Functional Coupling of Rab3-interacting Molecule 1 (RIM1) and L-type Ca2+ Channels in Insulin Release

Insulin release by pancreatic beta-cells is regulated by diverse intracellular signals, including changes in Ca2+ concentration resulting from Ca2+ entry through voltage-gated (Ca-V) channels. It has been reported that the Rab3 effector RIM1 acts as a functional link between neuronal Ca-V channels and the machinery for exocytosis. Here, we investigated whether RIM1 regulates recombinant and native L-type Ca-V channels (that play a key role in hormone secretion) and whether this regulation affects insulin release. Whole-cell patch clamp currents were recorded from HEK-293 and insulinoma RIN-m5F cells. RIM1 and Ca-V channel expression was identified by RT-PCR and Western blot. RIM1-Ca-V channel interaction was determined by co-immunoprecipitation. Knockdown of RIM1 and Ca-V channel subunit expression were performed using small interference RNAs. Insulin release was assessed by ELISA. Co-expression of Ca(V)1.2 and Ca(V)1.3 L-type channels with RIM1 in HEK-293 cells revealed that RIM1 may not determine the availability of L-type Ca-V channels but decreases the rate of inactivation of the whole cell currents. Co-immunoprecipitation experiments showed association of the Ca-V beta auxiliary subunit with RIM1. The lack of Ca-V beta expression suppressed channel regulation by RIM1. Similar to the heterologous system, an increase of current inactivation was observed upon knockdown of endogenous RIM1. Co-immunoprecipitation showed association of Ca-V beta and RIM1 in insulin-secreting RIN-m5F cells. Knockdown of RIM1 notably impaired high K+-stimulated insulin secretion in the RIN-m5F cells. These data unveil a novel functional coupling between RIM1 and the L-type Ca-V channels via the Ca-V beta auxiliary subunit that contribute to determine insulin secretion.