Finlin Brian S, Correll Robert N, Pang Chunyan, Crump Shawn M, Satin Jonathan, Andres Douglas A
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0509, USA.
J Biol Chem. 2006 Aug 18;281(33):23557-66. doi: 10.1074/jbc.M604867200. Epub 2006 Jun 21.
Voltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary beta-subunit (CaV beta) binds a conserved domain (the alpha-interaction domain (AID)) of the pore-forming CaV alpha1 subunit to modulate channel gating properties and promote cell surface trafficking. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. Here, we describe the Rem-association domain within CaV beta2a. The Rem interaction module is located in a approximately 130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaV beta mutants were identified that lost the ability to bind AID but retained their association with Rem, indicating that the AID and Rem association sites of CaV beta2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaV beta2a interaction is lower than that of AID for CaV beta2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaV beta2a with AID. Instead, CaV beta can simultaneously associate with both Rem and CaV alpha1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaV beta subunits with CaV alpha1 to inhibit plasma membrane trafficking. However, surface biotinylation studies in HIT-T15 cells indicate that Rem can acutely modulate channel function without decreasing the density of L-type channels at the plasma membrane. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a Rem x CaV beta x AID regulatory complex without the need to disrupt CaV alpha1 x CaV beta association or alter CaV alpha1 expression at the plasma membrane.
电压门控钙通道是调节钙内流的多蛋白复合体，对心脏兴奋性和收缩性起着重要作用。辅助β亚基（CaVβ）与形成孔道的CaVα1亚基的一个保守结构域（α相互作用结构域（AID））结合，以调节通道门控特性并促进细胞表面转运。最近，小GTP酶RGK家族成员（Rem、Rem2、Rad、Gem/Kir）已被确定为L型钙通道活性调节的新参与者。在此，我们描述了CaVβ2a内的Rem结合结构域。Rem相互作用模块位于高度保守的鸟苷酸激酶结构域内一个约130个残基的区域，该区域也指导AID结合。重要的是，已鉴定出CaVβ突变体，其失去了结合AID的能力，但仍保留与Rem的结合，这表明CaVβ2a的AID和Rem结合位点在结构上是不同的。体外结合研究表明，Rem与CaVβ2a相互作用的亲和力低于AID与CaVβ2a的亲和力。此外，体外结合研究表明，Rem结合并不抑制CaVβ2a与AID的相互作用。相反，CaVβ可以同时与Rem和CaVα1-AID结合。先前的研究表明，RGK蛋白可能通过阻断CaVβ亚基与CaVα1的结合来抑制质膜转运，从而调节Ca2+通道活性。然而，在HIT-T15细胞中的表面生物素化研究表明，Rem可以急性调节通道功能，而不会降低质膜上L型通道的密度。这些数据共同表明，Rem依赖的Ca2+通道调节涉及形成Rem x CaVβ x AID调节复合体，而无需破坏CaVα1 x CaVβ结合或改变质膜上CaVα1的表达。
