only possible one. Accordingly, some aspects of it may be interpreted differently or given a different weight. For example, the relative contributions of cacophony, CICR, IP3-induced Ca2+ depletion and TRP/TRPL to presynaptic Ca2+ for vesicle release can vary widely. Also, the activation of TRP/TRPL may rely on ER depletion and/or lipids associated to PLC activity. It may be thought that the Ca2+ influx through cacophony should be sufficient to account for exocytosis, making Ca2+ release redundant and rather unnecessary. However, in this graded synapse the level of cacophony activation will follow the graded depolarization. The amplitude attained by the receptor potential are most likely within a small voltage range above the threshold for cacophony activation (-20 or -40 mV), inconsistent with a massive cacophony-dependent Ca2+ influx. Therefore, additional Ca2+ sources amplifying this initial signal are likely to be required for light-induced synaptic transmission (Astorga, 2012).

This study has provide novel evidence for TRP/TRPL function in Drosophila photoreceptors. For the first time, it was shown that these channels have dual roles in separate regions of the same cell, namely the rhabdomere and the synapse, apparently involving different mechanisms. More generally, the observations reported herein shed light on the mechanism controlling presynaptic events in graded synapses (Astorga, 2012).

Motor neuron expression of the voltage-gated calcium channel cacophony restores locomotion defects in a Drosophila, TDP-43 loss of function model of ALS

Dysfunction of the RNA-binding protein, TDP-43, is strongly implicated as a causative event in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). TDP-43 is normally found in the nucleus and pathological hallmarks of ALS include the presence of cytoplasmic protein aggregates containing TDP-43 and an associated loss of TDP-43 from the nucleus. Loss of nuclear TDP-43 likely contributes to neurodegeneration. Using Drosophila melanogaster to model TDP-43 loss of function, this study shows that reduced levels of the voltage-gated calcium channel, Cacophony, mediate some of the physiological effects of TDP-43 loss. Null mutations in the Drosophila orthologue of TDP-43, named TBPH, resulted in defective larval locomotion and reduced levels of Cacophony protein in whole animals and at the neuromuscular junction. Restoring the levels of Cacophony in all neurons or selectively in motor neurons rescues these locomotion defects. Using TBPH immunoprecipitation, TBPH was shown to associate with cacophony transcript, indicating that it is likely to be a direct target for TBPH. Loss of TBPH leads to reduced levels of cacophony transcript, possibly due to increased degradation. In addition, TBPH also appears to regulate the inclusion of some alternatively spliced exons of cacophony. If similar effects of cacophony or related calcium channels are found in human ALS patients, these could be targets for the development of pharmacological therapies for ALS (Chang, 2013).


cacophony: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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