Biochimica et Biophysica Acta 862 (1986) 285-293 285 Elsevier BBA73295 Localization of cyanine dye binding to brush-border membranes by quenching of n-(9-anthroyloxy) fatty acid probes G. Cabrini and A.S. Vcrkman Department of Medicine and Division of Nephrologv, Cardiovascular Research Institute, Universi(v of California, San Francisco, CA (U.S.A.) (Received10 April 1986) (Revised manuscript received 3 July 1986) Key words: Cyaninedye; Fluorescence quenching; Stopped-flow;Brush-bordermembrane; Anthroyloxyst6aric acid The location and orientation of 3,3'-dipropylthiodicarbocyanine (diS-C3-(5)) binding sites in renal brush- border membrane vesicles was examined from the quenching of n-(9-anthroyioxy) fatty acid (n-AS) fluorescence. Based on previous kinetic studies (Cabrini, G. and Verkman, A.S. (1986) J. Membrane Biol. 90, 163-175) monomeric aqueous diS-C3-(5) binds to brush-border membrane vesicles (BBMV) by an initial 6 ms association to form bound monomer, a 30-40 ms equilibrium between bound monomer (M) and bound dimer (D), and a 1-1.3 s translocation of D from the outer to inner membrane leaflet. Based on Stern-Volmer and lifetime analyses, M and D quench the fluorescence of the n-AS probes by a collisional mechanism. At low [diS-C3-(5)]/[BBMV] (R), where M predominates, the n-AS quenching efficiencies (Q) are similar (n = 2-16); at high R, where D predominates, Q increases with n (16 > 12>>>6 > 2), suggesting that M is oriented parallel, and D perpendicular, to the phospholipid chains deep within the membrane. Mixture of diS-C3-(5) with brush-border membrane vesicles containing n-AS in a stopped-flow apparatus gave a biexponential fluorescence decrease (excitation 390 nm, emission above 450 nm) with time constants 30-40 ms and 1-1.5 s; there was no 6 ms quenching process. These findings are incorporated into a model in which diS-C3-(5) adheres loosely to the outer membrane surface in 6 ms, binds parallel to the membrane phospholipid in 30-40 ms, dimerizes and rotates by 90 ° in much less than 30 ms, and translocates to the opposite half of the bilayer in 1-1.5 s. Introduction The potential-sensitive carbocyanine dye diS- C3-(5 ) has been used extensively to study static and time-dependent membrane potentials in artifi- cial liposomes [1], cells [2], mitochondria [3], and Abbreviations: diS-C3-(5), 3,3'-dipropylthiocarbocyanine iodide; n-AS, n-(9-anthroyloxy)stearic acid; 16-AP, 16-(9-an- throyloxy)palmitic acid; (Me)2POPOP, 1,4-bis-2-(4-methyl-5- phenyloxazolyl)benzene; Mes, 2-(N-morpholino)ethanesulfonic acid. Correspondence address: Dr. Alan S. Verkman, 1065 Health Sciences East Tower, Division of Nephrology, University of California, San Francisco, CA 94143-0532, U.S.A. membrane vesicles isolated from sarcoplasmic re- ticulum [4] and renal proximal tubule [5-7]. DiS- C3-(5) is particularly useful to study ion transport in membrane vesicles because of its relatively rapid response to changes in membrane potential (less than 1 s) and its large signal intensity of up to 50% for a 100 mV change in membrane potential. The location and orientation of diS-C3-(5 ) binding sites within biological membranes, and the precise mechanism by which diS-C3-(5 ) responds to changes in membrane potential in vesicle systems have not been established. In the renal brush-border membrane vesicle, we have previously shown that aqueous diS-C3-(5 ) is 0005-2736/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)