Charles T. Yokoyama

Biochemical properties and subcellular distribution of the neuronal class E calcium channel alpha 1 subunit

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.

Reciprocal regulation of P/Q-type Ca2+ channels by SNAP-25, syntaxin and synaptotagmin.

Rapid synaptic transmission requires close proximity of docked neurotransmitter-containing synaptic vesicles and voltage-gated Ca2+ channels at presynaptic active zones. Here we show that the plasma membrane SNARE protein SNAP-25 specifically inhibited the activity of P/Q-type Ca2+ channels, and formation of a mature SNARE complex containing syntaxin and synaptotagmin reactivated them. In a nerve terminal, this mechanism would ensure that Ca2+ entry through P/Q-type Ca2+ channels occurs primarily near active zones with docked synaptic vesicles and efficiently evokes neurotransmitter release.

Phosphorylation of presynaptic and postsynaptic calcium channels by cAMP-dependent protein kinase in hippocampal neurons.

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.

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

Cav2.1 channels, which conduct P/Q-type Ca2+ currents, were expressed in superior cervical ganglion neurons in cell culture, and neurotransmission initiated by these exogenously expressed Ca2+ 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 Ca2+ 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 Ca2+ currents through the exogenous Cav1.2 channels. Substitution of the synprint site from Cav2.2 channels, which conduct N-type Ca2+ currents, was even more effective than Cav2.1. Our results show that localization and function of exogenous Ca2+ 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 Ca2+ channels.

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 Ca2+ currents through Cav2.2 channels. To test the ability of different Ca2+-channel subtypes to initiate synaptic transmission in these cells, SCGNs were injected with cDNAs encoding Cav1.2 channels, which conduct L-type currents, Cav2.1 channels, which conduct P/Q-type Ca2+ currents, and Cav2.3 channels, which conduct R-type Ca2+ 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 Cav2.3 channels yielded a lower level of presynaptic labeling and synaptic transmission, whereas injection with cDNA encoding Cav1.2 channels resulted in no presynaptic labeling and no synaptic transmission. Our results show that exogenously expressed Ca2+ channels can mediate synaptic transmission in SCGNs and that the specificity of reconstitution of neurotransmission (Cav2.1 > Cav2.3 ≫ Cav1.2) follows the same order as in neurons in vivo. The specificity of reconstitution of neurotransmission parallels the specificity of trafficking of these Cav channels to nerve terminals.

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.