The stimulatory G protein of adenylyl cyclase, Gs, also stimulates dihydropyridine-sensitive Ca2+ channels. Evidence for direct regulation independent of phosphorylation by cAMP-dependent protein kinase or stimulation by a dihydropyridine agonist.

A Yatani,Y Imoto,J Codina,S L Hamilton,A M Brown,L Birnbaumer

doi:10.1016/s0021-9258(19)81600-9

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https://doi.org/10.1016/s0021-9258(19)81600-9

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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 steady-state 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.

Highlights

EVIDENCE FOR DIRECT REGULATION INDEPENDENT OF PHOSPHORYLATION BY CAMP-DEPENDENT PROTEIN KINASE OR STIMULATION BY A DIHYDROPYRIDINE AGONIST*
We demonstrated recentlythat purified preparations We recently discoveredthat receptor-regulated K+channels of GB,the stimulatory G protein of adenylyl cyclase, of pacemaking atrial and cells secretory endocrine cells are can stabilize Ca2+channels in inside-out cardiac ven- under direct control of a pertussis toxin (PTX)’-sensitive G
Dihydropyridine agonist Bay K 8644 and that such activated Gprotein tothe cytoplasmic aspect of isolated preparations of G. canrestoreactivitytospontamembrane patches sealed onto the tipsof patch pipettes [7]

Summary

RESULTS

Probability density function with mean values of0.37-0.38 PA. Properties of Incorporated Channels-In most experiments, the dihydropyridine Ca2+ channel agonist Bay K 8466 was used to increase the opening probability,Po, of the incorporated channels and thereby facilitate their detection. All were blocked by the washoutof the cis chamber in the presenceof Bay K 8644 Such additional conductance states have been recently re- irregular cyclical activities characteristicof single DHP-senported [16, 31] and were proposed to represent subconduct- sitive Ca2+ channel(s13,14, 16, 17,33). Wewere unable to determineif the differ- long as 20 min or until the bilayer broke down This can be ent conductances observed by us represent such subconduct- seen by comparing the cumulatedNP, values between 0 and ance states or different channels because the frequency of 3 min t o cumulative NP, values between 15and 18min shown simultaneous opening was too low and because, due to the in Fig. 3 for a bilayer withBay K 8644-stimulated Ca2+.

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Findings

DISCUSSION