Functional coupling of Rab3-interacting molecule 1 (RIM1) and L-type Ca2+ channels in insulin release

Abstract

Insulin release by pancreatic β-cells is regulated by diverse intracellular signals, including changes in Ca2+ concentration resulting from Ca2+ entry through voltage-gated (CaV) channels. It has been reported that the Rab3 effector RIM1 acts as a functional link between neuronal CaV channels and the machinery for exocytosis. Here, we investigated whether RIM1 regulates recombinant and native L-type CaV 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 CaV channel expression was identified by RT-PCR and Western blot. RIM1-Ca V channel interaction was determined by co-immunoprecipitation. Knockdown of RIM1 and CaV channel subunit expression were performed using small interference RNAs. Insulin release was assessed by ELISA. Co-expression of CaV1.2 and CaV1.3 L-type channels with RIM1 in HEK-293 cells revealed that RIM1 may not determine the availability of L-type CaV channels but decreases the rate of inactivation of the whole cell currents. Coimmunoprecipitation experiments showed association of the CaVβ auxiliary subunit with RIM1. The lack of Ca Vβ 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 CaVβ 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 CaV channels via the CaVβ auxiliary subunit that contribute to determine insulin secretion. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.