Direct inhibition of P/Q-type voltage-gated Ca ²⁺ channels by Gem does not require a direct Gem/Ca _v β interaction

Abstract

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca 2+ channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca 2+ channel β subunits (Ca v β), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca 2+ channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca v β but not the Gem/Ca v β interaction, and that Gem coimmunoprecipitates with the P/Q channel α 1 subunit (Ca v α 1 ) in a Ca v β-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca v α 1 critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca v α 1 abolishes Gem inhibition of P/Q channels and confers Ca v β-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca v α 1 that binds Ca v β). Our results challenge the prevailing view regarding the role of Ca v β in RGK inhibition of high voltage-activated Ca 2+ channels and prompt a paradigm in which Gem directly binds and inhibits Ca v β-primed Ca v α 1 on the plasma membrane.

RGK regulation of voltage-gated calcium channels

“…Finally, a recent report studying Ca v 1.2 currents in cardiac myocytes from Rad knockout Gem/Kir Ca v 2.1 Directly inhibits channels in macropatces in a Ca v β-dependent but Ca v β-binding-independent manner. Xenopus oocytes [6] mice found that Ca v 1.2 activation is shifted to more negative voltages (channels are easier to open), which is consistent with a retarding effect of Rad on voltage sensor movement. It remains to be determined how universal this effect is [39].…”

“…Supporting this notion, mutating these critical residues individually or in combination severely weakens or abolishes in vitro binding of Gem and β 3 [6,40], while preserving calcium channel modulation by β 3 . Third, Ca v α 1 , Ca v β and RGK proteins can form a trimeric complex in vitro and in cells [5,6,14,30,40].…”

“…We coexpressed Ca v 2.1 channels with Ca v β 3 in Xenopus oocytes and excised large membrane patches containing these channel complexes. Application of a purified Gem protein to the intracellular face of the macropatches elicited partially reversible channel inhibition, demonstrating unequivocally that membrane-resident channels can be inhibited [6]. In addition, the speed of inhibition was relatively fast, reaching a maximum within 3 min of Gem application.…”

“…To overcome this problem, and to provide a direct answer to whether Ca v β is required for inhibition, we mutated the ABP of Ca v β 3 (M245A and L249A) to achieve two effects: (i) the mutation was mild enough to allow sufficient Ca v β 3 -Ca v α 1 binding to promote channel surface expression in Xenopus oocytes, in this case Ca v 2.1; (ii) at the same time, the weakened Ca v β 3 -Ca v α 1 binding allowed us to later wash away this mutant Ca v β from a macropatch preparation, leaving β-less channels on the plasma membrane [6]. In support of previous findings, β-less channels could not be inhibited by purified Gem perfused onto the intracellular side of the macropatch.…”

“…A, RGKs inhibit VGCC current completely and this inhibition is dependent on the presence of a Ca 2+ channel β subunit. Thus, β-less channels, which we could generate in macropatches [6], are insensitive to RGK inhibition. B, RGKs exert a dynamin-mediated inhibition of VGCC surface expression.…”