Muscarinic modulation of voltage-dependent Ca21 channels in insulin-secreting HIT-T15 cells

Love, Jeffrey A., Neil W. Richards, Chung Owyang, and David C. Dawson. Muscarinic modulation of voltagedependent Ca21 channels in insulin-secreting HIT-T15 cells. Am. J. Physiol. 274 (Gastrointest. Liver Physiol. 37): G397– G405, 1998.—Potentiation of insulin secretion from pancreatic b-cells by acetylcholine requires ongoing cyclic electrical activity initiated by other depolarizing secretagogues. Patchclamp recordings in glucose-free solutions were made from the clonal b-cell line HIT-T15 to determine whether the muscarinic agonist bethanechol (BCh) modulated voltagedependent Ca21 channels independent of effects on membrane potential. Only high-threshold, dihydropyridine-sensitive (L-type) Ca21 channels with a mean conductance of 26 pS were observed in cell-attached patches. BCh (100 μM) caused a twoto threefold increase in both fractional open time and mean current of single Ca21 channels. These changes resulted from a 44% decrease in the longer of two apparent mean closed times and a 25% increase in the mean open time. Similar BCh-stimulated increases in macroscopic Ca21 currents were recorded in whole cell, perforated-patch recordings. The role of protein kinase C (PKC) in the muscarinic activation of Ca21 channels was tested using a variety of PKC activators and inhibitors. Acute application of either the active phorbol ester phorbol 12-myristate 13-acetate (PMA) or the membrane-permeable diacylglycerol analog 1,2didecanoyl-rac-glycerol mimicked the effects of BCh, whereas an inactive phorbol (4a) had no effect. Depletion of PKC activity by chronic exposure to PMA or acute application of the PKC inhibitor staurosporine greatly reduced or abolished muscarinic activation of Ca21 channels. These results are consistent with muscarinic activation of L-type, voltagedependent Ca21 channels mediated in large part by PKC.