Poulsen J H, Fischer H, Illek B, Machen T E
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5340-4. doi: 10.1073/pnas.91.12.5340.
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 (delta F508-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 HCO3- transport across cell membranes. HCO3- permeability 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 delta F508-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 pHi regulatory mechanisms), pHi remained acidic (pH approximately 6.5) until the cells were treated with 20 microM forskolin (increases cellular [cAMP]); pHi then increased toward (but not completely to) control level (pHi 7.2) at a rate of 0.055 pH unit/min. Forskolin had no effect on rate of pHi recovery in delta F508 and mock-transfected cells. This Na(+)-independent, forskolin-dependent pHi recovery was not observed in HCO3-/CO2-free medium. Forskolin-treated WT-CFTR-transfected (but not delta F508-CFTR or mock-transfected) cells in Cl(-)-containing, HCO3(-)-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 [HCO3-] on the inside and outside, the Cl-/HCO3- permeability 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 HCO3-. We conclude that in acidified cells the WT-CFTR functions as a base loader by allowing a cAMP-dependent influx of HCO3- through channels that conduct HCO3- about one-quarter as efficiently as it conducts Cl-. Under physiological conditions, the electrochemical gradients for both Cl- and HCO3- are directed outward, so CFTR likely contributes to the epithelial secretion of both ions. HCO3- secretion 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 HCO3- may lead to decreased pH of the luminal fluid.
囊性纤维化跨膜传导调节因子（CFTR）是一种受蛋白激酶A调节的上皮细胞氯离子通道。囊性纤维化（CF）最常见的突变是苯丙氨酸508缺失（ΔF508 - CFTR），它会减少氯离子分泌，但CF的致命后果仅用这一缺陷难以解释。本研究的目的是确定CFTR在跨细胞膜的碳酸氢根（HCO₃⁻）转运中的作用。通过测量细胞内pH值（pHi；使用pH敏感染料2',7'-双（2 - 羧乙基）- 5 -（和 - 6）羧基荧光素的荧光分光光度法）以及通道活性（膜片钳；细胞贴附式和内面向外式膜片）来评估HCO₃⁻通透性，实验对象包括转染了野生型CFTR（WT - CFTR）或ΔF508 - CFTR的NIH 3T3成纤维细胞和C127乳腺上皮细胞，以及mock转染细胞。当用氯化铵脉冲使转染WT - CFTR的细胞酸化，并在无钠（用N - 甲基 - D - 葡糖胺替代）溶液中孵育（以阻断钠依赖的pHi调节机制）时，pHi保持酸性（pH约为6.5），直到用20 μM福斯高林处理（增加细胞内[cAMP]）；此时pHi以0.055 pH单位/分钟的速率向（但未完全达到）对照水平（pHi 7.2）升高。福斯高林对转染ΔF508和mock转染细胞的pHi恢复速率没有影响。在无HCO₃⁻/CO₂的培养基中未观察到这种不依赖钠、依赖福斯高林的pHi恢复。在含氯、无HCO₃⁻的溶液中，用福斯高林处理的转染WT - CFTR（但不是转染ΔF508 - CFTR或mock转染）的细胞显示出具有线性I/V关系且在对称的150 mM Cl⁻中电导为10.4±0.5 pS的氯离子通道。当通道在膜内和膜外与不同的[Cl⁻]和[HCO₃⁻]孵育时，Cl⁻/HCO₃⁻通透性比值（由I/V曲线的反转电位确定）为3.8±1.0（平均值±标准误；n = 9）；电导比值为3.9±0.5（在150 mM Cl⁻和127 mM HCO₃⁻时）。我们得出结论，在酸化细胞中，WT - CFTR作为一种碱装载器发挥作用，通过允许HCO₃⁻以依赖cAMP的方式通过通道内流，该通道传导HCO₃⁻的效率约为传导Cl⁻的四分之一。在生理条件下，Cl⁻和HCO₃⁻的电化学梯度都指向细胞外，因此CFTR可能有助于两种离子的上皮分泌。HCO₃⁻分泌对于控制含CFTR上皮细胞管腔液（但可能不是细胞质液）的pH值可能很重要。在CF中，HCO₃⁻分泌减少可能导致管腔液pH值降低。
