Quantitative fluorescence measurement of chloride transport mechanisms in phospholipid vesicles

A. S. Verkman, R. Takla, B. Sefton, C. Basbaum, Jonathan Widdicombe

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

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 (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°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°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 (JCl = 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.

Original languageEnglish (US)
Pages (from-to)4240-4244
Number of pages5
JournalBiochemistry
Volume28
Issue number10
StatePublished - 1989
Externally publishedYes

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Liposomes
Chlorides
Phospholipids
Fluorescence
Proton-Motive Force
Cholesterol
Valinomycin
Fluorometry
Sonication
Dialysis
Fluorophores
Clamping devices
Electric potential
Phosphatidylcholines
Baths
Detergents
Extrusion
Assays
Activation energy
Fluxes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Quantitative fluorescence measurement of chloride transport mechanisms in phospholipid vesicles. / Verkman, A. S.; Takla, R.; Sefton, B.; Basbaum, C.; Widdicombe, Jonathan.

In: Biochemistry, Vol. 28, No. 10, 1989, p. 4240-4244.

Research output: Contribution to journalArticle

Verkman, AS, Takla, R, Sefton, B, Basbaum, C & Widdicombe, J 1989, 'Quantitative fluorescence measurement of chloride transport mechanisms in phospholipid vesicles', Biochemistry, vol. 28, no. 10, pp. 4240-4244.
Verkman, A. S. ; Takla, R. ; Sefton, B. ; Basbaum, C. ; Widdicombe, Jonathan. / Quantitative fluorescence measurement of chloride transport mechanisms in phospholipid vesicles. In: Biochemistry. 1989 ; Vol. 28, No. 10. pp. 4240-4244.
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N2 - 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 (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°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°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 (JCl = 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.

AB - 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 (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°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°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 (JCl = 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.

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