THE STIMULATORY G-PROTEIN OF ADENYLYL CYCLASE, GS, ALSO STIMULATES DIHYDROPYRIDINE-SENSITIVE CA-2+ CHANNELS - EVIDENCE FOR DIRECT REGULATION INDEPENDENT OF PHOSPHORYLATION BY CAMP-DEPENDENT PROTEIN-KINASE OR STIMULATION BY A DIHYDROPYRIDINE AGONIST

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  • 15 July 1988
    • journal article
    • research article
    • Vol. 263  (20) , 9887-9895
Abstract
We demonstrated recently that purified preparations of Gs, the stimulatory G protein of adenylyl cyclase, can stabilize Ca2+ channels in inside-out cardiac ventricle membrane patches stimulated prior to excision by the .beta.-adrenergic agonist isoprenaline or by the dihydropyridine agonist Bay K 8644 and that such preparations of Gs can restore activity to spontaneously inactivated cardiac Ca2+ channels incorporated into planar lipid bilayers (Yatani, A., Codina, J., Reeves, J. P., Birnbaumer, L., and Brown, A. M. (1987) Science 238, 1288-1292). To test whether these effects represented true stimulation and to further identify the G protein responsible, we incorporated skeletal muscle T-tubule membranes into lipid bilayers and studied the response of their Ca2+ channels to G proteins, specifically Gs, and manipulations known to be specific for Gs. In contrast to cardiac channels, incorporated T-tubule Ca2+ channels exhibit stable average activities over prolonged periods of time (up to 20 min at room temperature), allowing assessment of possible effects of G proteins under steadystate assay conditions. We report that exogenously added human erythrocyte GTP.gamma.S (guanosine 5''-O-(3-thiotriphosphate))-activated Gs (Gs*) or its resolved GTP.gamma.S-activated .alpha. subunit (.alpha.s*) stimulate T-tubule Ca2+ channels by factors of 2-3 in the presence of Bay K 8644, and of 10-20 in the absence of Bay K 8644 and that they do so in a manner that is independent of concurrent or previous phosphorylation by cAMP-dependent protein kinase. Activation of purified Gs by cholera toxin increases both its adenylyl cyclase stimulatory and its Ca2+ channel stimulatory effects. Ca2+ channels previously stimulated by the combined actions of Bay K 8644 and cAMP-dependent protein kinase still respond to Gs. We conclude that the responses seen are due to Gs rather than a contaminant, that the effect on Ca2+ channel activity is that of a true stimulation, akin to that on adenylyl cyclase, and show that a given G protein may regulate more than one effector system.