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 language | English (US) |
|---|---|
| Pages (from-to) | 4240-4244 |
| Number of pages | 5 |
| Journal | Biochemistry |
| Volume | 28 |
| Issue number | 10 |
| State | Published - 1989 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - Quantitative fluorescence measurement of chloride transport mechanisms in phospholipid vesicles
AU - Verkman, A. S.
AU - Takla, R.
AU - Sefton, B.
AU - Basbaum, C.
AU - Widdicombe, Jonathan
PY - 1989
Y1 - 1989
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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M3 - Article
VL - 28
SP - 4240
EP - 4244
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 10
ER -