G-Proteins Modulate Cumulative Inactivation of N-Type (Ca_V2.2) Calcium Channels

Abstract

Precise regulation of N-type (Ca V 2.2) voltage-gated calcium channels (Ca-channels) controls many cellular functions including neurotransmitter and hormone release. One important mechanism that inhibits Ca 2ϩ entry involves binding of G-protein ␤␥ subunits (G␤␥) 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 G␤␥ from the Ca-channels and can occur during high-frequency bursts of action potential-like waveforms (APW). Inactivation of Ca-channels will also limit Ca 2ϩ 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 lowfrequency trains of APW to activate I Ca . This more closely mimics physiological stimuli and circumvents the problem of facilitation which does not occur at Յ5 Hz. Activation of endogenous G-proteins reduced both Ca 2ϩ -dependent, and voltage-dependent inactivation of recombinant I Ca in human embryonic kidney 293 cells. This was mimicked by expression of wild-type G␤␥, but not by a point mutant of G␤␥ 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 Ca 2ϩ entry and Ca 2ϩ-dependent events such as neurotransmitter and hormone release.

Novel Mechanism of Voltage-Gated N-type (Cav2.2) Calcium Channel Inhibition Revealed through α-Conotoxin Vc1.1 Activation of the GABAB Receptor

“…In the absence of an agonist, a prepulse (pp) to 1120 mV led to an increase in the current amplitude evoked by the test pulse (20.9 6 1.9%; n 5 6) compared with the current amplitude recorded in the absence of a prepulse. This observation suggests that some basal inhibition of Ca v 2.2 occurs in HEK293 cells, which is similar to previous reports of Gbg modulation of Ca v 2.2 in the absence of GPCR activation (Meza and Adams, 1998;McDavid and Currie, 2006). As expected, the prepulse to 1120 mV significantly relieved GABA-dependent inhibition of peak current from 64 6 4% (-pp) to 4 6 5% (1pp) (n 5 8; P , 0.05) (Fig.…”

Activation of Corticotropin-Releasing Factor Receptor 1 Selectively Inhibits CaV3.2 T-Type Calcium Channels

“…Besides, we used a 15-s protocol that is different from the 6-s voltage prepulses used by the Barrett's group . This difference in experimental protocol may be relevant as it has been demonstrated that a shorter duration of the prepulse resulted in a depolarizing shift in the steadystate inactivation in the G␤␥-mediated inhibition of Ca V 2.2 channels (McDavid and Currie, 2006). Nonetheless, besides the observed hyperpolarized shift in steady-state inactivation, we cannot exclude the possibility that the probability of opening of the Ca V 3.2 channels may also be reduced upon activation of CRFR1 receptors by UCN.…”

G protein inhibition of CaV2 calcium channels

“…Consistent with this, blocking ~50% of I Ca by ω-conotoxin GVIA (i.e., by decreasing global Ca 2+ entry) reduced calcium dependent inactivation, but a similar block of I Ca by Cd 2+ did not reduce inactivation. 76 This highlights the complexity of Ca 2+ -dependent regulation of Ca V channels and perhaps hints that the effects of Gβγ involve more than simply reducing global Ca 2+ entry. A recent study proposed that allosteric hindrance of channel activation underlies Ca 2+ -dependent inactivation of Ca V 1.3, 81 although it is not known if this holds true for Ca V 2 channels.…”

“…More recently, gating current modulation by G proteins has also been reported in rat sympathetic neurons. 74,75 Gβγ can also reduce inactivation of Ca V 2.2 channels, 76,77 although this is somewhat more subtle and can be masked by concomitant voltage-dependent reversal of Gβγ-mediated inhibition (Gβγ unbinding). Inactivation of Ca 2+ channels is complex and mediated by several voltage-dependent and Ca 2+ -dependent mechanisms.…”

“…Many effectors bind to the same surface of Gβ that interacts with Gα and a protein interaction "hot spot" on this face has been identified with overlapping subsets of residues that are involved in binding to different effectors. 133 Mutation of several residues on the Gα interacting surface disrupt inhibition of Ca V 2 channels, 76,122,134,135 but residues on the opposite face of Gβ 1 have also been implicated. [135][136][137] Moreover, Asn 35 and Asn 36 on Gβ 1 have been shown to underlie the ability of PKC to antagonize inhibition of Ca V 2.2.…”