Abstract
Voltage-dependent Ca2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40-47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents.
Highlights
Voltage-dependent Ca2؉ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs)
Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated voltage-dependent inactivation (VDI) suppression characteristic of RIM2␣. These results suggest that the interactions of the CaV2.1 C-terminal domain (CTD) with RIMs enable CaV2.1 proteins to distinguish ␣-RIM isoforms in VDI suppression of P/Q-type VDCC currents
We have previously demonstrated that the RIM C-terminal region containing the C2B domain interacts with VDCC  subunits (20, 39, 50)
Summary
We have previously demonstrated that the RIM C-terminal region containing the C2B domain interacts with VDCC  subunits (20, 39, 50). We performed sequence analysis of PCR products from a cDNA library of the human cerebellum, in which abundant expressions of ␣-RIMs and CaV2.1 mRNAs were reported (39, 59, 60). Agarose gel electrophoresis (1%) revealed a broad band of PCR products of ϳ1,000 bp consistent with the predicted sizes ranging from 834 to 989 bp (Fig. 1C) This DNA band was subcloned into a vector, and the relative levels of splice variants were determined by counting the number of clones containing each exon. CaV2.1-containing regions encoded by exons 44 and 47 (ϩ44 ,47) were detected at the highest relative proportion (56%) (Fig. 1A and Table 1). Characterization of CaV2.1 C-terminal splice variation in the human cerebellum n means number of clones sequenced
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