Cu2+ reveals amiloride-induced activation and blocking of non-selective cation channel in larval bullfrog skin

The non-selective cation channel (NSCC) in the larval bullfrog skin contrib utes to the short-circuit current (SCC) across the skin. The effects of ami loride and acetylcholine on the SCC were examined in the presence or absenc e of Cu2+ to determine whether the amiloride binding site mediating activat ion or that mediating inhibition of the channel is blocked by Cu2+ and whet her amiloride and acetylcholine share a common binding site on the NSCC. Th e skin of tadpoles raised in aldosterone was examined with K-Ringer present on the apical side to potentiate the SCC. Amiloride (10(-4) M) transiently increased SCC in the absence of Cu2+. Apical application of 500 muM Cu2+ i ncreased the SCC. In the presence of CU2+, amiloride decreased the SCC. In contrast, acetylcholine (1 mM) transiently increased SCC whether CU2+ was p resent or not. These results suggest that there are two binding sites for a miloride on the NSCC, whereby the site that activates the channel is blocke d by Cu2+ while the site that inhibits it is not, and that the binding site s for acetylcholine and amiloride may be different.