Analysis of the complex between Ca 2+ channel β-subunit and the Rem GTPase

Abstract

Voltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary β-subunit (Ca Vβ) binds a conserved domain (the α-interaction domain (AID)) of the pore-forming Ca Vα1 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 Ca Vβ 2a. The Rem interaction module is located in a ∼130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, Ca Vβ 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 Ca Vβ 2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for Ca Vβ 2a interaction is lower than that of AID for Ca Vβ 2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of Ca Vβ 2a with AID. Instead, Ca Vβ can simultaneously associate with both Rem and Ca Vα 1- AID. Previous studies had suggested that RGK proteins may regulate Ca 2+ channel activity by blocking the association of Ca Vβ subunits with Ca Vα 1 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 Ca 2+ channel modulation involves formation of a Rem·Ca Vβ·AID regulatory complex without the need to disrupt Ca Vα 1·Ca Vβ association or alter Ca Vα 1 expression at the plasma membrane. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.