Divalent cations regulate epithelial Na channel (ENaC) activity in A6 cells

Abstract

Introduction Heavy metals are known to affect Na+ transport across epithelial cells, but the specific effects of heavy metals on single epithelial Na channels (ENaC) are not known. We tested six divalent cations (Cd2+, Hg2+, Pb2+, Cu2+, Zn2+, and Ni2+) on ENaC activity in A6 cells. Methods Metals were applied by inclusion in pipette solution; the single channel activity was recorded by patch clamp in a cell-attached configuration with channels exposed to metals compared to untreated channels. Results Pb2+ which inhibits neuronal Ca and K channels has no significant effect on ENaC, the metals with thiol affinity, Cd2+ and Hg2+, strongly inhibit ENaC activity. The Cu2+, Zn2+ and Ni2+ increase ENaC activity, but by different mechanisms. Zn2+ increases the number of active channels; Ni2+ increases channel open probability, Cu2+ increases both. We also observed that the activity of ENaC is voltage dependent, but exposure to Ni2+ or the His modifying reagent, diethyl pyrocarbonate (DEPC), attenuates this voltage dependence. The effect of Zn2+ is imitated by the thiol affinity reagent, [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), but DEPC or MTSET only partially mimiced the effects of Ni2+ or Cu2+. Conclusions The effects of different metals on ENaC activity imply that the binding sites for these metals in the extracellular loops of ENaC are different. Since MTSET and DEPC partially imitate the effects of some metals suggests that His and Cys are involved with metal binding, but the different effects of Cd2+ and Hg2+ from that of MTSET may imply that these two metal ions interact with structures deep within the channel inaccessible to MTSET. Supported by DK 37963 and DK 061521