RIM modulates CaV1.3 Ca2+ channels

Abstract

Calcium channel β subunits (CaVβs) are essential cytoplasmic components of voltage-gated calcium channels (VGCCs) affecting their gating and targeting. CaVβs bind with high affinity to the cytoplasmic loop between transmembrane segments I and II of the α1 subunit (loop-I-II). To identify new proteins that modulate VGCCs by interaction with CaVβs we performed a yeast two-hybrid screen using CaVβ2a as bait. Screening of a human fetal brain cDNA library identified a C-terminal fragment of RIM1α (Rab3-interacting molecule) which contains a highly conserved C2B domain as potential interaction partner. To proof the interaction between RIM and CaVβs we developed a protein targeting assay in tsA-201 cells heterologously expressing the loop-I-II of CaV1.3 channels with diverse CaVβ subunits. The CaV1.3-loop-I-II was transported to the plasma membrane and co-targeted all CaVβ subunits indicating that the CaV1.3-loop-I-II and the CaVβ subunits formed a functional complex. The C-terminal fragment of RIM1α or the full-length form of RIM2β exhibited a cytoplasmic distribution but when co-expressed with CaVβs in presence of the CaV1.3-loop-I-II both were co-localized at the plasma membrane. Using qualitative RT-PCR analysis we detected various RIM isoforms in the total organ of Corti and RIM2α in cochlea inner hair cells (IHCs) at an early developmental stage, before the onset of hearing. As RIM is a presynaptic active zone protein involved in Ca2+-induced neurotransmitter release, we asked the question whether the association of RIM with CaV1.3 could account for the slow CaV1.3 current inactivation seen in IHCs. In whole-cell patch-clamp analysis of tsA-201 cells using 15 mM Ca2+ as charge carrier the C2B domain containing fragments of RIM1α and RIM2α caused a significant depolarizing shift of the activation-curve of CaV1.3 (7–12 mV) and slowed the inactivation of both Ca2+ and Ba2+ currents (p < 0.05) albeit to a lesser extent as found in native IHCs. To investigate if a slowly inactivating CaV1.3 spliceform (1b) could contribute to this effect we examined its expression with RT-PCR analysis. However, we did not detect CaV1.3(1b) transcripts in the total organ of Corti at the same developmental stage as we found RIM. Taken together these data showed that indeed RIM modulated CaV1.3 Ca2+ channels. However, we assume that a mixture of diverse proteins and/or CaV1.3 splice variants probably accounts for the slow current inactivation of these channels in native IHCs.