Identification of a Na+,K+, Cl--cotransport protein of Mr 34 000 from kidney by photolabeling with [3H]bumethanide. The protein is associated with cytoskeleton components

Abstract

A polypeptide of Mr 34 000 is photolabeled with [3H]bumethanide after binding of this drug to membranes from the outer renal medulla and irradiation at 345 nm, a wavelength where bumethanide has an absorption maximum. Our data show that the polypeptide of Mr 34 000 is a component of the Na+/K+/Cl--cotransport system. The [3H]bumethanide binding protein is not extracted by concentrations of the nonionic detergent C12E8 that solubilizes 67% of the protein of the membranes including (Na+ + K+)-ATPase. This step increases the capacity for binding of [3H]bumethanide to 681 pmol/mg protein. Extraction of the binding protein requires high ionic strength suggesting that the Na+/K+/Cl--cotransport protein is associated with cytoskeleton components. This association may be important for control of the entry of NaCl into the cytoplasm and for cellular regulation of the rate of active transport of NaCl across the tubule cells in the thick ascending limb of Henles loop.