Rem a member of the RGK GTPases inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase

摘要

Rad/Rem/Gem/Kir (RGK) GTPases potently inhibit CaV1 and CaV2 (CaV1-2) channels, a paradigm of ion channel regulation by monomeric G-proteins with significant physiological ramifications and potential biotechnology applications. The mechanism(s) underlying how RGK proteins inhibit ICa is unknown, and it is unclear how key structural and regulatory properties of these GTPases (such as the role of GTP binding to the nucleotide binding domain (NBD), and the C-terminus which contains a membrane-targeting motif) feature in this effect. Here, we show that Rem inhibits CaV1.2 channels by three independent mechanisms that rely on distinct configurations of the GTPase: (1) a reduction in surface density of channels is accomplished by enhancing dynamin-dependent endocytosis, (2) a diminution of channel open probability (Po) that occurs without impacting on voltage sensor movement, and (3) an immobilization of CaV channel voltage sensors. The presence of both the Rem NBD and C-terminus (whether membrane-targeted or not) in one molecule is sufficient to reconstitute all three mechanisms. However, membrane localization of the NBD by a generic membrane-targeting module reconstitutes only the decreased Po function (mechanism 2). A point mutation that prevents GTP binding to the NBD selectively eliminates the capacity to immobilize voltage sensors (mechanism 3). The results reveal an uncommon multiplicity in the mechanisms Rem uses to inhibit ICa, predict new physiological dimensions of the RGK GTPase–CaV channel crosstalk, and suggest original approaches for developing novel CaV channel blockers.