MECHANISM OF ACTIVATION OF NONSELECTIVE CATION CHANNELS BY PUTATIVE M(4) MUSCARINIC RECEPTOR IN GUINEA-PIG CHROMAFFIN CELLS

1 Mechanisms involved in the generation of nonselective cation currents (I-NS) by muscarinic agonists in the chromaffin cell were investigated by the perforated patch method. 2 Bath application of muscarine (0.1-30 mu M) produced an inward I-NS With Or without a transient outward current at -40 mV, whereas oxotremorine (0.06-60 mu M) induced I-NS alone. Rectangular hyperbolas with EC(50)s of 2.01 and 0.21 mu M were fitted to muscarine- and oxotremorine-induced INSS, respectively, and the maximal amplitude of the former was about 3.4 times larger than that of the latter. 3 In 36% of the cells exposed to Ca2+-free solution, muscarine I-NS was suppressed, being 53% of control 20 min after the perfusion, and in four cells that were incubated with Ca2+-free solution for 2 h or more, the I-NS averaged 44% of that induced subsequently in normal solution. In contrast, muscarine I-NS was enhanced by about 30% when A-23187 was added to normal solution. 4 W-7 and W-5, calmodulin-related agents, were almost equally potent in inhibiting muscarine I-NS, whereas compound 5, a potent inhibitor of calmodulin-dependent kinase II (CaM kinase II), produced no evident inhibition. 5 HA1004, a weak kinase C inhibitor, induced a reversible suppression of muscarine I-NS With an IC50 of 163 mu M, whereas H-8, another kinase inhibitor, produced an even small degree of inhibition. Administration of phorbol 12, 13-dibutyrate did not mimic muscarinic stimulation of NS channels; rather, it led to a progressive inhibition of I-NS and this inhibition was almost complete within 20 min. An inactive phorbol ester had no such effect. 6 The muscarinic antagonists, pirenzepine and AF-DX 116, shifted the dose-response curve for the muscarine I-NS to the right in a parallel manner. The K(D)s for pirenzepine and AF-DX 116 were estimated to be 13 nM (95% confidence interval, 11-16 nM) and 365 nM (283-470 nM), respectively. 7 These results suggest that muscarine efficiently produces I-NS, probably through binding to the M(4) Subtype, that intracellular Ca2+ has a facilitating, but not an essential role in the generation of I-NS, and that neither CaM kinase II nor protein kinase C is involved.