Department of Cell Biology, Center for Research and Advanced Studies, National Polytechnic Institute (Cinvestav-IPN), Colonia Zacatenco, México DF, México.
J Biol Chem. 2011 May 6;286(18):15757-65. doi: 10.1074/jbc.M110.187757. Epub 2011 Mar 14.
Insulin release by pancreatic β-cells is regulated by diverse intracellular signals, including changes in Ca(2+) concentration resulting from Ca(2+) 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)β auxiliary subunit with RIM1. The lack of Ca(V)β 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)β 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)β auxiliary subunit that contribute to determine insulin secretion.
胰岛β细胞的胰岛素分泌受多种细胞内信号调节，包括通过电压门控（Ca(V)）通道进入 Ca(2+)引起的 Ca(2+)浓度变化。据报道，Rab3 效应物 RIM1 作为神经元 Ca(V)通道和胞吐机制之间的功能连接物发挥作用。在这里，我们研究了 RIM1 是否调节重组和天然 L 型 Ca(V)通道（在激素分泌中起关键作用），以及这种调节是否影响胰岛素分泌。使用全细胞膜片钳技术从 HEK-293 和胰岛素瘤 RIN-m5F 细胞中记录电流。通过 RT-PCR 和 Western blot 鉴定 RIM1 和 Ca(V)通道的表达。通过共免疫沉淀确定 RIM1-Ca(V)通道相互作用。使用小干扰 RNA 进行 RIM1 和 Ca(V)通道亚基表达的敲低。通过 ELISA 评估胰岛素释放。在 HEK-293 细胞中共表达 Ca(V)1.2 和 Ca(V)1.3 L 型通道与 RIM1 表明，RIM1 可能不会决定 L 型 Ca(V)通道的可用性，而是降低整个细胞电流的失活速率。共免疫沉淀实验表明 Ca(V)β 辅助亚基与 RIM1 相关。缺乏 Ca(V)β 表达抑制了 RIM1 对通道的调节。与异源系统相似，内源性 RIM1 敲低后观察到电流失活增加。共免疫沉淀显示 Ca(V)β 和 RIM1 在胰岛素分泌的 RIN-m5F 细胞中相关。RIM1 的敲低显著损害了 RIN-m5F 细胞中高 K(+)刺激的胰岛素分泌。这些数据揭示了 RIM1 通过 Ca(V)β 辅助亚基与 L 型 Ca(V)通道之间的新的功能偶联，有助于决定胰岛素分泌。
