Distinct RGK GTPases Differentially Use α1- and Auxiliary β-Binding-Dependent Mechanisms to Inhibit CaV1.2/CaV2.2 Channels

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.

RGK regulation of voltage-gated calcium channels

“…This experiment was remarkable for another reason: cells expressing Rem and the dominant negative dynamin still had strongly reduced VGCC currents, suggesting that membrane-resident channels were inhibited via an alternative mechanism. Similar dual mechanisms were also found for Rem2 inhibition of Ca v 1.2 [2]. Thus, we now know that RGK proteins inhibit VGCCs simultaneously using a slow, trafficking-dependent mechanism and a fast mechanism that inhibits membrane-resident channels (see below and Figure 2).…”

“…Interestingly, Gem and Rem2 do not seem to inhibit voltage sensor movement in Ca v 1.2 channels. As will be discussed later, this difference is thought to result from their inability to bind to the channel's N-terminus [2]. The mode of inhibition in this case likely involves a decrease in surface expression coupled with a decrease in channel P o [1,2].…”

“…The mode of inhibition in this case likely involves a decrease in surface expression coupled with a decrease in channel P o [1,2]. Indeed, the comparison between Ca v 1.2 gating currents and tail currents (the latter quantify the total ionic flow through open channels), revealed that channel P o is reduced in the presence of Rem [2]. Similarly, Rem2 was found to inhibit Ca v 2.2 channels by rendering them nonconducting, the molecular mechanism of which is yet to be studied [22].…”

“…The voltage sensors' movements elicit minuscule "gating currents" that can be measured independently from the larger Ca 2+ currents flowing through the channel's pore. The "gating current" concept is noted here because RGK proteins can restrict the movement of the voltage sensors in some instances [1,2]. The four homologous Ca v α 1 repeats are connected by three intracellular connecting loops: the III loop, IIIII loop and IIIIV loop.…”

“…As will be discussed later, this difference is thought to result from their inability to bind to the channel's N-terminus [2]. The mode of inhibition in this case likely involves a decrease in surface expression coupled with a decrease in channel P o [1,2]. Indeed, the comparison between Ca v 1.2 gating currents and tail currents (the latter quantify the total ionic flow through open channels), revealed that channel P o is reduced in the presence of Rem [2].…”