Photoaffinity labelling of a 150 kDa (Na + K + Cl)-cotransport protein from duck red cells with an analog of bumetanide

Abstract

We have used a radiolabelled, benzophenone analog of bumetanide, 4-[ 3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ([ 3H]BSTBA) to photolabel plasma membranes from duck red blood cells. BSTBA, like bumetanide, is a loop diuretic and a potent inhibitor of (Na + K + Cl) cotransport, and ([ 3H]BSTBA) binds to intact duck red cells with a high affinity similar to that of [ 3H]bumetanide ( K 1 2 ≌ 0.1 gmM ). We incubated duck red cells with ([ 3H]BSTBA), then lysed the cells and exposed the ghosts to ultraviolet light. The ghosting and photolysis was done at 0°C to prevent dissociation of the ([ 3H]BSTBA). The ghosts were then sonicated to remove the nuclei and run on SDS-polyacrylamide gels. Analysis of H 2O 2-digested gel slices revealed ([ 3H]BSTBA) to be incorporated into a protein of approx. 150 kDa. This is the same molecular weight we obtain for a protein from dog kidney membranes which is photolabelled by ([ 3H]BSTBA) in a manner highly consistent with labelling of the (Na + K + Cl) cotransporter (Haas and Forbush (1987) Am. J. Physiol. 253, C243-C252). Several lines of evidence strongly suggest that the 150 kDa protein from duck red cell membranes is an integral component of the (Na + K + Cl)-cotransport system in these cells: (1)Photolabelling of this protein by [ 3H]BSTBA is blocked when μM unlabelled bumetanide is included in the initial incubation medium with [ 3H]BSTBA; (2) Photoincorporation of [ 3H]BSTBA into the 150 kDa protein is markedly increased when the initial incubation medium is hypertonic or contains norepinephrine, conditions which similarly stimulate both (Na + K + Cl) cotransport and saturable [ 3H]bumetanide binding in duck red cells; (3) The photolabelling of this protein shows a saturable dependence on [ 3H]BSTBA concentration, with a K 1 2 (0.06 μM) similar to that for the reversible, saturable binding of [ 3H]BSTBA and [ 3H]bumetanide to duck red cells; and (4) [ 3H]BSTBA photoincorporation into the 150 kDa protein, like saturable [ 3H]bumetanide binding to intact cells, requires the simultaneous presence of Na +, K +, and Cl − in the medium containing the radiolabelled diuretic.