Quantitative fluorescence measurement of chloride transport mechanisms in phospholipid vesicles

A quantitative fluorescence assay has been developed to measure Cl flux across liposomal membranes for use in chloride transporter reconstitution studies. A Cl-sensitive fluorophore [6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ] was entrapped into phospholipid/cholesterol liposomes formed by bath sonication, high-pressure extrusion, and detergent dialysis. Liposomes containing entrapped SPQ were separated from external SPQ by passage down a Sephadex G25 column. There was <10% leakage of SPQ from liposomes in 8 h at 4°C and in 2 h at 23°C. Cl influx (J_Cl in millimolar per second or nanomoles per second per centimeter squared) was determined from the time course of SPQ fluorescence, measured by cuvette or stopped-flow fluorometry, in response to inward Cl gradients. In 90% phosphatidylcholine/10% cholesterol liposomes at 23°C, J_Cl in response to a 50 mM inward Cl gradient was 0.06 ± 0.01 mM·s^-1 (SD, n = 3) in the absence and 0.27 ± 0.02 mM·s^-1 in the presence of a K/valinomycin voltage clamp (0 mV), showing that the basal Cl "leak" is conductive; J_Cl increased (1.7 ± 0.1)-fold in the presence of a 60-mV inside-positive diffusion potential. Accuracy of chloride influx rates determined by the SPQ method was confirmed by measurement of ³⁶Cl uptake. In liposomes voltage-clamped to 0 mV, J_Cl was linear with external [Cl] (0-100 mM), independent of pH gradients, and strongly dependent on temperature (activation energy 18 ± 1 kcal/mol, 12-42°C) as predicted for channel-independent Cl diffusion. To test this method for measurement of rapid Cl transport rates, liposomes were reconstituted with the Cl/OH exchanger tributyltin. Tributyltin incorporation gave rapid, pH gradient driven Cl influx (J_Cl = 13 mM·s^-1, no pH gradient; 24 mM·s^-1, 1.5-unit pH gradient). These results establish a rapid and accurate method for measurement of Cl influx in liposomes suitable for reconstitution studies.