G-proteins modulate cumulative inactivation of N-type (Ca_V2.2) calcium channels

Precise regulation of N-type (Ca_V2.2) voltage-gated calcium channels (Ca-channels) controls many cellular functions including neurotransmitter and hormone release. One important mechanism that inhibits Ca²⁺ entry involves binding of G-protein βγ subunits (Gβγ) to the Ca-channels. This shifts the Ca-channels from "willing" to "reluctant" gating states and slows activation. Voltage-dependent reversal of the inhibition (facilitation) is thought to reflect transient dissociation of Gβγ from the Ca-channels and can occur during high-frequency bursts of action potential-like waveforms (APW). Inactivation of Ca-channels will also limit Ca²⁺ entry, but it remains unclear whether G-proteins can modulate inactivation. In part this is because of the complex nature of inactivation, and because facilitation of Ca-channel currents (I_Ca) masks the extent and kinetics of inactivation during typical stimulation protocols. We used low-frequency trains of APW to activate I_Ca. This more closely mimics physiological stimuli and circumvents the problem of facilitation which does not occur at ≤5 Hz. Activation of endogenous G-proteins reduced both Ca ²⁺-dependent, and voltage-dependent inactivation of recombinant I_Ca in human embryonic kidney 293 cells. This was mimicked by expression of wild-type Gβγ, but not by a point mutant of Gβγ with reduced affinity for Ca-channels. A similar decrease in the inactivation of I_Ca was produced by P2Y receptors in adrenal chromaffin cells. Overall, our data identify and characterize a novel effect of G-proteins on I_Ca, and could have important implications for understanding how G-protein-coupled receptors control Ca²⁺ entry and Ca²⁺-dependent events such as neurotransmitter and hormone release.