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

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.