Abstract
Modulation of the Ca- and voltage-dependent K channel—KCa—by receptors coupled to the G proteins G i /G o and G s has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the KCa channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous α subunits of either G i or G o -type G proteins also inhibited the KCa channel (IC50 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses KCa channel activity via a membrane-delimited pathway that involves the α subunits of PTX-sensitive G proteins G i and/or G o . In outside-out patches, activation of G s either by β-agonists or with cholera toxin (CTX) increased KCa channel activity, consistent with a membrane-delimited stimulatory pathway linking the β-adrenergic receptor to the KCa channel via G s . In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of β-agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous α i or α o . Taken together these data suggest that KCa channel activity is enhanced by activation of G s and blocked by activated G i and/or G o . Further, KCa channel stimulation by activated G s may be “direct,” while inhibition by G i /G o may involve deactivation of G s . In inside-out patches KCa channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the KCa channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous α i or α o . These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G i /G o -induced deactivation of G s .
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The authors would like to thank Dr. S. Ciani for many helpful discussions, Dr. A.E. Boyd III for supplying the HIT cells, Drs. J. Codina and L. Birnbaumer for supplying the alpha subunits of the G proteins G i and G o , and Mrs. Satoko Hagiwara for preparing and maintaining the cell cultures.
This work was supported by grant DCB-8919368 from the National Science Foundation and a research grant (W-P 880513) from the American Diabetes Association to B.R., and by grant RO1-DK39652 from the National Institutes of Health to G.T.E.
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Ribalet, B., Eddlestone, G.T. Characterization of the G protein coupling of SRIF and β-adrenergic receptors to the maxi KCa channel in insulin-secreting cells. J. Membarin Biol. 148, 111–125 (1995). https://doi.org/10.1007/BF00207268
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DOI: https://doi.org/10.1007/BF00207268