RIM modulates Ca_V1.3 Ca²⁺ channels

Calcium channel β subunits (Ca_Vβs) are essential cytoplasmic components of voltage-gated calcium channels (VGCCs) affecting their gating and targeting. Ca_Vβs 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 Ca_Vβs we performed a yeast two-hybrid screen using Ca_Vβ2a 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 Ca_Vβs we developed a protein targeting assay in tsA-201 cells heterologously expressing the loop-I-II of Ca_V1.3 channels with diverse Ca_Vβ subunits. The Ca_V1.3-loop-I-II was transported to the plasma membrane and co-targeted all Ca_Vβ subunits indicating that the Ca_V1.3-loop-I-II and the Ca_Vβ 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 Ca_Vβs in presence of the Ca_V1.3-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 Ca_V1.3 could account for the slow Ca_V1.3 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 Ca_V1.3 (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 Ca_V1.3 spliceform (1b) could contribute to this effect we examined its expression with RT-PCR analysis. However, we did not detect Ca_V1.3(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 Ca_V1.3 Ca²⁺ channels. However, we assume that a mixture of diverse proteins and/or Ca_V1.3 splice variants probably accounts for the slow current inactivation of these channels in native IHCs.