Bidirectional modulation of transmitter release by calcium channel/syntaxin interactions in vivo.

Protein interactions within the active zone of the nerve terminal are critical for regulation of transmitter release. The SNARE protein syntaxin 1A, primarily known for important interactions that control vesicle fusion, also interacts with presynaptic voltage-gated calcium channels. Based on recordings of calcium channel function in vitro, it has been hypothesized that syntaxin 1A-calcium channel interactions could alter calcium channel function at synapses. However, results at synapses in vitro suggest two potentially opposing roles: enhancement of neurotransmitter release by positioning docked vesicles near calcium channels and inhibition of calcium channel function by interaction with SNARE proteins. We have examined the possibility that these two effects of syntaxin can occur at synapses by studying the effects on transmitter release of manipulating syntaxin 1A-calcium channel interactions at Xenopus tadpole tail neuromuscular synapses in vivo. Introduction of synprint peptides, which competitively perturb syntaxin 1A-calcium channel interactions, decreased quantal content at these synapses and increased paired-pulse and tetanic facilitation. In contrast, injecting mRNA for mutant (A240V, V244A) syntaxin 1A, which reduces calcium channel modulation but not binding in vitro, increased quantal content and decreased paired-pulse and tetanic facilitation. Injection of wild-type syntaxin 1A mRNA had no effect. The opposing effects of synprint peptides and mutant syntaxin 1A provide in vivo support for the hypothesis that these interactions serve both to colocalize calcium channels with the release machinery and to modulate the functional state of the calcium channel. As such, these two effects of syntaxin on calcium channels modulate transmitter release in a bidirectional manner.

Highly cooperative control of endocytosis by clathrin.

Clathrin assembles into a dynamic two-dimensional lattice on the plasma membrane where it plays a critical role in endocytosis. To probe the regulation of this process, we used siRNA against clathrin, in combination with single cell assays for transferrin uptake as well as total internal reflection microscopy, to examine how endocytic rates and membrane dynamics depend upon cellular clathrin concentration ([Clathrin]). We find that endocytosis is tightly controlled by [Clathrin] over a very narrow dynamic range such that small changes in [Clathrin] can lead to large changes in endocytic rates, indicative of a highly cooperative process (apparent Hill coefficient, n > 6). The number of clathrin assemblies at the cell surface was invariant over a wide range of [Clathrin]; however, both the amount of clathrin in each assembly and the subsequent membrane dynamics were steeply dependent on [Clathrin]. Thus clathrin controls the structural dynamics of membrane internalization via a strongly cooperative process. We used this analysis to show that one important regulator of endocytosis, the actin cytoskeleton, acts noncompetitively as a modulator of clathrin function.

Mechanism of SNARE protein binding and regulation of Cav2 channels by phosphorylation of the synaptic protein interaction site.

Ca(v)2.1 and Ca(v)2.2 channels conduct P/Q-type and N-type Ca(2+) currents that initiate neurotransmission and bind SNARE proteins through a synaptic protein interaction (synprint) site. PKC and CaMKII phosphorylate the synprint site and inhibit SNARE protein binding in vitro. Here we identify two separate microdomains that each bind syntaxin 1A and SNAP-25 in vitro and are regulated by PKC phosphorylation at serines 774 and 898 and CaMKII phosphorylation at serines 784 and 896. Activation of PKC resulted in its recruitment to and phosphorylation of Ca(V)2.2 channels, but PKC phosphorylation did not dissociate Ca(V)2.2 channel/syntaxin 1A complexes. Chimeric Ca(V)2.1a channels containing the synprint site of Ca(v)2.2 gain modulation by syntaxin 1A, which is blocked by PKC phosphorylation at the sites identified above. Our results support a bipartite model for the synprint site in which each SNARE-binding microdomain is controlled by a separate PKC and CaMKII phosphorylation site that regulates channel modulation by SNARE proteins.

Subtype-selective reconstitution of synaptic transmission in sympathetic ganglion neurons by expression of exogenous calcium channels.

Fast cholinergic neurotransmission between superior cervical ganglion neurons (SCGNs) in cell culture is initiated by N-type Ca(2+) currents through Ca(v)2.2 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(v)1.2 channels, which conduct L-type currents, Ca(v)2.1 channels, which conduct P/Q-type Ca(2+) currents, and Ca(v)2.3 channels, which conduct R-type Ca(2+) currents. Exogenously expressed Ca(v)2.1 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(v)2.3 channels yielded a lower level of presynaptic labeling and synaptic transmission, whereas injection with cDNA encoding Ca(v)1.2 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(v)2.1 > Ca(v)2.3 >> Ca(v)1.2) 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.

Requirement for the synaptic protein interaction site for reconstitution of synaptic transmission by P/Q-type calcium channels.

Ca(v)2.1 channels, which conduct P/Q-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 Ca(v)2.1 channels reduced their effectiveness in synaptic transmission. Surprisingly, this effect was correlated with loss of presynaptic localization of the exogenously expressed channels. Ca(v)1.2 channels, which conduct L-type Ca(2+) currents, are ineffective in supporting synaptic transmission, but substitution of the synprint site from Ca(v)2.1 channels in Ca(v)1.2 was sufficient to establish synaptic transmission initiated by L-type Ca(2+) currents through the exogenous Ca(v)1.2 channels. Substitution of the synprint site from Ca(v)2.2 channels, which conduct N-type Ca(2+) currents, was even more effective than Ca(v)2.1. 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.