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

Abstract

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...

CaV2 channel subtype expression in rat sympathetic neurons is selectively regulated by α2δ subunits

“…This subtype is the dominant channel in adult SCG neurons, and primarily induces neurotransmission. 17 Therefore it is plausible that there are additional requirements for this channel to be trafficked to the plasma membrane, or the channel is segregated to be trafficked to the presynapse exclusively. In dorsal root ganglia (DRG) sensory neurons it was established that splice isoforms have differential membrane expression patterns.…”

“…16 In exploring how neurotransmission is affected by recombinant channel expression, Mochida et al demonstrated that recombinant Ca V 2 subtypes reconstituted synaptic transmission similarly to native Ca V 2 channels in rat superior cervical ganglion. 17 What remained to be identified was how the recombinant channels were limited at the presynapse, as overexpression of the channels did not enhance the synaptic transmission. In 2004, Cao and Tsien hypothesized that "slots," or expression limits due to some unidentified mechanisms, regulated the relative amount of recombinant channels at the presynapse in a subtype dependent manner.…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…1). 27,50 In N-type channels (rat Ca V 2.2), the synprint site is an 265aa residue stretch spanning residues 718-983, 51,52 although smaller subregions within this motif are in fact involved in binding to SNARE proteins such as syntaxin 1A and SNAP-25. 52 In the rabbit brain P/Q-type channel (BI) isoform, the synprint motif encompasses residues 722-1036.…”

“…74 A third regulatory modality linked to the synprint motif is subcellular targeting of the channel. Work from Mochida et al 50,75 shows that the presence of the synaptic protein interaction site is an important determinant of subcellular targeting of P/Q-type channels. Similarly, splice variants of human N-type channels lacking the synprint region are still targeted to axonal compartments, but are excluded from the active zone.…”

T-type channels-secretion coupling: evidence for a fast low-threshold exocytosis

“…The two Ca v 2 channel types bind to syntaxin and synaptotagmin through a "synprint" region [45] and are closely associated to the SNARE complex. They mediate vesicle docking, fusion, and neurotransmitter release in highly specialized presynaptic regions (active zones).…”