Elise F. Stanley

A Syntaxin 1, Gαo, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

Presynaptic CaV2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive GαO G-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of CaV2.2 with syntaxin 1 and GαO. CaV2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with CaV2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the CaV2.2 puncta, whereas the bulk colocalized with MUNC18-1. GαO, whether in its trimeric or monomeric state, did not coimmunoprecipitate with CaV2.2, MUNC18-1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18-1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with CaV2.2 and GαO, as well as in large clusters remote from the release site with MUNC18-1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.

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

Presynaptic terminals favor intermediate-conductance CaV2.2 (N type) over high-conductance CaV1 (L type) channels for single-channel, Ca2+ nanodomain–triggered synaptic vesicle fusion. However, the standard CaV1>CaV2>CaV3 conductance hierarchy is based on recordings using nonphysiological divalent ion concentrations. We found that, with physiological Ca2+ gradients, the hierarchy was CaV2.2>CaV1>CaV3. Mathematical modeling predicts that the CaV2.2 Ca2+ nanodomain, which is ∼25% more extensive than that generated by CaV1, can activate a calcium-fusion sensor located on the proximal face of the synaptic vesicle.

The Ca2+ release-activated Ca2+ current (ICRAC) 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 (SOCE) pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca2+-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, Ca2+-dependent current potentiation, and block by SKF-96365, DES and 50 μM 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.

A unified model of presynaptic release site gating by calcium channel domains.

Calcium ions enter through discrete ion channels at presynaptic nerve terminals before binding to and activating transmitter release sites. Opposing models hold that release sites are gated either by calcium domains of single, closely associated channels or by extensive, overlapping domains from many remote channels. At the chick calyx synapse we find a linear relation between transmitter release and the number of open calcium channels, favouring single domain activation. This finding is consistent with results from the squid giant synapse but contrasts with steep power dependences reported in rodent synapses, suggestive of activation by extensive overlapping domains. These different reports were reconciled by plotting ‘per cent domain overlap’ against the external calcium concentration used for each species. This relationship predicts the involvement of local channels in the activation of release sites in all species. Further, it suggests that each release site is activated by calcium ions from its immediately associated channels and not by ions that enter through channels associated with a neighbouring release site.

The presynaptic CaV2.2 channel-transmitter release site core complex.

CaV2.2 channels play a key role in the gating of transmitter release sites (TRS) at presynaptic terminals. Physiological studies predict that the channels are linked directly to the TRS but the molecular composition of this complex remains poorly understood. We have used a high‐affinity anti‐CaV2.2 antibody, Ab571, to test a range of proteins known to contribute to TRS function for both an association in situ and a link in vitro. CaV2.2 clusters were isolated intact on immunoprecipitation beads and coprecipitated with a number of these proteins. Quantitative staining covariance analysis (ICA/ICQ method) was applied to the transmitter release face of the giant calyx terminal in the chick ciliary ganglion to test for TRS proteins with staining intensities that covary in situ with CaV2.2, resulting in a covariance sequence of NSF > RIM > spectrin > Munc18 > VAMP > α‐catenin, CASK > SV2 > Na+–K+ ≈ 0. A high‐NaCl dissociation challenge applied to the immunoprecipitated complex, using the fractional recovery (FR) method [ Khanna, R., Li, Q. & Stanley, E.F. (2006 ) PLoS.ONE., 1, e67], was used to test which proteins were most intimately associated with the channel, generating an FR sequence for CaV2.2 of: VAMP ≥ actin > tubulin, NSF, Munc18, syntaxin 1 > spectrin > CASK, SNAP25 > RIM, Na+–K+ pump, v‐ATPase, β‐catenin ≈ 0. Proteins associated with endocytosis are considered in a companion paper [ Khanna et al. (2007)Eur. J. Neurosci., 26, 560–574]. With the exception of VAMP and RIM, the ICQ and FR sequences were consistent, suggesting that proteins that covary the most strongly with CaV2.2 in situ are also the most intimately attached. Our findings suggest that the CaV2.2 cluster is an integral element of a multimolecular vesicle‐fusion module that forms the core of a multifunctional TRS.

N type Ca2+ channels and RIM scaffold protein covary at the presynaptic transmitter release face but are components of independent protein complexes

Fast neurotransmitter release at presynaptic terminals occurs at specialized transmitter release sites where docked secretory vesicles are triggered to fuse with the membrane by the influx of Ca2+ ions that enter through local N type (CaV2.2) calcium channels. Thus, neurosecretion involves two key processes: the docking of vesicles at the transmitter release site, a process that involves the scaffold protein RIM (Rab3A interacting molecule) and its binding partner Munc-13, and the subsequent gating of vesicle fusion by activation of the Ca2+ channels. It is not known, however, whether the vesicle fusion complex with its attached Ca2+ channels and the vesicle docking complex are parts of a single multifunctional entity. The Ca2+ channel itself and RIM were used as markers for these two elements to address this question. We carried out immunostaining at the giant calyx-type synapse of the chick ciliary ganglion to localize the proteins at a native, undisturbed presynaptic nerve terminal. Quantitative immunostaining (intensity correlation analysis/intensity correlation quotient method) was used to test the relationship between these two proteins at the nerve terminal transmitter release face. The staining intensities for CaV2.2 and RIM covary strongly, consistent with the expectation that they are both components of the transmitter release sites. We then used immunoprecipitation to test if these proteins are also parts of a common molecular complex. However, precipitation of CaV2.2 failed to capture either RIM or Munc-13, a RIM binding partner. These findings indicate that although the vesicle fusion and the vesicle docking mechanisms coexist at the transmitter release face they are not parts of a common stable complex.

Molecular scaffold reorganization at the transmitter release site with vesicle exocytosis or botulinum toxin C1.

Neurotransmitter release sites at the freeze‐fractured frog neuromuscular junction are composed of inner and outer paired rows of large membrane particles, the putative calcium channels, anchored by the ribs of an underlying protein scaffold. We analysed the locations of the release site particles as a reflection of the scaffold structure, comparing particle distributions in secreting terminals with those where secretion was blocked with botulinum toxin A, which cleaves a small segment off SNAP‐25, or botulinum toxin C1, which cleaves the cytoplasmic domain of syntaxin. In the idle terminal the inner and outer paired rows were located ≈25 and ≈44 nm, respectively, from the release site midline. However, adjacent to vesicular fusion sites both particle rows were displaced towards the midline by ≈25%. The intervals between the particles along each row were examined by a nearest‐neighbour approach. In control terminals the peak interval along the inner row was ≈17 nm, consistent with previous reports and the spacing of the scaffold ribs. While the average distance between particles in the outer row was also ≈17 nm, a detailed analysis revealed short ‘linear clusters’ with a ≈14 nm interval. These clusters were enriched at vesicle fusion sites, suggesting an association with the docking sites, and were eliminated by botulinum C1, but not A. Our findings suggest, first, that the release site scaffold ribs undergo a predictable, and possibly active, shortening during exocytosis and, second, that at the vesicle docking site syntaxin plays a role in the cross‐linking of the rib tips to form the vesicle docking sites.

Reversed Na+/Ca2+ Exchange Contributes to Ca2+ Influx and Respiratory Burst in Microglia

Phagocytosis and the ensuing NADPH-mediated respiratory burst are important aspects of microglial activation that require calcium ion (Ca2+) influx. However, the specific Ca2+ entry pathway(s) that regulates this mechanism remains unclear, with the best candidates being surface membrane Ca2+-permeable ion channels or Na+/Ca2+ exchangers. In order to address this issue, we used quantitative real-time RT-PCR to assess mRNA expression of the Na+/Ca2+ exchangers, Slc8a1-3/NCX1-3, before and after phagocytosis by rat microglia. All three Na+/Ca2+ exchangers were expressed, with mRNA levels of NCX1 > NCX3 > NCX2, and were unaltered during the one hour phagocytosis period. We then carried out a biophysical characterization of Na+/Ca2+ exchanger activity in these cells. To investigate conditions under which Na+/Ca2+ exchange was functional, we used a combination of perforated patch-clamp analysis, fluorescence imaging of a Ca2+ indicator (Fura-2) and a Na+ indicator (SBFI), and manipulations of membrane potential and intracellular and extracellular ions. Then, we used a pharmacological toolbox to compare the contribution of Na+/Ca2+ exchange with candidate Ca2+-permeable channels, to the NADPH-mediated respiratory burst that was triggered by phagocytosis. We find that inhibiting the reversed mode of the Na+/Ca2+ exchanger with KB-R7943, dose dependently reduced the phagocytosis-stimulated respiratory burst; whereas, blockers of store-operated Ca2+ channels or L-type voltage-gated Ca2+ channels had no effect. These results provide evidence that Na+/Ca2+ exchangers are potential therapeutic targets for reducing the bystander damage that often results from microglia activation in the damaged CNS.

Long splice variant N type calcium channels are clustered at presynaptic transmitter release sites without modular adaptor proteins

The presynaptic N type Ca channel (CaV2.2) is associated with the transmitter release site apparatus and plays a critical role in the gating of transmitter release. It has been suggested that a distinct CaV2.2 long C terminal splice variant is targeted to the nerve terminal and is anchored at the release face by calcium/calmodulin-dependent serine protein kinase (CASK) and Munc-18-interacting protein (MINT), two modular adaptor proteins. We used the isolated chick ciliary ganglion calyx terminal together with two new antibodies (L4569, L4570) selective for CaV2.2 long C terminal splice variant to test these hypotheses. CaV2.2 long C terminal splice variant was present at the presynaptic transmitter release sites, as identified by Rab3a-interacting molecule (RIM) co-staining and quantitative immunocytochemistry. CASK was also present at the terminal both in conjunction with, and independent of its binding partner, MINT. Immunoprecipitation of CaV2.2 long C terminal splice variant from brain lysate coprecipitated CASK, confirming that these two proteins can form a complex. However, CASK was not colocalized either with CaV2.2 long C terminal splice variant or the transmitter release site marker RIM at the calyx terminal release face. Neither was MINT colocalized with CaV2.2 long C terminal splice variant. Our results show that native CaV2.2 long C terminal splice variant is targeted to the transmitter release sites at an intact presynaptic terminal. However, the lack of enrichment of CASK at the release site combined with the failure of this protein or its partner MINT to colocalize with CaV2.2 argues against the idea that these modular adaptor proteins anchor CaV2.2 at presynaptic nerve terminals.

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.