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 less than 10% leakage of SPQ from liposomes in 8 h at 4 degrees C and in 2 h at 23 degrees C. Cl influx (JCl 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 degrees C, JCl 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; JCl 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, JCl 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 degrees C) as predicted for channel-independent Cl diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Cardiovascular Research Institute, University of California, San Francisco 94143.
A S
AS
Verkman
R
R
Takla
B
B
Sefton
C
C
Basbaum
J H
JH
Widdicombe
D001693:Biological Transport, Active; D002712:Chlorides / Q000378:metabolism*; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008081:Liposomes; D010743:Phospholipids; D011806:Quinolinium Compounds; D013050:Spectrometry, Fluorescence
D002712:Chlorides; D005456:Fluorescent Dyes; D008081:Liposomes; D010743:Phospholipids; D011806:Quinolinium Compounds; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium