CHARACTERIZATION OF THE G-PROTEIN COUPLING OF A GLUCAGON RECEPTOR TO THE K-ATP CHANNEL IN INSULIN-SECRETING CELLS

The G-protein-mediated coupling of a glucagon receptor to ATP-dependen t K channels-K-ATP-has been studied in insulin-secreting cells using t he patch clamp technique. In excised outside-out patches, K-ATP channe l activity was inhibited by low concentrations of glucagon (IC50 = 2.4 nM); the inhibitory effect vanished at concentrations greater than 50 nM. In cell-attached patches, inhibition by bath-applied glucagon was seen most often, although stimulation was observed in a few cases. A dual action of the hormone is proposed to resolve these apparently div ergent results. In excised inside-out patches, K-ATP channel activity was inhibited by addition of beta gamma subunits purified from either erythrocyte or retina (IC50 = 50 pM and 1 nM, respectively). Subsequen t exposure of the patch to alpha(i) or alpha(o) reversed this effect. In excised inside-out patches, increasing Mg2+ in the bath stimulated the channel activity between 0 and 0.5 mM, but blocked it at higher co ncentrations (IC50 = 2.55 mM). In most cases (70%), GTP had a stimulat ory effect at concentrations up to 100 mu M. However, in three cases, similar GTP levels had clear inhibitory effects. In excised inside-out patches, cholera toxin (CTX) caused channel inhibition. Although the effect could not be reversed by removal of the toxin, the activity was restored by subsequent addition of purified alpha(i) or alpha(o). The se results are compatible with a model whereby channel inhibition by a ctivated G(s)-coupled receptors occurs, at least in part, via associat ion of the beta gamma subunits of G(s) with alpha(i)/alpha(o) subunits and deactivation of the alpha(i)/alpha(o)-, dependent stimulatory pat hway. On the basis of this hypothesis, a model is developed to describ e the effects of G proteins on the K-ATP channel, as well as to accoun t for the concentration-dependent stimulation and inhibition of K-ATP channel by Mg2+. An interpretation of the ability of glucagon to poten tiate, but not initiate, insulin release is also given in terms of thi s model and the effects of ATP on K-ATP channels.