Role of phospholipids in the binding of bumetanide to the rabbit parotid Na/K/Cl cotransporter

Summary It was recently reported (Turner, R.J., George, J.N., 1990,J. Membrane Biol.113:203–210) that the high affinity bumetanide binding site of the rabbit parotid Na/K/Cl cotransporter could be extracted from a basolateral membrane preparation from this gland using relatively low concentrations of the non-ionic detergent Triton X-100. At the detergent: protein ratios required for complete membrane solubilization bumetanide binding activity in this extract was lost but could be recovered by the addition of crude soybean lipids. In the present paper the ability of various purified lipids to restore high affinity bumetanide binding activity in detergent solubilized rabbit parotid basolateral membranes is studied. We show that the effect of exogenous lipid on the detergent-inactivated bumetanide binding site is to increase the affinity of binding without affecting the number of binding sites. Of the 11 lipid species tested, several relatively minor, negatively charged membrane phospholipids are the most effective in restoring binding activity (phosphatidylserine phosphatidylglycerol > phosphatidylinositol > cardiolipin). while the major mammalian plasma membrane lipid components phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol are without effect. In addition, we show that in the presence of these minor lipids the affinity of bumetanide binding is considerably increased over that observed in the native membrane (e.g.,K_d0.06 m in membranes extracted with 0.3% Triton and treated with 0.15% wt/vol phosphatidylserine,vs. K_d3 m in native basolateral membranes). This dramatic dependence of bumetanide binding affinity on the presence of certain lipid species suggests that the properties of the bumetanide binding proteinin situ may be quite dependent on the minor lipid content of the plasma membrane. This effect may account for the relatively large variations in bumetanide binding affinity observed from tissue to tissue.