Interaction and inactivation of the rabbit parotid basolateral sodium/potassium/chloride cotransport protein with N-ethylmaleimide

Inactivation of the rabbit parotid Na$\sp+$/K$\sp+$/Cl$\sp-$ cotransporter by the irreversible sulfhydryl reagent N-ethylmaleimide (NEM) was studied by monitoring its effect on high affinity bumetanide (3-Aminosulfonyl-5-butylamino-4-phenoxybenzoic acid) binding (K$\sb{\rm d}$ $\simeq$ 3 $\mu$M) to the carrier. NEM reduces the number of bumetanide binding sites without changing the affinity of the remaining sites. $\sp{22}$Na flux via the cotransporter was reduced by the same factor as the reduction in bumetanide binding sites. Bumetanide at concentrations $\simeq$ K$\sb{\rm d}$ protected against inactivation by NEM, indicating an association of the high affinity bumetanide binding site with the site of action of NEM. Additionally, sodium and potassium, both required for bumetanide binding, increased the rate of inactivation of binding by NEM. Chloride, a competitive inhibitor of bumetanide binding, protected the cotransporter against NEM. The results indicate that the presence of a reduced sulfhydryl group at or closely associated with the bumetanide binding site is essential for the operation of the parotid Na$\sp+$/K$\sp+$/Cl$\sp-$ cotransporter.