UTILIZATION OF MONENSIN FOR DETECTION OF MICRODOMAINS IN CHOLESTEROL CONTAINING MEMBRANE

The effect of cholesterol on the monensin mediated proton-cation excha nge reaction was measured in the time-resolved domain. The experimenta l system consisted of a black lipid membrane equilibrated with monensi n (Nachliel, E., Finkelstein, Y, and Gutman, M. (1996) Biochim. Biophy s. Acta 1285, 131-145). The membrane separated two compartments contai ning electrolyte solutions and pyranine (8-hydroxypyrene 1,3,6-trisulf onate) was added on to one side of the membrane. A short laser pulse w as used to cause a brief transient acidification of the pyranine-conta ining solution and the resulting electric signal, derived from proton- cation exchange, was measured in the microsecond time domain. Incorpor ation of cholesterol had a clear effect on the electric transients as measured with Na+ or K+ as transportable cations. The measured transie nts were subjected to rigorous analysis based on numeric integration o f coupled, non-linear, differential rate equations which correspond wi th the perturbed multi-equilibria state between all reactants present in the system. The various kinetic parameters of the reaction and thei r dependence on the cholesterol content had been determined. On the ba sis of these observations we can draw the following conclusions: (1) C holesterol perturbed the homogeneity of the membrane and microdomains were formed, having a composition that differed from the average value . The ionophore was found in domains which were practically depleted o f phosphatidylserine. (2) The diffusivity of the protonated monensin ( MoH) was not affected by the presence of cholesterol, indicating that the viscosity of the central layer of the membrane was unaltered. (3) The diffusivity of the monensin metal complexes (MoNa and MoK) was sig nificantly increased upon addition of cholesterol. As the viscosity al ong the cross membranal diffusion route is unchanged, the enhanced mot ion of the MoNa and MoK is attributed to variations of the electrostat ic potential within the domains.