Bicarbonate Conductance and pH Regulatory Capability of Cystic Fibrosis Transmembrane Conductance Regulator
The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl-channel regulated by protein kinase A. The most common mutation in cystic fibrosis (CF), deletion of Phe-508 (ΔF508-CFTR), reduces Cl-secretion, but the fatal consequences of CF have been difficult to rationalize sole...
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Published in | Proceedings of the National Academy of Sciences - PNAS Vol. 91; no. 12; pp. 5340 - 5344 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
National Academy of Sciences of the United States of America
07.06.1994
National Acad Sciences National Academy of Sciences |
Subjects | |
Online Access | Get full text |
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Summary: | The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl-channel regulated by protein kinase A. The most common mutation in cystic fibrosis (CF), deletion of Phe-508 (ΔF508-CFTR), reduces Cl-secretion, but the fatal consequences of CF have been difficult to rationalize solely in terms of this defect. The aim of this study was to determine the role of CFTR in HCO-
3transport across cell membranes. HCO-
3permeability was assessed from measurements of intracellular pH [pHi; from spectrofluorimetry of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5-(and -6)carboxyfluorescein] and of channel activity (patch clamp; cell attached and isolated, inside-out patches) on NIH 3T3 fibroblasts and C127 mammary epithelial cells transfected with wild-type CFTR (WT-CFTR) or ΔF508-CFTR, and also on mock-transfected cells. When WT-CFTR-transfected cells were acidified (pulsed with NH4Cl) and incubated in Na+-free (N-methyl-D-glucamine substitution) solutions (to block Na+-dependent pHiregulatory mechanisms), pHiremained acidic (pH ≈ 6.5) until the cells were treated with 20 μM forskolin (increases cellular [cAMP]); pHithen increased toward (but not completely to) control level (pHi7.2) at a rate of 0.055 pH unit/min. Forskolin had no effect on rate of pHirecovery in ΔF508 and mock-transfected cells. This Na+-independent, forskolin-dependent pHirecovery was not observed in HCO-
3/CO2-free medium. Forskolin-treated WT-CFTR-transfected (but not ΔF508-CFTR or mock-transfected) cells in Cl--containing, HCO-
3-free solutions showed Cl-channels with a linear I/V relationship and a conductance of 10.4 ± 0.5 pS in symmetrical 150 mM Cl-. When channels were incubated with different [Cl-] and [HCO-
3] on the inside and outside, the Cl-/HCO-
3permeability ratio (determined from reversal potentials of I/V curves) was 3.8 ± 1.0 (mean ± SEM; n = 9); the ratio of conductances was 3.9 ± 0.5 (at 150 mM Cl-and 127 mM HCO-
3. We conclude that in acidified cells the WT-CFTR functions as a base loader by allowing a cAMP-dependent influx of HCO-
3through channels that conduct HCO-
3about one-quarter as efficiently as it conducts Cl-. Under physiological conditions, the electrochemical gradients for both Cl-and HCO-
3are directed outward, so CFTR likely contributes to the epithelial secretion of both ions. HCO-
3secretion may be important for controlling pH of the luminal, but probably not the cytoplasmic, fluid in CFTR-containing epithelia. In CF, a decreased secretion of HCO-
3may lead to decreased pH of the luminal fluid. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.91.12.5340 |