Abstract 739: Identification of the RGK Interaction Domain of Calcium Channel Beta-subunits
Recent studies have identified members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) as novel contributors to the regulation of L-type calcium channel activity via an association with auxiliary Beta-subunits in a variety of excitable cells. Here, we describe the Rem-association domain within CaVBeta2a. The Rem interaction module is located within the highly conserved guanylate kinase (GK) domain which also directs binding to the conserved alpha-interaction domain, AID, within the pore forming CaV1 subunit. Importantly, Beta-subunit deletion mutants were generated which lacked the conserved SH3-like module and had lost the ability to bind AID, but retained their association with Rem (CaVBeta260–604), indicating that the AID and Rem association sites of CaVBeta are structurally distinct. In vitro binding studies indicate that the affinity of Rem/Beta association is significantly weaker than that of AID for Beta-subunits. Furthermore, Rem binding does not inhibit the interaction of Beta2a with AID. Instead, Beta-subunits can simultaneously associate with both Rem and AID. While co-expression of Rem with CaV1.2 and Beta2a subunits in tsA201 cells results in a complete absence of detectable ionic current expression, and CaVBeta260–604 expression alone fails to modulate Ca-channel activity, co-expression of CaVBeta260–604 partially relieved Rem-mediated inhibition of current activity, consistent with a requirement for CaVBeta-binding in Rem-mediated channel regulation. Earlier work has suggested that RGK proteins may regulate Ca2+ channel activity by blocking CaV1/Beta complex assembly to inhibit trafficking to the cell surface. However, Rem can inhibit whole-cell currents at 24 hrs post-transfection without decreasing the density of L-type channels at the plasma membrane as measured by surface biotinylation. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a higher order Rem/Beta/AID regulatory complex without the need to disrupt CaV1/beta association or alter the steady-state level of surface expressed Ca2+ channel.