Transmitter Release Sites Research Articles

The Role of Cav2.2 C-Terminus in Calcium Channel Anchoring at the Presynaptic Transmitter Release Sites

Voltage gated neuronal calcium channels (Cav2.2) gate neurotransmitter release at specialized regions within the presynaptic nerve terminals, known as transmitter release sites (TRS). Published studies have predicted that calcium channels are closely attached to the TRS in an organized structure, supporting a channel-channel anchoring mechanism. Although calcium channels have been linked to a broad range of presynaptic proteins, molecular components of the channel-channel anchor are poorly understood. Our objective is to identify the mechanism of Ca2+ channel anchoring at the TRS and will explore the role of channel cytoplasmic regions. GST or strep tagged Cav2.2 cytoplasmic region fusion proteins were generated and used for pull-down assays from which distal C-terminus region (C3strep) captured the channel from brain lysate. In order to determine the specific region responsible for Cav2.2 pull-down, GST fusion proteins comprising overlapping proximal (C3proximal) and distal (C3distal) regions of C3strep were generated. Our data show that C3distal region captures the channel from avian synaptosome membrane lysate. This finding was supported by a reduction of channel pull-down from brain lysate in the presence of a blocking peptide mimicking the last four amino acids of long splice variant Cav2.2 distal C-terminus which code a PDZ-ligand domain. C3strep protein was also able to capture transfected Cav2.2 from TSA201 cell lysate, narrowing down the possibilities of a channel-channel bridging protein(s). Overall, these data suggest a key role for Cav2.2 distal C-terminus in channel anchoring at TRS.

Lack of Brain-Derived Neurotrophic Factor Hampers Inner Hair Cell Synapse Physiology, But Protects against Noise-Induced Hearing Loss

The precision of sound information transmitted to the brain depends on the transfer characteristics of the inner hair cell (IHC) ribbon synapse and its multiple contacting auditory fibers. We found that brain derived neurotrophic factor (BDNF) differentially influences IHC characteristics in the intact and injured cochlea. Using conditional knock-out mice (BDNF(Pax2) KO) we found that resting membrane potentials, membrane capacitance and resting linear leak conductance of adult BDNF(Pax2) KO IHCs showed a normal maturation. Likewise, in BDNF(Pax2) KO membrane capacitance (ΔC(m)) as a function of inward calcium current (I(Ca)) follows the linear relationship typical for normal adult IHCs. In contrast the maximal ΔC(m), but not the maximal size of the calcium current, was significantly reduced by 45% in basal but not in apical cochlear turns in BDNF(Pax2) KO IHCs. Maximal ΔC(m) correlated with a loss of IHC ribbons in these cochlear turns and a reduced activity of the auditory nerve (auditory brainstem response wave I). Remarkably, a noise-induced loss of IHC ribbons, followed by reduced activity of the auditory nerve and reduced centrally generated wave II and III observed in control mice, was prevented in equally noise-exposed BDNF(Pax2) KO mice. Data suggest that BDNF expressed in the cochlea is essential for maintenance of adult IHC transmitter release sites and that BDNF upholds opposing afferents in high-frequency turns and scales them down following noise exposure.

Cell Type-Dependent Trafficking of Neuropeptide Y-Containing Dense Core Granules in CNS Neurons

Neuropeptide transmitters are synthesized throughout the CNS and play important modulatory roles. After synthesis in the neuronal cell body, it is generally assumed that peptides are transported to nonspecialized sites of release. However, apart from a few cases, this scenario has not been thoroughly examined. Using wild-type and NPY(GFP) transgenic mice, we have studied the subcellular distribution of neuropeptide Y (NPY), a prototypical and broadly expressed neuropeptide. NPY puncta were found in the dendrites and axons of hippocampal GABAergic interneurons in situ. In contrast in hypothalamic GABAergic interneurons, NPY was restricted to the axon. Surprisingly this differential trafficking was preserved when the neurons were maintained in vitro. When hippocampal and hypothalamic neurons were transfected with NPY-Venus, the distribution of the fluorescent puncta replicated the cell type-specific distribution of endogenous neuropeptide Y. The NPY puncta in the axons of hippocampal and hypothalamic neurons colocalized with the sites of classical transmitter release (identified by staining for synapsin and the vesicular GABAergic transporter, VGAT). In hippocampal neurons, most of the postsynaptic NPY puncta were clustered opposite synapsin-containing varicosities. When neurons were stained for a second neuropeptide, agouti-related protein, immunoreactivity was found in the axon and dendrites of hippocampal neurons but only in the axons of hypothalamic neurons, thus mimicking the polarized distribution of NPY. These results indicate that the trafficking of neuropeptide-containing dense core granules is markedly cell type specific and is not determined entirely by the characteristics of the particular peptide per se.

The Calcium Channel Single Channel Conductance Heirarchy is N>L>T at Physiological External Calcium: Implications for Presynaptic Transmitter Release Site Gating

A number of studies support the conclusion that single CaV channels Ca2+ nanodomains gate molecular signaling pathways. Thus, at presynaptic terminals single CaV2.2 channels trigger fusion of synaptic vesicle (SVs) by saturating a nearby calcium sensor.2 It is generally accepted that CaV1, CaV2, and CaV3 families (L, N and T, respectively) exhibit a decreasing order of single channel conductance.1 Since nanodomain dimensions are proportional to single channel current amplitude (i), high-conductance L type channels would be expected to be favored over the intermediate conductance N-type. Since the L>N>T hierarchy was determined with high Ba2+EXT, we tested the idea that this sequence may differ at physiological Ca2+EXT.We recorded i values for all three CaV families across a broad range of Ca2+EXT, spanning the physiological range. We focused on i-65mV to avoid non-linear current-to-voltage relationship complications and for direct relevance to the gating of synaptic transmission.3 A CaV2.2>CaV1>CaV3.2 hierarchy was determined for i-65mV at 1-2 mM Ca2+EXT. Mathematical modeling predicts that the CaV2.2 Ca2+ nanodomain is ∼25% more extensive than that generated by CaV1. We also calculated single channel ‘SV fusion’ domains, defined as the radii where the channel would saturate ≥50% of 5-binding site calcium sensors. With a sensor binding affinity of 10μM a single CaV2.2 can activate a calcium-fusion sensor located on the proximal face of the synaptic vesicle.4 These findings may explain why CaV2 family channels are preferred for transmitter release site gating.1. Fox AP, Nowycky MC & Tsien RW JP 394:173(1987).2. Stanley EF Neuron 11:1007(1993).3. Llinas RR, Sugimori M & Simon SM PNAS 79:2415(1982).4. Weber AM,∗ Wong FK∗, Tufford AR, Schlichter LC, Matveev V & Stanley EF Nature Neurosci in press (2010).

The mossy fiber bouton: the "common" or the "unique" synapse?

Synapses are the key elements for signal processing and plasticity in the brain. They are composed of nearly the same structural subelements, an apposition zone including a pre- and postsynaptic density, a cleft and a pool of vesicles. It is, however, their actual composition that determines their different behavior in synaptic transmission and plasticity. Here, we describe and discuss the structural factors underlying the unique functional properties of the hippocampal mossy fiber (MF) synapse. Two membrane specializations, active zones (AZs; transmitter release sites), and puncta adherentia (PA), putative adhesion complexes were found. On average, individual boutons had ∼20 AZs with a mean surface area of 0.1 μm2 and a short distance of 0.45 μm between individual AZs. Mossy fiber boutons (MFBs) and their target structures were isolated from each other by astrocytes, but fine glial processes never reached the AZs. Therefore, two structural factors are likely to promote synaptic cross-talk: the short distance and the absence of fine glial processes between individual AZs. Thus, synaptic crosstalk may contribute to the high efficacy of hippocampal MF synapses. On average, an adult bouton contained ∼16,000 synaptic vesicles; ∼600 vesicles were located within 60 nm from the AZ, ∼4000 between 60 nm and 200 nm, and the remaining beyond 200 nm, suggesting large readily releasable, recycling, and reserve pools. Thus, the size of the three pools together with the number and distribution of AZs underlie the unique extent of synaptic efficacy and plasticity of the hippocampal MF synapse.

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.

Neuroligin 2 Drives Postsynaptic Assembly at Perisomatic Inhibitory Synapses through Gephyrin and Collybistin

In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.

Single Channel Conductance Of CaV2.2 At Physiological [Ca2+]Ext

Ca2+ that enters through voltage-gated CaV2.2 channels and binds to a calcium sensor at the transmitter release site links membrane depolarization to activation of synaptic vesicle discharge. Recent evidence supports the hypothesis that the release site calcium sensor is within the single CaV2.2 channel domain. Thus, modeling presynaptic nanophysiology requires knowledge of the channel transport rate at physiological [Ca2+]ext. However, this value has only been determined previously for the non-presynaptic CaV1.x (L type) channel with a conductance of ∼2.4 pS at [Ca2+]ext=2 mM (Church and Stanley, JP 1996). Since at [Ba2+]ext=100 mM CaV1.x has a conductance of ∼24 pS while CaV2.2 has one of ∼14 pS we predicted that at [Ca2+]ext=2 mM the latter channel would have a conductance of ∼1.2 pS.Single calcium channels were recorded using low noise, quartz electrodes from freshly isolated chick dorsal root ganglion neurons which express virtually entirely CaV2.2 current. In the presence of [Ca2+]ext=2 mM and 2 μM nifedipine, to block CaV1.x, and 0.1 mM Ni+ to block CaV3.X, together with standard Na+ and K+ channel blockers and n-methyl-D glucamine+ as the primary cation, we noted two single inward channel conductances: ∼1.4 pS and ∼2.5 pS (N=4). The larger channel was identified as CaV2.2 since it was absent in 4 of 4 patches with ω-conotoxin GIVA (2.5 μM), a specific CaV2.2 blocker, but was present in 7 out of 8 patches with 2 mM [Ca2+ext] or [Ba2+ext] whereas the small channel remained (N=3). Thus, our data indicate that at physiological [Ca2+]ext, CaV2.2 has a much higher conductance, and hence larger single channel domain, than predicted.

Rab3a interacting molecule (RIM) and the tethering of presynaptic transmitter release site-associated Cav2.2 calcium channels

Event Abstract Back to Event Rab3a interacting molecule (RIM) and the tethering of presynaptic transmitter release site-associated Cav2.2 calcium channels Fiona K. Wong1* and Elise F. Stanley1 1 Toronto Western Research Institute, Laboratory of Synaptic Transmission, Genes and Development, Canada Current studies suggest that presynaptic Cav2.2 calcium channels are attached to the transmitter release site within the active zone by a molecular tether. ‘Rab3a Interacting Molecule’ (RIM) is known to play a role in synaptic vesicle docking but has recently been proposed (Kiyonaka et al. Nature Neurosci. 2007) to also fulfill the channel tethering role. However, this suggestion is at variance with results published in our earlier study (Khanna et al., Neurosci. 2006). The main difference in these studies was that we failed to demonstrate co-immunoprecipitation (co-IP) of RIM and CaV2.2 whereas this was observed in the later study. Objective: Three hypotheses were explored to account for the different conclusions between Kiyonaka et al and Khanna et al reports, respectively: 1) the use of different anti-RIM antibodies, mRIM and pRIM, 2) the stringency of the lysate solubilization buffer: digitonin or RIPA and, 3) the type of brain fraction used to generate the lysate: brain microsomes or purified synaptosome membrane. Materials and Methods: Immunoprecipitation (IP) and Western blotting of avian brain lysates. Results: We could not distinguish between the two studies based on antibody selectivity or solubilization buffers. While IP of Cav2.2 (Ab571) from whole brain lysate did co-IP RIM, the two proteins failed to co-IP from purified brain synaptosome membrane lysate using anti-CaV2.2 or either anti-RIM antibody. We also ruled out occlusion of the Ab571 binding site (CaV2.2 II-III loop) as two antibodies against a very different region of the channel (distal C terminal) also failed to co-IP RIM. Conclusions: While our findings are consistent with the existence of a Cav2.2/RIM protein complex in brain we conclude that this complex is not significant at presynaptic terminals. Thus, it is highly unlikely that RIM serves as a key scaffold protein for active zone Cav2.2 calcium channels. Conference: B.R.A.I.N. platform in Physiology poster day 2009, Toronto, ON, Canada, 16 Dec - 16 Dec, 2009. Presentation Type: Poster Presentation Topic: Poster presentations Citation: Wong FK and Stanley EF (2009). Rab3a interacting molecule (RIM) and the tethering of presynaptic transmitter release site-associated Cav2.2 calcium channels. Front. Neurosci. Conference Abstract: B.R.A.I.N. platform in Physiology poster day 2009. doi: 10.3389/conf.neuro.03.2009.17.050 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Dec 2009; Published Online: 18 Dec 2009. * Correspondence: Fiona K Wong, Toronto Western Research Institute, Laboratory of Synaptic Transmission, Genes and Development, Toronto, Canada, fwong@uhnres.utoronto.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Fiona K Wong Elise F Stanley Google Fiona K Wong Elise F Stanley Google Scholar Fiona K Wong Elise F Stanley PubMed Fiona K Wong Elise F Stanley Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Role of group-II and -III metabotropic glutamate receptors in the modulation of miniature synaptic activity in frog spinal-cord motoneurons

The results of present work demonstrate significant modulating effects mediated by group II and III mGluRs on miniature postsynaptic potentials (mPSP) of the frog spinal motoneurons. The mode of group II and III mGluRs ligands influences, i. e. the changes in the mPSPs average frequency without significant changes in their average amplitude, suggests the presynaptic mechanism of modulation by the change in transmitter release. Selective antagonists of group II and III mGluRs (EGLU and MAP4) increased the average frequency of mPSPs by 52.8 +/- 30.2% (in 4 of 6 motoneurons) and by 54.7 +/- 23.7% (in all 7 motoneurons), respectively. Application of the group III mGluRs agonist LAP4 decreased the mPSPs frequency by 21.8 +/- 5.2% in 3 of 5 motoneurons. The efficiency of the antagonist usage and comparative low efficiency of the agonist suggest that presynaptic mGluRs at motoneuronal synapses under normal condition possess some level of tonic activity. The lack of group II mGluR antagonist effect on some motoneurons appears to be explained by specific localization of the group II mGluRs in preterminal area which is distant from the transmitter release site. The hetero-receptor modulation of pharmacologically isolated inhibitory miniature activity and its glycine- and GABAergic fractions by group III mGluRs was investigated. MAP4 application has been shown to increase the glycine-mediated mlPSPs frequency more than GABA-mediated mlPSPs frequency: in average by 97.6 +/- 20.7% (n = 7) and 54.6 +/- 20.8% (n = 5), respectively. This difference may be due to the segregation of the postsynaptic glycine- and GABA-receptors. The preliminary examination of the convergence of the presynaptic mGluRs and metabotropic GABA(B) receptors influences on GABA-mediated IPSPs was undertaken. It has been shown that presynaptic GABA(B) receptors are tonically active under normal condition. Under condition of GABA(B) receptor blockage by phaclofen, the application of group III mGluR agonist L-AP4 elicited typical effect which was completely taken off by subsequent application of the group III mGluRs antagonist MAP4. This result is in accordance with the assumption that the effects mediated by GABA(B) receptors and mGluRs are independent.

Induction of GABAergic Postsynaptic Differentiation by α-Neurexins

Beta-neurexin and neuroligin cell adhesion molecules contribute to synapse development in the brain. The longer alpha-neurexins function at both glutamate and gamma-aminobutyric acid (GABA) synapses in coupling to presynaptic calcium channels. Binding of alpha-neurexins to neuroligins was recently reported, but the role of the alpha-neurexins in synapse development has not been well studied. Here we report that in COS cell neuron coculture assays, all three alpha-neurexins induce clustering of the GABAergic postsynaptic scaffolding protein gephyrin and neuroligin 2 but not of the glutamatergic postsynaptic scaffolding protein PSD-95 or neuroligin 1/3/4. alpha-Neurexins also induce clustering of the GABA(A) receptor gamma2 subunit. This synapse promoting activity of alpha-neurexins is mediated by the sixth LNS (laminin neurexin sex hormone-binding protein) domain and negatively modulated by upstream sequences. Although inserts at splice site 4 (S4) in beta-neurexins promote greater clustering activity for GABA than glutamate proteins in coculture assay, alpha-neurexin-specific sequences confer complete specificity for GABA proteins. We further report a developmental increase in the ratio of -S4 to +S4 forms of neurexins correlating with an increase in glutamate relative to GABA synaptogenesis and activity regulation of splicing at S4. Thus, +S4 beta-neurexins and, even more selectively, alpha-neurexins may be mediators of GABAergic synaptic protein recruitment and stabilization.

Structural Determinants of Transmission at Large Hippocampal Mossy Fiber Synapses

Synapses are the key elements for signal processing and plasticity in the brain. To determine the structural factors underlying the unique functional properties of the hippocampal mossy fiber synapse, the complete quantitative geometry was investigated, using electron microscopy of serial ultrathin sections followed by computer-assisted three-dimensional reconstruction. In particular, parameters relevant for transmitter release and synaptic plasticity were examined. Two membrane specializations were found: active zones (AZs), transmitter release sites, and puncta adherentia, putative adhesion complexes. Individual boutons had, on average, 25 AZs (range, 7-45) that varied in shape and size (mean, 0.1 microm2; range, 0.07-0.17 microm2). The mean distance between individual AZs was 0.45 microm. Mossy fiber boutons and their target structures were mostly ensheathed by astrocytes, but fine glial processes never reached the active zones. Two structural factors are likely to promote synaptic cross talk: the short distance between AZs and the absence of fine glial processes at AZs. Thus, synaptic cross talk may contribute to the efficacy of hippocampal mossy fiber synapses. On average, a bouton contained 20,400 synaptic vesicles; approximately 900 vesicles were located within 60 nm from the active zone, approximately 4400 between 60 and 200 nm, and the remaining beyond 200 nm, suggesting large readily releasable, recycling, and reserve pools. The organization of the different pools may be a key structural correlate of presynaptic plasticity at this synapse. Thus, the mossy fiber bouton differs fundamentally in structure and function from the calyx of Held and other central synapses.

P2 receptor-mediated modulation of neurotransmitter release—an update

Presynaptic nerve terminals are equipped with a number of presynaptic auto- and heteroreceptors, including ionotropic P2X and metabotropic P2Y receptors. P2 receptors serve as modulation sites of transmitter release by ATP and other nucleotides released by neuronal activity and pathological signals. A wide variety of P2X and P2Y receptors expressed at pre- and postsynaptic sites as well as in glial cells are involved directly or indirectly in the modulation of neurotransmitter release. Nucleotides are released from synaptic and nonsynaptic sites throughout the nervous system and might reach concentrations high enough to activate these receptors. By providing a fine-tuning mechanism these receptors also offer attractive sites for pharmacotherapy in nervous system diseases. Here we review the rapidly emerging data on the modulation of transmitter release by facilitatory and inhibitory P2 receptors and the receptor subtypes involved in these interactions.

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>alpha-catenin, CASK>SV2>Na+-K+ approximately 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>or=actin>tubulin, NSF, Munc18, syntaxin 1>spectrin>CASK, SNAP25>RIM, Na+-K+ pump, v-ATPase, beta-catenin approximately 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.

The transmitter release‐site CaV2.2 channel cluster is linked to an endocytosis coat protein complex

Synaptic vesicles (SVs) are triggered to fuse with the surface membrane at the presynaptic transmitter release site (TRSs) core by Ca2+ influx through nearby attached CaV2.2 channels [see accompanying paper: Khanna et al. (2007)Eur. J. Neurosci., 26, 547-559] and are then recovered by endocytosis. In this study we test the hypothesis that the TRS core is linked to an endocytosis-related protein complex. This was tested by immunostaining analysis of the chick ciliary ganglion calyx presynaptic terminal and biochemical analysis of synaptosome lysate, using CaV2.2 as a marker for the TRS. We noted that CaV2.2 clusters abut heavy-chain (H)-clathrin patches at the transmitter release face. Quantitative coimmunostaining analysis (ICA/ICQ method) demonstrated a strong covariance of release-face CaV2.2 staining with that for the AP180 and intersectin endocytosis adaptor proteins, and a moderate covariance with H- or light-chain (L)-clathrin and dynamin coat proteins, consistent with a multimolecular complex. This was supported by coprecipitation of these proteins with CaV2.2 from brain synaptosome lysate. Interestingly, the channel neither colocalized nor coprecipitated with the endocytosis cargo-capturing adaptor AP2, even though this protein both colocalized and coprecipitated with H-clathrin. Fractional recovery analysis of the immunoprecipitated CaV2.2 complex by exposure to high NaCl (approximately 1 m) indicated that AP180 and S-intersectin adaptors are tightly bound to CaV2.2 while L-intersectin, H- and L-clathrin and dynamin form a less tightly linked subcomplex. Our results are consistent with two distinct clathrin endocytosis complexes: an AP2-containing, remote, non-TRS complex and a specialised, AP2-lacking, TRS-associated subcomplex linked via a molecular bridge. The most probable role of this subcomplex is to facilitate SV recovery after transmitter release.

Munc18: A Presynaptic Transmitter Release Site N type (CaV2.2) Calcium Channel Interacting Protein

Munc18 is a presynaptic protein that is essential for transmitter release. Recent studies have indicated that this protein is involved in secretory vesicle docking but its binding partners in this role remain a mystery. We demonstrate using the isolated calyx-type presynaptic terminal of the chick ciliary ganglion that staining for Munc18 co-localizes and covaries with that for transmitter release site N type calcium channels (CaV2.2), consistent with elements of a common release site complex. Biochemical analysis demonstrated that the protein co-precipitates with CaV2.2 from lysates of rat or chick brain, including the synaptic, long-splice variant; presynaptic terminal surface membrane proteins, and a cell line co-expressing Munc18 and CaV2.2. Munc18 bound with high affinity to the CaV2.2 II-III intracellular loop, low affinity to the I-II loop but not to other channel intracellular regions. Over-expression of Munc18 in dorsal root ganglion neurons did not affect CaV2.2 current amplitude or fast kinetics but siRNA-knockdown resulted in a negative shift in the steady state inactivation curve, an effect attributed to an indirect action via syntaxin 1. Recombinant Munc18 also co-precipitated strongly with the v-SNARE synaptotagmin but only weakly with other SNAREs. Thus, the calcium channel may serve as a surface membrane platform anchoring a Munc18-containing bridge to synaptotagmin and the synaptic vesicle.

Differential expression of otoferlin in brain, vestibular system, immature and mature cochlea of the rat

Mutations of the human otoferlin gene lead to an autosomal recessive nonsyndromic form of prelingual, sensorineural deafness (deafness autosomal recessive 9, DFNB9). Several studies have demonstrated expression of otoferlin in the inner ear and brain, and suggested a role of otoferlin in Ca(2+)-triggered exocytosis. So far, otoferlin expression profiles were solely based on the detection of mRNA. Here, we analysed the expression of otoferlin protein and mRNA using immunohistochemistry, in situ hybridization and RT-PCR in neonatal and mature Wistar rat tissue. In agreement with previous studies, otoferlin expression was found in the brain and in inner and vestibular hair cells. Otoferlin mRNA and protein was, however, also detected in mature outer hair cells of low-frequency processing cochlear turns and in auditory nerve fibres. In outer, inner and vestibular hair cells, otoferlin was subcellularly localized at a considerable distance from the presumed active release sites. Double-staining with the synaptic ribbon marker, C-terminal binding protein 2 (CtBP2), or the presynaptic Ca(2+)-channel, Ca(v)1.3, both assumed to mark the sites of vesicle fusion and transmitter release, did not colocalize with otoferlin expression and thus do not necessarily support a selected role of otoferlin in Ca(2+)-triggered exocytosis. The widespread distribution of otoferlin in neurons, nerve fibres and hair cells, and its subcellular distribution extending beyond the regions of synaptic vesicle fusion, i.e. coenrichment with the cytosolic Golgi matrix protein 130 (GM130) in inner hair cells or the early endosomal autoantigen 1 (EEA1) in outer hair cells support instead the idea of a more ubiquitous role of otoferlin in early/recycling endosome trans-Golgi network dynamics.

Regulation of Gephyrin Assembly and Glycine Receptor Synaptic Stability

Gephyrin is required for the formation of clusters of the glycine receptor (GlyR) in the neuronal postsynaptic membrane. It can make trimers and dimers through its N- and C-terminal G and E domains, respectively. Gephyrin oligomerization could thus create a submembrane lattice providing GlyR-binding sites. We investigated the relationships between the stability of cell surface GlyR and the ability of gephyrin splice variants to form oligomers. Using truncated and full-length gephyrins we found that the 13-amino acid sequence (cassette 5) prevents G domain trimerization. Moreover, E domain dimerization is inhibited by the gephyrin central L domain. All of the gephyrin variants bind GlyR beta subunit cytoplasmic loop with high affinity regardless of their cassette composition. Coexpression experiments in COS-7 cells demonstrated that GlyR bound to gephyrin harboring cassette 5 cannot be stabilized at the cell surface. This gephyrin variant was found to deplete synapses from both GlyR and gephyrin in transfected neurons. These data suggest that the relative expression level of cellular variants influence the overall oligomerization pattern of gephyrin and thus the turnover of synaptic GlyR.

Controlling the Synapse

Synapses in the neuromuscular junction are key components involved in control of muscle movement. Kittel et al . (see the Perspective by Atwood) describe the role of Drosophila Bruchpilot (BRP), a coiled-coil domain protein, in the establishment and maintenance of synapses. BRP was localized to donut-shaped structures centered at the transmitter release sites (active zones) of Drosophila neuromuscular synapses. In mutants lacking BRP, presynaptic membranes were defective. The authors suggest that BRP is needed to form a fully functional synapse and might mediate presynaptic changes in vivo by establishing a proximity between Ca 2+ channels and vesicles at release sites. R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, S. J. Sigrist, Bruchpilot promotes active zone assembly, Ca 2+ channel clustering, and vesicle release. Science 312 , 1051-1054 (2006). [Abstract] [Full Text] H. L. Atwood, Gatekeeper at the synapse. Science 312 , 1008-1009 (2006). [Summary] [Full Text]

Transmitter release face Ca2+ channel clusters persist at isolated presynaptic terminals

Ca(2+) influx through N-type Ca(2+) channels (CaV2.2) is known to be critical for transmitter release at many synapses. These channels are known to be localized to transmitter release sites, but their anchoring mechanism remains unknown. Recent studies have demonstrated that presynaptic organization is subject to interactions with the postsynaptic cell or the intervening extracellular matrix. We used a previously described high-affinity antibody against the N-type Ca(2+) channels, Ab571, to localize Ca(2+) channel clusters at the release face of an isolated giant calyx-type synapse to test whether the maintenance of these clusters requires an intact extracellular matrix or contact with the postsynaptic cell. Because the number of Ca(2+) channel clusters was unchanged after extracellular matrix dispersal or nerve terminal isolation, we conclude that presynaptic transmitter release face Ca(2+) clusters can be maintained independently of extracellular influences. Our results suggest that a presynaptic molecular scaffold is responsible for the maintenance of release site Ca(2+) channel clusters.