Effect of divalent heavy metals on epithelial Na+ channels in A6 cells

tter understand how renal Na+ reabsorption is altered by heavy metal poisoning, we examined the effects of several divalent heavy metal ions (Zn2+, Ni2+, Cu2+, Pb2+, Cd2+, and Hg2+) on the activity of single epithelial Na+ channels (ENaC) in a renal epithelial cell line (A6). None of the cations changed the single-channel conductance. However, ENaC activity [measured as the number of channels (N) x open probability (P-o)] was decreased by Cd2+ and Hg2+ and increased by Cu2+, Zn2+, and Ni2+ but was not changed by Pb2+. Of the cations that induced an increase in Na+ channel function, Zn2+ increased N, Ni2+ increased P-o, and Cu2+ increased both. The cysteine modification reagent [2-(trimethylammonium) ethyl] methanethiosulfonate bromide also increased N, whereas diethylpyrocarbonate, which covalently modifies histidine residues, affected neither P-o nor N. Cu2+ increased N and stimulated P-o by reducing Na+ self-inhibition. Furthermore, we observed that ENaC activity is slightly voltage dependent and that the voltage dependence of ENaC is insensitive to extracellular Na+ concentration; however, apical application of Ni2+ or diethylpyrocarbonate reduced the channel voltage dependence. Thus the voltage sensor of Xenopus ENaC is different from that of typical voltagegated channels, since voltage appears to be sensed by histidine residues in the extracellular loops of ENaC, rather than by charged amino acids in a transmembrane domain.