Presynaptic N-type calcium channels interact with syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) through a binding site in the intracellular loop connecting domains II and III of the ␣ 1 subunit. This binding region was loaded into embryonic spinal neurons of Xenopus by early blastomere injection. After culturing, synaptic transmission of peptide-loaded and control cells was compared by measuring postsynaptic responses under different external Ca 2ϩ concentrations. The relative transmitter release of injected neurons was reduced by ϳ25% at physiological Ca 2ϩ concentration, whereas injection of the corresponding region of the L-type Ca 2ϩ channel had virtually no effect. When applied to a theoretical model, these results imply that 70% of the formerly linked vesicles have been uncoupled after action of the peptide. Our data suggest that severing the physical interaction between presynaptic calcium channels and synaptic proteins will not prevent synaptic transmission at this synapse but will make it less efficient by shifting its Ca 2ϩ dependence to higher values.

Rapid synaptic transmission requires close proximity of docked neurotransmitter-containing synaptic vesicles and voltage-gated Ca 2+ channels at presynaptic active zones. Here we show that the plasma membrane SNARE protein SNAP-25 specifically inhibited the activity of P/Q-type Ca 2+ channels, and formation of a mature SNARE complex containing syntaxin and synaptotagmin reactivated them. In a nerve terminal, this mechanism would ensure that Ca 2+ entry through P/Q-type Ca 2+ channels occurs primarily near active zones with docked synaptic vesicles and efficiently evokes neurotransmitter release.At many synapses in the central nervous system, Ca 2+ influx through P/Q-type Ca 2+ channels densely localized in presynaptic nerve terminals triggers rapid neurotransmitter release 1,2 . The synaptic vesicle proteins synaptotagmin and synaptobrevin/VAMP bind to the synaptic plasma membrane SNARE proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) to form a core complex implicated in synaptic vesicle docking and/or membrane fusion 3,4 . This core complex associates with N-type and P/Qtype Ca 2+ channels via the synaptic protein interaction ('synprint') site within the intracellular loop connecting homologous domains II and III of their α 1B and α 1A subunits 2 . Disrupting interactions between SNARE proteins and Ca 2+ channels inhibits neurotransmission, demonstrating that such interaction is required for efficient neurotransmitter release 2 , and co-expression of syntaxin with Fig. 1. Modulation of P/Q-type Ca 2+ -channel activity by SNARE proteins.Recombinant rat cDNAs for syntaxin 1A, SNAP-25, synaptotagmin I-CT (the cytoplasmic domain containing residues 80-421) and VAMP 2-NT (the cytoplasmic domain containing residues 1-92) were subcloned into the mammalian expression vector pEBV-his (Invitrogen, San Diego, California). cDNA encoding the Ca 2+ channel subunits α 1A (rbA isoform) and β 1b were cloned in pMT2, α 2 δ in pZEM228 and CD8 in EBO-pcD. Cells of the tsA-201 subclone of HEK 293 cells were transfected with the α 1A , β 1b and α2δ cDNAs in a 1:1:1 molar ratio, plus the indicated SNARE proteins and CD8, by the calcium phosphate method and incubated for 24 h. Transfection-positive cells were identified by labeling with a fluorophore-tagged anti-CD8 antibody and analyzed by whole-cell patch clamp as described 14 . (a) Expression of SNARE proteins. Cells transfected with P/Q-type Ca 2+ channel and a SNARE protein were harvested and lysed in hypotonic buffer. A P2 membrane fraction was solubilized in 1% CHAPS for syntaxin and SNAP-25 immunoblotting and for immunoprecipitation with anti-CNA2, an antibody against the carboxyl terminal sequence ([KY]RRAPGPREPLANDSPGR) of the rbA isoform of α 1A . The remaining S2 fraction was used for synaptotagmin and VAMP/synaptobrevin immunoblotting. Lane 1, SNAP-25; lane 2, syntaxin; lane 3, synaptotagmin; lane 4, VAMP/synaptobrevin. (b) Co-immunoprecipitation of Ca 2+ channels and SNARE proteins. Cells transfected with P/Q-type Ca 2+ channel and SNARE proteins w...

N-type Ca 2؉ channels mediate Ca 2؉ inf lux, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their ␣ 1B subunits. Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission. Here we report a direct interaction of the synprint peptide from N-type Ca 2؉ channels with synaptotagmin I, an important Ca 2؉ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay. This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca 2؉ . Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin. This interaction is Ca 2؉ -dependent because of the Ca 2؉ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca 2؉ channels and synaptotagmin, and suggest that N-type Ca 2؉ channels may undergo a complex series of Ca 2؉ -dependent interactions with multiple presynaptic proteins during neurotransmission.During synaptic transmission, exocytosis of synaptic vesicles in neurons is initiated by the rapid influx of Ca 2ϩ through voltage-gated Ca 2ϩ channels within 200 sec. Major progress has been made toward the understanding of the molecular mechanisms that underlie Ca 2ϩ -dependent exocytosis by identifying proteins that are involved in this process and analyzing their interactions (1, 2). Vesicle docking and fusion are mediated by a core complex of proteins including the synaptic vesicle protein VAMP͞synaptobrevin (3) and the plasmalemmal proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) (4-11). The synaptic vesicle protein p65 or synaptotagmin (12) binds Ca 2ϩ (13,14) and interacts with syntaxin in a Ca 2ϩ -dependent manner (15,16). It is thought to serve as a Ca 2ϩ sensor for fast, Ca 2ϩ -dependent neurotransmitter release (17)(18)(19)(20)(21)(22).N-type Ca 2ϩ channels are localized in nerve terminals (23-25) and participate in neurotransmitter release in central and peripheral synapses (26-28). They are coimmunoprecipitated and copurified with proteins of the synaptic core complex in immunochemical studies (4,5,29), and they are closely associated with sites of transmitter release in physiological experiments (30). The ␣ 1B subunits of N-type Ca 2ϩ channels (31, 32) interact with the synaptic core complex through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III (33, 34) in a Ca 2ϩ -dependent manner with maximum binding in the range of 10-30 M (35). Peptides containing th...

Cav2.1 channels, which conduct P͞Q-type Ca 2؉ currents, were expressed in superior cervical ganglion neurons in cell culture, and neurotransmission initiated by these exogenously expressed Ca 2؉ channels was measured. Deletions in the synaptic protein interaction (synprint) site in the intracellular loop between domains II and III of Cav2.1 channels reduced their effectiveness in synaptic transmission. Surprisingly, this effect was correlated with loss of presynaptic localization of the exogenously expressed channels. Cav1.2 channels, which conduct L-type Ca 2؉ currents, are ineffective in supporting synaptic transmission, but substitution of the synprint site from Cav2.1 channels in Cav1.2 was sufficient to establish synaptic transmission initiated by L-type Ca 2؉ currents through the exogenous Cav1.2 channels. Substitution of the synprint site from Cav2.2 channels, which conduct N-type Ca 2؉ currents, was even more effective than Cav2.1. Our results show that localization and function of exogenous Ca 2؉ channels in nerve terminals of superior cervical ganglion neurons require a functional synprint site and suggest that binding of soluble NSF attachment protein receptor (SNARE) proteins to the synprint site is a necessary permissive event for nerve terminal localization of presynaptic Ca 2؉ channels. E lectrophysiological and pharmacological studies have defined a diverse array of native Ca 2ϩ currents having different functions in neurons (1, 2). Voltage-gated Ca 2ϩ channels are complexes of a pore-forming ␣ 1 subunit with associated ␣ 2 ␦, ␤, and ␥ subunits (3-5). Ca v 2.1 channels that conduct P͞Q-type Ca 2ϩ currents and Ca v 2.2 channels that conduct N-type Ca 2ϩ currents are the primary initiators of fast synaptic transmission in vertebrate neurons (1, 6-12). These Ca 2ϩ channels bind directly to soluble NSF attachment protein (SNAP) receptor (SNARE) proteins involved in neurotransmitter release through a synaptic protein interaction (synprint) site in the large intracellular loop connecting domains II and III (L II-III ) of their ␣ 1 subunits (13-15). Disruption of this interaction by peptide inhibitors injected into presynaptic neurons reduces the efficiency of Ca 2ϩ entry in stimulating exocytosis (16,17). These results implicate the interaction of SNARE proteins with the synprint site in determining the efficiency of fast synaptic transmission, possibly by organizing docked synaptic vesicles close to the site of Ca 2ϩ entry.The molecular basis for the specific role of Ca v 2 channels in initiation of fast neurotransmission is not well understood. It may involve specific localization of Ca v 2 channels in nerve terminals, specific interactions with SNARE proteins or other proteins in the nerve terminal, or both. Results presented in the accompanying paper (18) show that exogenous Ca v 2.1 channels can be functionally expressed in superior cervical ganglion neurons (SCGNs) and can reconstitute synaptic transmission in neurons whose endogenous Ca v 2.2 channels have been blocked by -conotoxin GVIA. Here w...

Anti-peptide antibodies specific for the neuronal calcium channel alpha 1E subunit (anti-CNE1 and anti-CNE2) were produced to study the biochemical properties and subcellular distribution of the alpha 1E polypeptide from rat brain. Immunoblotting identified a single size form of 245-255 kDa which was a substrate for phosphorylation by cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II. Ligand-binding studies of alpha 1E indicate that it is not a high affinity receptor for the dihydropyridine isradipine or the peptide toxins omega-conotoxin GVIA or omega-conotoxin MVIIC at concentrations which elicit high affinity binding to other channel types in the same membrane preparation. The alpha 1E subunit is widely distributed in the brain with the most prominent immunocytochemical staining in deep midline structures such as caudate-putamen, thalamus, hypothalamus, amygdala, cerebellum, and a variety of nuclei in the ventral midbrain and brainstem. Staining is primarily in the cell soma but is also prominent in the dendritic field of a discrete subset of neurons including the mitral cells of the olfactory bulb and the distal dendritic branches of the cerebellar Purkinje cells. Our observations indicate that the 245-255 kDa alpha 1E subunit is localized in cell bodies, and in some cases in dendrites, of a broad range of central neurons and is potentially modulated by multiple second messenger-activated protein kinase.

Phosphorylation by cAMP‐dependent protein kinase (PKA) and other second messenger‐activated protein kinases modulates the activity of a variety of effector proteins including ion channels. Anti‐peptide antibodies specific for the alpha 1 subunits of the class B, C or E calcium channels from rat brain specifically recognize a pair of polypeptides of 220 and 240 kDa, 200 and 220 kDa, and 240 and 250 kDa, respectively, in hippocampal slices in vitro. These calcium channels are localized predominantly on presynaptic and dendritic, somatic and dendritic, and somatic sites, respectively, in hippocampal neurons. Both size forms of alpha 1B and alpha 1E and the full‐length form of alpha 1C are phosphorylated by PKA after solubilization and immunoprecipitation. Stimulation of PKA in intact hippocampal slices also induced phosphorylation of 25‐50% of the PKA sites on class B N‐type calcium channels, class C L‐type calcium channels and class E calcium channels, as assessed by a back‐phosphorylation method. Tetraethylammonium ion (TEA), which causes neuronal depolarization and promotes repetitive action potentials and neurotransmitter release by blocking potassium channels, also stimulated phosphorylation of class B, C and E alpha 1 subunits, suggesting that these three classes of channels are phosphorylated by PKA in response to endogenous electrical activity in the hippocampus. Regulation of calcium influx through these calcium channels by PKA may influence calcium‐dependent processes within hippocampal neurons, including neurotransmitter release, calcium‐activated enzymes and gene expression.

The synaptic protein interaction (synprint) site on the N-type calcium channel ␣ 1B subunit binds to the soluble N-ethylmaleimide-sensitive attachment factor receptor (SNARE) proteins syntaxin and synaptosomal protein of 25 kDa , and this association may be required for efficient fast synaptic transmission. Protein kinase C (PKC) and calcium and calmodulin-dependent protein kinase type II (CaM KII) phosphorylated a recombinant his-tagged synprint site polypeptide rapidly to a stoichiometry of 3-4 mol of phosphate/ mol, whereas cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) phosphorylated the synprint peptide more slowly to a stoichiometry of Ͻ1 mol/mol. Two-dimensional phosphopeptide mapping revealed similar patterns of phosphorylation of synprint polypeptides and native rat brain N-type calcium channel ␣ 1B subunits by PKC and Cam KII. Phosphorylation of the synprint peptide with PKC or CaM KII, but not PKA or PKG, strongly inhibited binding of recombinant syntaxin or SNAP-25, even at a level of free calcium (15 M) that stimulates maximal binding. In contrast, phosphorylation of syntaxin and SNAP-25 with PKC and CaM KII did not affect interactions with the synprint site. Binding assays with polypeptides representing the N-and C-terminal halves of the synprint site indicate that the PKC-and CaM KII-mediated inhibition of binding involves multiple, disperse phosphorylation sites. PKC or CaM KII phosphorylation of the synprint peptide also inhibited its interactions with native rat brain SNARE complexes containing syntaxin and SNAP-25. These results suggest that phosphorylation of the synprint site by PKC or CaM KII may serve as a biochemical switch for interactions between N-type calcium channels and SNARE protein complexes.

Fast cholinergic neurotransmission between superior cervical ganglion neurons (SCGNs) in cell culture is initiated by N-type Ca 2؉ currents through Cav2.2 channels. To test the ability of different Ca 2؉ -channel subtypes to initiate synaptic transmission in these cells, SCGNs were injected with cDNAs encoding Ca v1.2 channels, which conduct L-type currents, Ca v2.1 channels, which conduct P͞Q-type Ca 2؉ currents, and Ca v 2.3 channels, which conduct R-type Ca 2؉ currents. Exogenously expressed Cav2.1 channels were localized in nerve terminals, as assessed by immunocytochemistry with subtype-specific antibodies, and these channels effectively initiated synaptic transmission. Injection with cDNA encoding Ca v2.3 channels yielded a lower level of presynaptic labeling and synaptic transmission, whereas injection with cDNA encoding Ca v1.2 channels resulted in no presynaptic labeling and no synaptic transmission. Our results show that exogenously expressed Ca 2؉ channels can mediate synaptic transmission in SCGNs and that the specificity of reconstitution of neurotransmission (Ca v2.1 > Cav2.3 Ͼ Ͼ Cav1.2) follows the same order as in neurons in vivo. The specificity of reconstitution of neurotransmission parallels the specificity of trafficking of these Ca v channels to nerve terminals.