IDENTIFICATION OF ORGANIC CATION TRANSPORTER IN RAT RENAL BRUSH-BORDER MEMBRANE BY PHOTOAFFINITY-LABELING

As an approach to identification of the organic ration transport syste m in brush-border membranes, we designed a photoaffinity probe, (5-met hyl-4-imidazolyl)methyl]thio]ethyl]guanidine ([H-3]AMC) based on the m olecular structure of cimetidine, which is taken up by the organic cat ion transport system in brush-border membrane vesicles. The effect of nonradioactive (5-methyl-4-imidazolyl)methyl]thio]ethyl]guanidine (AMC ) on tetraethylammonium uptake was investigated in rat renal brush-bor der membrane vesicles. We examined the photolysis of AMC in which the azido group was converted to an active nitrene group using UV light at a wavelength of 254 nm and established a half-life of 7 s. This half- life duration did not significantly impair brush-border membrane vesic les during the exposure to light for photo-labeling. Photoaffinity lab eling of brush-border membrane vesicles from the rat renal cortex with [H-3]AMC resulted in the covalent incorporation of radioactivity into membrane polypeptides; an apparent 36 kDa polypeptide was predominant ly labeled. Photolabeling specificity was shown by a reduction in the labeling of the 36 kDa polypeptide in the presence of organic cations, cimetidine, tetraethylammonium and N-methylnicotinamide whereas the o rganic anion, furosemide, had no effect on labeling patterns. These da ta demonstrate that AMC, as well as organic cations, cimetidine, tetra ethylammonium and N-methylnicotinamide, interact with a common 36 kDa membrane polypeptide, which may be the transport system or one of its brush-border membrane components.