Solubilization of thiazide diuretic receptors from rat kidney membranes.
Thiazide-type diuretics act at receptors to inhibit NaCl transport in the renal distal tubule. We solubilized high-affinity [3H]metolazone binding sites from rat kidney membranes with Triton X-100, which was more effective than several other detergents. Phosphatidylcholine and a mixture of proteinase inhibitors were needed to stabilize binding so that 57% of solubilized binding remained after 72 h at 4 degrees C. The affinities of solubilized (Kd = 11.4 +/- 0.5 nM) and membrane-bound receptors (Kd = 12.0 +/- 1.7 nM) were similar. The maximal number of binding sites/mg protein of solubilized receptors was 46 +/- 3% (n = 5) of membrane-bound receptors. Diuretics with a wide range of affinities had similar affinities for the solubilized and membrane-bound sites. Chloride inhibited and sodium stimulated the binding of [3H]metolazone to solubilized receptors, as they do with membrane-bound receptors. These studies demonstrate that, as judged by ligand binding, thiazide receptors can be solubilized in an active conformation and provide the basis for future purification and reconstitution.