Inactivation of the Rabbit Parotid Na/K/Cl Cotransporter by N-ethylmaleimide

The inactivation of the rabbit parotid Na/K/Cl cotransporter by the irreversible sulfhydryl reagent N-ethylmaleimide (NEM) is studied by monitoring its effect on high affinity bumetanide binding to the carrier. NEM reduces the number of bumetanide binding sites with no significant change in the affinity of those remaining. NEM also reduces KCl-dependent 22Na flux via the cotransporter by the same factor as the reduction in bumetanide binding sites. Both bumetanide and its analogue furosemide can protect against the effect of NEM. The concentration range over which this protection occurs is in good agreement with affinities of these two compounds for the high affinity bumetanide binding site (2.6 and 8.5 microM, respectively), indicating an association of this site with the site of action of NEM. Also consistent with this hypothesis are the observations that (i) sodium and potassium, both of which are required for high affinity bumetanide binding, increase the rate of inactivation of binding by NEM and (ii) chloride, at concentrations previously shown to competitively inhibit bumetanide binding, protects the cotransporter against NEM. The effects of NEM on bumetanide binding are mimicked by another highly specific sulfhydryl reagent, methyl methanethiolsulfonate. The apparent rate constant for inactivation of high affinity bumetanide binding by NEM is a hyperbolic function of NEM concentration consistent with a model in which the inactivation reaction is first order in [NEM] and proceeds through an intermediate adsorptive complex. The data indicate that the presence of a reduced sulfhydryl group at or closely related to the bumetanide binding site is essential for the operation of the parotid Na/K/Cl cotransporter.