Abstract
CaV1/CaV2 channels, comprised of pore-forming α1 and auxiliary (β,α2δ) subunits, control diverse biological responses in excitable cells. Molecules blocking CaV1/CaV2 channel currents (I Ca) profoundly regulate physiology and have many therapeutic applications. Rad/Rem/Rem2/Gem GTPases (RGKs) strongly inhibit CaV1/CaV2 channels. Understanding how RGKs block I Ca is critical for insights into their physiological function, and may provide design principles for developing novel CaV1/CaV2 channel inhibitors. The RGK binding sites within CaV1/CaV2 channel complexes responsible for I Ca inhibition are ambiguous, and it is unclear whether there are mechanistic differences among distinct RGKs. All RGKs bind β subunits, but it is unknown if and how this interaction contributes to I Ca inhibition. We investigated the role of RGK/β interaction in Rem inhibition of recombinant CaV1.2 channels, using a mutated β (β2aTM) selectively lacking RGK binding. Rem blocked β2aTM-reconstituted channels (74% inhibition) less potently than channels containing wild-type β2a (96% inhibition), suggesting the prevalence of both β-binding-dependent and independent modes of inhibition. Two mechanistic signatures of Rem inhibition of CaV1.2 channels (decreased channel surface density and open probability), but not a third (reduced maximal gating charge), depended on Rem binding to β. We identified a novel Rem binding site in CaV1.2 α1C N-terminus that mediated β-binding-independent inhibition. The CaV2.2 α1B subunit lacks the Rem binding site in the N-terminus and displays a solely β-binding-dependent form of channel inhibition. Finally, we discovered an unexpected functional dichotomy amongst distinct RGKs— while Rem and Rad use both β-binding-dependent and independent mechanisms, Gem and Rem2 use only a β-binding-dependent method to inhibit CaV1.2 channels. The results provide new mechanistic perspectives, and reveal unexpected variations in determinants, underlying inhibition of CaV1.2/CaV2.2 channels by distinct RGK GTPases.
Highlights
Ca2+ influx via high-voltage-activated CaV1/CaV2 Ca2+ channels links electrical signals to physiological responses in excitable cells, and regulates myriad biological functions ranging from muscle contraction to hormone and neurotransmitter release [1,2]
Cells expressing mutant CaV1.2 channels reconstituted with a1C+b2a to generate a mutant (b2aTM) yielded strong ICa,L with amplitude and voltagedependence indistinguishable from wild-type CaV1.2 (Fig. 1 D and E), demonstrating that the mutations did not adversely affect the structure and functional interaction of b with a1C
It was subsequently shown that RGKs do not disrupt the a1-b interaction leading to revised models invoking a ternary a1/b/RGK complex in which bs bridge a1 subunits and RGKs to initiate ICa inhibition [11,16,20,34,35]
Summary
Ca2+ influx via high-voltage-activated CaV1/CaV2 Ca2+ channels links electrical signals to physiological responses in excitable cells, and regulates myriad biological functions ranging from muscle contraction to hormone and neurotransmitter release [1,2]. CaV1/CaV2 channel activity is modulated by various intracellular signaling molecules, and this serves as a powerful method to alter physiology [1,3]. Rad/Rem/Rem2/Gem (RGK) proteins are a four-member subfamily of the Ras superfamily of monomeric GTPases [9], and are the most potent known intracellular inhibitors of CaV1/CaV2 channels [10,11,12]. RGK proteins are present in excitable tissue— including skeletal/cardiac muscle, nerve, and endocrine cells— suggesting that their inhibition of CaV1/CaV2 channels has physiological significance. Consistent with this notion, suppression of basal Rad expression in heart increases L-type CaV1.2 calcium current (ICa,L) and leads to cardiac hypertrophy [13,14].
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