Conserved biophysical features of the CaV2 presynaptic Ca2+ channel homologue from the early-diverging animal Trichoplax adhaerens.

The dominant role of CaV2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of CaV2 and CaV1 channels, and less so CaV3 channels, it is unclear why there have not been major shifts toward dependence on other CaV channels for synaptic transmission. Here, we provide a structural and functional profile of the CaV2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possesses single gene homologues for CaV1-CaV3 channels. Remarkably, the highly divergent channel possesses similar features as human CaV2.1 and other CaV2 channels, including high voltage-activated currents that are larger in external Ba2+ than in Ca2+; voltage-dependent kinetics of activation, inactivation, and deactivation; and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax CaV2 suggests that the core features of presynaptic CaV2 channels were established early during animal evolution, after CaV1 and CaV2 channels emerged via proposed gene duplication from an ancestral CaV1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian CaV2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA and to metal cation blockers Cd2+ and Ni2+ Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gßγ subunits, which nevertheless inhibited the human CaV2.1 channel, suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G proteins. Last, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior and other likely secretory cells, suggesting contributions to regulated exocytosis.

Molecular Characterization of an SV Capture Site in the Mid-Region of the Presynaptic CaV2.1 Calcium Channel C-Terminal.

Neurotransmitter is released from presynaptic nerve terminals at fast-transmitting synapses by the action potential-gating of voltage dependent calcium channels (CaV), primarily of the CaV2.1 and CaV2.2 types. Entering Ca2+ diffuses to a nearby calcium sensor associated with a docked synaptic vesicle (SV) and initiates its fusion and discharge. Our previous findings that single CaVs can gate SV fusion argued for one or more tethers linking CaVs to docked SVs but the molecular nature of these tethers have not been established. We recently developed a cell-free, in vitro biochemical assay, termed SV pull-down (SV-PD), to test for SV binding proteins and used this to demonstrate that CaV2.2 or the distal third of its C-terminal can capture SVs. In subsequent reports we identified the binding site and characterized an SV binding motif. In this study, we set out to test if a similar SV-binding mechanism exists in the primary presynaptic channel type, CaV2.1. We cloned the chick variant of this channel and to our surprise found that it lacked the terminal third of the C-terminal, ruling out direct correlation with CaV2.2. We used SV-PD to identify an SV binding site in the distal half of the CaV2.1 C-terminal, a region that corresponds to the central third of the CaV2.2 C-terminal. Mutant fusion proteins combined with motif-blocking peptide strategies identified two domains that could account for SV binding; one in an alternatively spliced region (E44) and a second more distal site. Our findings provide a molecular basis for CaV2.1 SV binding that can account for recent evidence of C-terminal-dependent transmitter release modulation and that may contribute to SV tethering within the CaV2.1 single channel Ca2+ domain.

The Calcium Channel C-Terminal and Synaptic Vesicle Tethering: Analysis by Immuno-Nanogold Localization.

At chemical synapses the incoming action potential triggers the influx of Ca2+ through voltage-sensitive calcium channels (CaVs, typically CaV2.1 and 2.2) and the ions binds to sensors associated with docked, transmitter filled synaptic vesicles (SVs), triggering their fusion and discharge. The CaVs and docked SVs are located within the active zone (AZ) region of the synapse which faces a corresponding neurotransmitter receptor-rich region on the post-synaptic cell. Evidence that the fusion of a SV can be gated by Ca2+ influx through a single CaV suggests that the channel and docked vesicle are linked by one or more molecular tethers (Stanley, 1993). Short and long fibrous SV-AZ linkers have been identified in presynaptic terminals by electron microscopy and we recently imaged these in cytosol-vacated synaptosome 'ghosts.' Using CaV fusion proteins combined with blocking peptides we previously identified a SV binding site near the tip of the CaV2.2 C-terminal suggesting that this intracellular channel domain participates in SV tethering. In this study, we combined the synaptosome ghost imaging method with immunogold labeling to localize CaV intracellular domains. L45, raised against the C-terminal tip, tagged tethered SVs often as far as 100 nm from the AZ membrane whereas NmidC2, raised against a C-terminal mid-region peptide, and C2Nt, raised against a peptide nearer the C-terminal origin, resulted in gold particles that were proportionally closer to the AZ. Interestingly, the observation of gold-tagged SVs with NmidC2 suggests a novel SV binding site in the C-terminal mid region. Our results implicate the CaV C-terminal in SV tethering at the AZ with two possible functions: first, capturing SVs from the nearby cytoplasm and second, contributing to the localization of the SV close to the channel to permit single domain gating.

Characterization of a Synaptic Vesicle Binding Motif on the Distal CaV2.2 Channel C-terminal.

Neurotransmitter is released from synaptic vesicles (SVs) that are gated to fuse with the presynaptic membrane by calcium ions that enter through voltage-gated calcium channels (CaVs). There is compelling evidence that SVs associate closely with the CaVs but the molecular linking mechanisms remain poorly understood. Using a cell-free, synaptic vesicle-pull-down assay method (SV-PD) we have recently demonstrated that SVs can bind both to the intact CaV2.2 channel and also to a fusion protein comprising the distal third, C3 segment, of its long C-terminal. This site was localized to a 49 amino acid region just proximal to the C-terminal tip. To further restrict the SV binding site we generated five, 10 amino acid mimetic blocking peptides spanning this region. Of these, HQARRVPNGY effectively inhibited SV-PD and also inhibited SV recycling when cryoloaded into chick brain nerve terminals (synaptosomes). Further, SV-PD was markedly reduced using a C3 fusion protein that lacked the HQARRVPNGY sequence, C3HQless. We zeroed in on the SV binding motif within HQARRVPNGY by means of a palette of mutant blocking peptides. To our surprise, peptides that lacked the highly conserved VPNGY sequence still blocked SV-PD. However, substitution of the HQ and RR amino acids markedly reduced block. Of these, the RR pair was essential but not sufficient as the full block was not observed without H suggesting a CaV2.2 SV binding motif of HxxRR. Interestingly, CaV2.1, the other primary presynaptic calcium channel, exhibits a similar motif, RHxRR, that likely serves the same function. Bioinformatic analysis showed that variations of this binding motif, +(+) xRR (where + is a positively charged aa H or R), are conserved from lung-fish to man. Further studies will be necessary to identify the C terminal motif binding partner on the SV itself and to determine the role of this molecular interaction in synaptic transmission. We hypothesize that the distal C-terminal participates in the capture of the SVs from the cytoplasm, initiating their delivery to the active zone where additional tethering interactions secure the vesicle within range of the CaV single Ca(2+) domains.

The Nanophysiology of Fast Transmitter Release.

Action potentials invading the presynaptic terminal trigger discharge of docked synaptic vesicles (SVs) by opening voltage-dependent calcium channels (CaVs) and admitting calcium ions (Ca(2+)), which diffuse to, and activate, SV sensors. At most synapses, SV sensors and CaVs are sufficiently close that release is gated by individual CaV Ca(2+) nanodomains centered on the channel mouth. Other synapses gate SV release with extensive Ca(2+) microdomains summed from many, more distant CaVs. We review the experimental preparations, theories, and methods that provided principles of release nanophysiology and highlight expansion of the field into synaptic diversity and modifications of release gating for specific synaptic demands. Specializations in domain gating may adapt the terminal for roles in development, transmission of rapid impulse frequencies, and modulation of synaptic strength.

Single calcium channel domain gating of synaptic vesicle fusion at fast synapses; analysis by graphic modeling.

At fast-transmitting presynaptic terminals Ca(2+) enter through voltage gated calcium channels (CaVs) and bind to a synaptic vesicle (SV) -associated calcium sensor (SV-sensor) to gate fusion and discharge. An open CaV generates a high-concentration plume, or nanodomain of Ca(2+) that dissipates precipitously with distance from the pore. At most fast synapses, such as the frog neuromuscular junction (NMJ), the SV sensors are located sufficiently close to individual CaVs to be gated by single nanodomains. However, at others, such as the mature rodent calyx of Held (calyx of Held), the physiology is more complex with evidence that CaVs that are both close and distant from the SV sensor and it is argued that release is gated primarily by the overlapping Ca(2+) nanodomains from many CaVs. We devised a 'graphic modeling' method to sum Ca(2+) from individual CaVs located at varying distances from the SV-sensor to determine the SV release probability and also the fraction of that probability that can be attributed to single domain gating. This method was applied first to simplified, low and high CaV density model release sites and then to published data on the contrasting frog NMJ and the rodent calyx of Held native synapses. We report 3 main predictions: the SV-sensor is positioned very close to the point at which the SV fuses with the membrane; single domain-release gating predominates even at synapses where the SV abuts a large cluster of CaVs, and even relatively remote CaVs can contribute significantly to single domain-based gating.

Synaptic vesicle tethering and the CaV2.2 distal C-terminal.

Evidence that synaptic vesicles (SVs) can be gated by a single voltage sensitive calcium channel (CaV2.2) predict a molecular linking mechanism or "tether" (Stanley, 1993). Recent studies have proposed that the SV binds to the distal C-terminal on the CaV2.2 calcium channel (Kaeser et al., 2011; Wong et al., 2013) while genetic analysis proposed a double tether mechanism via RIM: directly to the C terminus PDZ ligand domain or indirectly via a more proximal proline rich site (Kaeser et al., 2011). Using a novel in vitro SV pull down binding assay, we reported that SVs bind to a fusion protein comprising the C-terminal distal third (C3, aa 2137-2357; Wong et al., 2013). Here we limit the binding site further to the last 58 aa, beyond the proline rich site, by the absence of SV capture by a truncated C3 fusion protein (aa 2137-2299). To test PDZ-dependent binding we generated two C terminus-mutant C3 fusion proteins and a mimetic blocking peptide (H-WC, aa 2349-2357) and validated these by elimination of MINT-1 or RIM binding. Persistence of SV capture with all three fusion proteins or with the full length C3 protein but in the presence of blocking peptide, demonstrated that SVs can bind to the distal C-terminal via a PDZ-independent mechanism. These results were supported in situ by normal SV turnover in H-WC-loaded synaptosomes, as assayed by a novel peptide cryoloading method. Thus, SVs tether to the CaV2.2 C-terminal within a 49 aa region immediately prior to the terminus PDZ ligand domain. Long tethers that could reflect extended C termini were imaged by electron microscopy of synaptosome ghosts. To fully account for SV tethering we propose a model where SVs are initially captured, or "grabbed," from the cytoplasm by a binding site on the distal region of the channel C-terminal and are then retracted to be "locked" close to the channel by a second attachment mechanism in preparation for single channel domain gating.

Cryoloading: introducing large molecules into live synaptosomes.

Neurons communicate with their target cells primarily by the release of chemical transmitters from presynaptic nerve terminals. The study of CNS presynaptic nerve terminals, isolated as synaptosomes (SSMs) has, however, been hampered by the typical small size of these structures that precludes the introduction of non-membrane permeable test substances such as peptides and drugs. We have developed a method to introduce large alien compounds of at least 150 kDa into functional synaptosomes. Purified synaptosomes are frozen in cryo-preserving buffer containing the alien compound. Upon defrosting, many of the SSMs contain the alien compound presumably admitted by bulk buffer-transfer through the surface membranes that crack and reseal during the freeze/thaw cycle. ~80% of the cryoloaded synaptosomes were functional and recycled synaptic vesicles (SVs), as assessed by a standard styryl dye uptake assay. Access of the cryoloaded compound into the cytoplasm and biological activity were confirmed by block of depolarization-induced SV recycling with membrane-impermeant BAPTA (a rapid Ca(2+)-scavenger), or botulinum A light chain (which cleaves the soluble NSF attachment protein receptor (SNARE) protein SNAP25). A major advantage of the method is that loaded frozen synaptosomes can be stored virtually indefinitely for later experimentation. We also demonstrate that individual synaptosome types can be identified by immunostaining of receptors associated with its scab of attached postsynaptic membrane. Thus, cryoloading and scab-staining permits the examination of SV recycling in identified individual CNS presynaptic nerve terminals.

Low voltage-activated calcium channels gate transmitter release at the dorsal root ganglion sandwich synapse.

A subpopulation of dorsal root ganglion (DRG) neurons are intimately attached in pairs and separated solely by thin satellite glial cell membrane septa. Stimulation of one neuron leads to transglial activation of its pair by a bi-, purinergic/glutamatergic synaptic pathway, a transmission mechanism that we term sandwich synapse (SS) transmission. Release of ATP from the stimulated neuron can be attributed to a classical mechanism involving Ca(2+) entry via voltage-gated calcium channels (CaV) but via an unknown channel type. Specific blockers and toxins ruled out CaV1, 2.1 and 2.2. Transmission was, however, blocked by a moderate depolarization (-50 mV) or low-concentration Ni(2+) (0.1 mM). Transmission persisted using a voltage pulse to -40 mV from a holding potential of -80 mV, confirming the involvement of a low voltage-activated channel type and limiting the candidate channel type to either CaV3.2 or a subpopulation of inactivation- and Ni(2+)-sensitive CaV2.3 channels. Resistance of the neuron calcium current and SS transmission to SNX482 argue against the latter. Hence, we conclude that inter-somatic transmission at the DRG SS is gated by CaV3.2 type calcium channels. The use of CaV3 family channels to gate transmission has important implications for the biological function of the DRG SS as information transfer would be predicted to occur not only in response to action potentials but also to sub-threshold membrane voltage oscillations. Thus, the SS synapse may serve as a homeostatic signalling mechanism between select neurons in the DRG and could play a role in abnormal sensation such as neuropathic pain.

Synaptic vesicle capture by CaV2.2 calcium channels.

The fusion of synaptic vesicles (SVs) at the presynaptic transmitter release face is gated by Ca(2) (+) influx from nearby voltage-gated calcium channels (CaVs). Functional studies favor a direct molecular "tethering" attachment and recent studies have proposed a direct link to the channel C-terminal. To test for direct CaV-SV attachment we developed an in vitro assay, termed SV pull-down (SV-PD), to test for capture of purified, intact SVs. Antibody-immobilized presynaptic or expressed CaV2.2 channels but not plain beads, IgG or pre-blocked antibody successfully captured SVs, as assessed byWestern blot for a variety of protein markers. SV-PD was also observed with terminal fusion proteins of the distal half of the C-terminal, supporting involvement of this CaV region in tethering. Thus our results support a model in which the SV tethers directly to the CaV. Since the tip of the C-terminal could extend as far as 200 nm into the cytoplasm, we hypothesize that this link may serve as the initial SV capture mechanism by the release site. Further studies will be necessary to evaluate the molecular basis of C-terminal tethering and whether the SV binds to the channel by additional, shorter-range attachments.

Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain.

Calcium entry through CaV2.2 calcium channels clustered at the active zone (AZ) of the presynaptic nerve terminal gates synaptic vesicle (SV) fusion and the discharge of neurotransmitters, but the mechanism of channel scaffolding remains poorly understood. Recent studies have implicated the binding of a PDZ ligand domain (PDZ-LD) at the tip of the channel C terminal to a partner PDZ domain on RIM1/2, a synaptic vesicle-associated protein. To explore CaV2.2 scaffolding, we created intracellular region fusion proteins and used these to test for binding by 'fishing' for native CaV2.2 channels from cell lysates. Fusion proteins mimicking the distal half of the channel C terminal (C3strep) reliably captured CaV2.2 from whole brain crude membrane or purified synaptosome membrane lysates, whereas channel I-II loop or the distal half of the II-III loop proteins were negative. This capture could be replicated in a non-synaptic environment using CaV2.2 expressed in a cell line. The distal tip PDZ-LD, DDWC-COOH, was confirmed as the critical binding site by block of pull-down with mimetic peptides. Pull-down experiments using brain crude membrane lysates confirmed that RIM1/2 can bind to the DDWC PDZ-LD. However, robust CaV2.2 capture was observed from synaptosome membrane or in the cell line expression system with little or no RIM1/2 co-capture. Thus, we conclude that CaV2.2 channels can scaffold to each other via an interaction that involves the PDZ-LD by an inter-channel linkage bridged by an unknown protein.

Transglial transmission at the dorsal root ganglion sandwich synapse: glial cell to postsynaptic neuron communication.

The dorsal root ganglion (DRG) contains a subset of closely-apposed neuronal somata (NS) separated solely by a thin satellite glial cell (SGC) membrane septum to form an NS-glial cell-NS trimer. We recently reported that stimulation of one NS with an impulse train triggers a delayed, noisy and long-lasting response in its NS pair via a transglial signaling pathway that we term a 'sandwich synapse' (SS). Transmission could be unidirectional or bidirectional and facilitated in response to a second stimulus train. We have shown that in chick or rat SS the NS-to-SGC leg of the two-synapse pathway is purinergic via P2Y2 receptors but the second SGC-to-NS synapse mechanism remained unknown. A noisy evoked current in the target neuron, a reversal potential close to 0 mV, and insensitivity to calcium scavengers or G protein block favored an ionotropic postsynaptic receptor. Selective block by D-2-amino-5-phosphonopentanoate (AP5) implicated glutamatergic transmission via N-methyl-d-aspartate receptors. This agent also blocked NS responses evoked by puff of UTP, a P2Y2 agonist, directly onto the SGC cell, confirming its action at the second synapse of the SS transmission pathway. The N-methyl-d-aspartate receptor NR2B subunit was implicated by block of transmission with ifenprodil and by its immunocytochemical localization to the NS membrane, abutting the glial septum P2Y2 receptor. Isolated DRG cell clusters exhibited daisy-chain and branching NS-glial cell-NS contacts, suggestive of a network organization within the ganglion. The identification of the glial-to-neuron transmitter and receptor combination provides further support for transglial transmission and completes the DRG SS molecular transmission pathway.

Purinergic transmission and transglial signaling between neuron somata in the dorsal root ganglion.

Most dorsal root ganglion neuronal somata (NS) are isolated from their neighbours by a satellite glial cell (SGC) sheath. However, some NS are associated in pairs, separated solely by the membrane septum of a common SGC to form a neuron-glial cell-neuron (NGlN) trimer. We reported that stimulation of one NS evokes a delayed, noisy and long-duration inward current in both itself and its passive partner that was blocked by suramin, a general purinergic antagonist. Here we test the hypothesis that NGlN transmission involves purinergic activation of the SGC. Stimulation of the NS triggered a sustained current noise in the SGC. Block of transmission through the NGlN by reactive blue 2 or thapsigargin, a Ca(2+) store-depletion agent, implicated a Ca(2+) store discharge-linked P2Y receptor. P2Y2 was identified by simulation of the NGlN-like transmission by puffing UTP onto the SGC and by immunocytochemical localization to the SGC membrane septum. Block of the UTP effect by BAPTA, an intracellular Ca(2+) scavenger, supported the involvement of SGC Ca(2+) stores in the signaling pathway. We infer that transmission through the NGlN trimer involves secretion of ATP from the NS and triggering of SGC Ca(2+) store discharge via P2Y2 receptors. Presumably, cytoplasmic Ca(2+) elevation leads to the release of an as-yet unidentified second transmitter from the glial cell to complete transmission. Thus, the two NS of the NGlN trimer communicate via a 'sandwich synapse' transglial pathway, a novel signaling mechanism that may contribute to information transfer in other regions of the nervous system.

Slow chemical transmission between dorsal root ganglion neuron somata.

Somatic sensory neuron somata are located within the dorsal root ganglia (DRG) and are mostly ensheathed by individual satellite glial cell sheets. It has been noted, however, that a subpopulation of these DRG somata are intimately associated, separated only by a single thin satellite glial cell membrane septum. We set out to test whether such neuron-glial cell-neuron trimers (NGlNs) are also linked functionally. The presence of NGlNs in chick DRGs was confirmed by electron microscopy. Selective satellite glial cell immunostains were identified and were used to image the inter-neuron septa in DRG frozen sections. We used a gentle, dispase-based enzymatic method to isolate chick and rat NGlNs in vitro for double patch clamp recordings. In the majority of pairs tested, an action potential-like stimulus train delivered to one soma resulted in a delayed, noisy and long-duration response in its idle partner. The response to a second stimulus train given minutes later was markedly facilitated. Both bidirectional and unidirectional transmission was observed between the paired neurons. Transmission was chemical and block by the general purinergic blocker suramin implicated ATP as a neurotransmitter. We conclude that the two neuronal somata in the NGlN can communicate by chemical transmission, which may involve a transglial, bi-synaptic pathway. This novel soma-to-soma transmission reflects a novel form of processing that may play a role in sensory disorders in the DRG and interneuron communication in the central nervous system.

N-type Ca2+ channels carry the largest current: implications for nanodomains and transmitter release.

Presynaptic terminals favor intermediate-conductance Ca(V)2.2 (N type) over high-conductance Ca(V)1 (L type) channels for single-channel, Ca(2+) nanodomain-triggered synaptic vesicle fusion. However, the standard Ca(V)1>Ca(V)2>Ca(V)3 conductance hierarchy is based on recordings using nonphysiological divalent ion concentrations. We found that, with physiological Ca(2+) gradients, the hierarchy was Ca(V)2.2>Ca(V)1>Ca(V)3. Mathematical modeling predicts that the Ca(V)2.2 Ca(2+) nanodomain, which is ∼25% more extensive than that generated by Ca(V)1, can activate a calcium-fusion sensor located on the proximal face of the synaptic vesicle.

Long C terminal splice variant CaV2.2 identified in presynaptic membrane by mass spectrometric analysis.

Ca(V)2.2 voltage-gated calcium channels play a key role in the gating of transmitter release at presynaptic terminals. Recently we used mass spectrometry (MS) to analyze the protein complex associated with Ca(V)2.2 in purified presynaptic terminal membranes. A number of known and new Ca(V)2.2-associated proteins were identified, but not the channel itself. Here we set out to explore this anomaly. As previously, we used antibody Ab571 to capture the channel from purified synaptosome membrane lysate. We prepared a brain membrane lysate enriched for presynaptic active zones using standard methods to fractionate purified synaptosomes. These were osmotically lysed to generate a fraction enriched in presynaptic surface membranes. The lysate was solubilized in modified RIPA buffer and was passed over anti-Ca(V)2.2 antibody covalently bonded to immunoprecipitation beads. Captured complexes on the beads were then stripped of weakly-bound proteins by exposure to high salt to enrich the channel fraction. Proteins remaining bound to the sample were recovered in high concentration urea and the sample was subjected to standard enzyme digestion and MS analysis. We identified 12 distinct Ca(V)2.2 peptides, but no other ion channel peptides, in the lysate-exposed bead sample but no other ion channel peptides were recovered. Interestingly one of the channel peptides was derived from the alternatively spliced, long-C terminal region. Hence, confidence in identification of Ca(V)2.2 was beyond reasonable doubt. The identification of the long-splice Ca(V)2.2 provides compelling evidence that this variant is targeted to the presynaptic terminal, as we and others have suggested.

Rab3a interacting molecule (RIM) and the tethering of pre-synaptic transmitter release site-associated CaV2.2 calcium channels.

Biochemical and physiological evidence suggest that pre-synaptic calcium channels are attached to the transmitter release site within the active zone by a molecular tether. A recent study has proposed that 'Rab3a Interacting Molecule' (RIM) serves as the tether for CaV2.1 channels in mouse brain, based in part on biochemical co-immunoprecipitation (co-IP) using a monoclonal antibody, mRIM. We previously argued against this idea for CaV2.2 calcium channel at chick synapses based on experiments using a different anti-RIM antibody, pRIM1,2: while staining for the two proteins co-localized and co-varied at the transmitter release face, consistent with an association, they failed to co-IP from a synaptosome membrane lysate. RIM is, however, a family of proteins and we tested the possibility that the mRIM antibody used in the more recent study identifies a particular channel-tethering variant. We find that co-immunostaining with mRIM and anti-CaV2.2 antibody neither co-localized nor co-varied at the transmitter release face and the two proteins did not co-IP, arguing against a common protein complex and a key CaV2.2 scaffolding role for RIM at the active zone. The differing results might be reconciled, however, in a model where a RIM family member contributes to a protein bridge that anchors the pre-fusion secretory vesicle to the calcium channel protein complex.

The Ca2+ release-activated Ca2+ current (I(CRAC)) mediates store-operated Ca2+ entry in rat microglia.

Ca2+ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca2+ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca2+ permeable channels, and limited electrophysiological analyses of Ca2+ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are 'transient receptor potential' (TRP) channels and the recently cloned Orai1, which produces a Ca2+-release-activated Ca2+ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca2+ entry pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 microM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 microM), and exhibited Ca(2+)-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca2+ current with the same properties, including high selectivity for Ca2+ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca(2+)-dependent current potentiation, and block by SKF-96365, DES and 50 microM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.