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 contributes to the short-circuit current (SCC) across the skin. The effects of amiloride and acetylcholine on the SCC were examined in the presence or absence of Cu2+ to determine whether the amiloride binding site mediating activation or that mediating inhibition of the channel is blocked by Cu2+ and whether amiloride and acetylcholine share a common binding site on the NSCC. The 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 microM Cu2+ increased the SCC. In the presence of Cu2+, amiloride decreased the SCC. In contrast, acetylcholine (1 mM) transiently increased SCC whether Cu2+ was present or not. These results suggest that there are two binding sites for amiloride on the NSCC, whereby the site that activates the channel is blocked by Cu2+ while the site that inhibits it is not, and that the binding sites for acetylcholine and amiloride may be different.