Inactivation Regulates RGK-Mediated Inhibition of Voltage-Gated Calcium Channels

Abstract

Voltage-gated Ca2+ channels (VGCC) directly control muscle contraction and neurotransmitter release, and slower processes such as cell differentiation, migration, and death. They are potently inhibited by RGK GTP-ases (Rem1, Rem2, Rad and Gem/Kir), which decrease Ca2+ channel membrane expression, as well as directly inhibit membrane-resident channels. The mechanisms of membrane-resident channel inhibition are difficult to study because RGK-overexpression causes complete or near complete channel inhibition. Using titrated levels of RGK expression in Xenopus oocytes that inhibit WT P/Q-type calcium channels by ∼50%, we show that inhibition depends on channel inactivation. Interestingly, fast-inactivating channels, including Familial Hemiplegic Migraine mutants, are more potently inhibited than WT channels, while slow-inactivating channels, such as those expressed with the β2a auxiliary subunit, are spared. We found similar results in L-type channels, and, remarkably, an insensitivity of Timothy Syndrome mutants to RGK inhibition. Further results suggest that RGKs slow channel recovery from inactivation and identify RGKs as potential modulating factors in channelopathies. Finally, our results confirm a previously proposed immobilization of calcium channel voltage sensors by Rad, which we were now able to observe in electrophysiological experiments with titrated levels of Rad. Thus, RGK-mediated inhibition is a much subtler affair than previously thought, and physiological conditions can control the levels of inhibition.