Effects of NEM on voltage-activated chloride conductance in toad skin

The regulation of the voltage-activated chloride current conductance (G(Cl)) in toad skin was investigated by the use of the SH reagents N-ethylmaleimide (NEM) and p-chloro-mercuricbenzenesulfonic acid PCMBS. This anion pathway is controlled by a voltage-sensitive gating regulator, Mucosal application of NEM decreased the voltage-activation in a time and concentration dependent manner, half-maximal inhibition being exerted at a concentration of 30 mu M within 20 min. At concentrations higher than 100 mu M, the voltage-activated G(Cl) was near-completely and irreversibly inhibited in less than 10 min. Resting, deactivated conductance was essentially unaffected, NEM had no effect on active sodium transport (measured as I-SC) under conditions, which fully dissipated the voltage-activated G(Cl). After complete inhibition of the voltage-activated G(Cl) with NEM, chloride conductance could still be stimulated by CPT-cAMP as in control tissues. Under these conditions, NEM at concentrations above 1 mM decreased G(Cl) reversibly. Mucosal application of PCMBS at 500 mu M inhibited the activated conductance by 35%, which was slightly reversible. Inhibition of voltage-activated G(Cl), which was observed after mucosal addition of the membrane-impermeable NEM analogue, eosin-5-maleimide, was completely reversible after washout, This suggests that the binding site for the maleimide is not accessible from the external face of the apical membrane. Brief application of NEM at lower concentrations (1-3 min, less than or equal to 100 mu M) led to partial inhibition of G(Cl), followed by occasionally complete recovery upon washout of NEM. Recovery of voltage-activated G(Cl) was progressively attenuated and eventually disappeared after subsequent brief applications of NEM. This could reflect recruitment of permeation/control sites from a finite pool. The data are discussed in the frame of a working model for the voltage-activated Cl--pathway, that contains two principle components, i.e., an anion-selective permeation path which is controlled by regulatory protein(s).