| Summary: | In excitable cells, high-voltage activated calcium channels (CaV1/CaV2) are essential for translating electrical signals into biological responses. These channels are multimeric subunit protein complexes composed of a main pore-forming α1 subunit, which associates with auxiliary β, γ, and α2δ subunits. CaV1/CaV2 channels are critical for the initiation of many vital physiological processes. Molecules that modulate CaV1/CaV2 channel activity can dramatically alter cellular physiology and are used to treat cardiovascular and neurological disorders. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like monomeric GTPases that potently and non-selectively inhibit all CaV1/CaV2 channels. Despite being studied by several groups, the mechanism underlying this inhibition has yet to be fully understood. All RGK proteins bind directly to the auxiliary β subunits, and it was generally assumed that this RGK/β interaction is required for calcium channel inhibition. A comprehensive understanding of how RGK proteins inhibit calcium channels will not only enhance our perspectives on their (patho)physiological roles but could also advance their practical use as calcium channel blockers (CCBs). Using a mutated β subunit (βTM), which selectively loses the ability to interact with RGKs, we show that the RGK protein, Rem, inhibits CaV1.2 channels by utilizing both β-binding-dependent (BBD) and direct α1C-binding-dependent mechanisms (ABD). Previous studies have demonstrated that Rem inhibits CaV1.2 channels by 1) decreasing the number of channels on the cell surface, 2) reducing the channel’s open probability and 3) immobilizing their voltage sensors. Here, we identify a novel Rem binding site on the distal end of the α1C N-terminus that mediates ABD inhibition by reducing CaV1.2 voltage sensor movement, without significantly affecting the other two mechanistic signatures of Rem inhibition of CaV1.2. The molecular determinants of Rem required for BBD CaV1.2 inhibition are the distal C-terminus and the guanine nucleotide-binding domain (G-domain), which interact with the plasma membrane and CaVβ, respectively. Here, we determine that the Rem G-domain and distal C-terminus also mediate ABD CaV1.2 inhibition. For this mode of inhibition the Rem distal C-terminus interacts with α1C N-terminus to anchor the G-domain, which presumably interacts with an as-yet-unidentified site. To profile the relative prevalence of BBD and ABD of inhibition across the RGK and CaV1/CaV2 channel families we compared the impact of all four RGKs on currents through recombinant CaV1.3, CaV2.1, CaV2.2 channels, reconstituted with either wt β2a or β2aTM, respectively. When reconstituted with β2aTM, CaV1.3 and CaV2.1 were completely refractory to all four RGKs indicating these channels display only β-binding-dependent mechanisms of inhibition. CaV2.2 channels reconstituted with β2aTM displayed a strong inhibition solely to Rad, identifying another example of β-binding-independent regulation of a CaV channel by an RGK protein. The results reveal latent capabilities of distinct RGKs to selectively inhibit particular CaV1/CaV2 channels in an isoform-specific manner. These dormant capabilities may be exploitable to develop novel genetically-encoded isoform-selective CaV1/CaV2 channel inhibitors.
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