Cu2+ reveals different binding sites of amiloride and CDPC on the apical Na channel of frog skin.

The effect of Cu2+ ions, present in the mucosal bathing solution, on the transepithelial short-circuit current (Isc) and conductance (Gt) and on the blocker-induced noise of apical Na channels, was studied on the isolated ventral skin of the frog Rana temporaria. Cu2+ effects were concentration-dependent, the full effect being reached at 50 micromol/l. Cu2+ increased Isc and Gt; this effect was eliminated by high concentrations of amiloride (30 micromol/l) and of CDPC (150 micromol/l). Cu2+ markedly reduced the corner frequency (fc) of the Na channel noise, while having virtually no effect on the fc of CDPC-induced noise. Cu2+ reduces the association rate constant of amiloride to the Na channel to one third; this effect is interpreted as indicating competition between Cu2+ and amiloride for the same (negatively charged) binding site on the channel, while CDPC appears to bind on a different site.