Calcium Channel β Subunit Promotes Voltage-Dependent Modulation of α1B by Gβγ

Abstract

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.

Dominant-Negative Calcium Channel Suppression by Truncated Constructs Involves a Kinase Implicated in the Unfolded Protein Response

“…Whole-cell patch-clamp recording was performed and analyzed as described (Meir et al, 2000), with 1-20 mM Ba 2ϩ as charge carrier (as stated) and a holding potential of Ϫ100 mV. Currents were measured 10 msec after the onset of the test pulse, and the average over a 2 msec period was calculated and used for analysis.…”

G protein inhibition of CaV2 calcium channels

“…In particular, there appears to be an absolute requirement for Ca V β subunit binding to the AID in the I-II linker. 120,123,124,145 The role of Ca V β subunits in voltage-dependent inhibition has been controversial with several seemingly opposing data sets and interpretations that have been reviewed in depth elsewhere. 20,38,39 Briefly, one of the central questions that arose was whether or not Ca V β and Gβγ can both bind to the channel at the same time, or whether they compete for binding to the I-II linker in a mutually exclusive manner.…”

The Ducky Mutation in Cacna2d2 Results in Altered Purkinje Cell Morphology and Is Associated with the Expression of a Truncated α2δ-2 Protein with Abnormal Function

“…Transfection was performed as described above, using the ratios for ␣ 1 , ␤, ␣2␦, and GFP of 3:1:1:0.1. In the single channel experiments cDNA for ␤-adrenergic receptor kinase 1 (␤-ARK1), G␤␥ binding domain was included in the transfections, at the same concentration as the ␤ subunit cDNA (23). For expression in Xenopus oocytes, cDNAs encoding rabbit Ca V 2.1 (X57689), rat ␤ 4 and mouse ␣2␦-2 or du-mut1 ␣ 2 cDNAs were injected intranuclearly as described previously (24), except that 4 nl of the 1:1:1 ratio cDNA mixture was injected at 1 g⅐l Ϫ1 .…”

“…Latency to first opening was measured in 2-ms bins. First latency (FL) histograms from each experiment were divided by the number of episodes collected, and the plots were then accumulated and divided by the number of stimulations, to express the data as the FL probability (23,27). Two-and three-channel patches were also corrected for the apparent number of channels in the patch, according to Equation 2,…”