BICARBONATE CONDUCTANCE AND PH REGULATORY CAPABILITY OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR
BICARBONATE CONDUCTANCE AND PH REGULATORY CAPABILITY OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR
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DOI：
10.1073/pnas.91.12.5340
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发表时间：
1994-06-07
影响因子：
11.1
通讯作者：
MACHEN, TE
中科院分区：
文献类型：
--
作者：
POULSEN, JH;FISCHER, H;MACHEN, TE
囊性纤维化跨膜传导调节因子（CFTR）是一种由蛋白激酶A调节的上皮细胞Cl-通道。囊性纤维化（CF）中最常见的突变是Phe-508缺失（Delta F508-CFTR），可减少Cl-分泌，但CF的致命性后果难以仅根据该缺陷合理化。本研究的目的是确定CFTR在HCO 3-跨细胞膜转运中的作用。通过测量细胞内pH [pH（i）;通过荧光分光光度法测定pH敏感染料2 '，7'-bis（2-羧乙基）-5-（和-6）羧基荧光素]和通道活性（膜片钳;细胞附着和分离，由内而外的贴片）在用野生型CFTR（WT-CFTR）或Delta F508-CFTR转染的NIH 3 T3成纤维细胞和C127乳腺上皮细胞上，以及模拟转染的细胞。当WT-CFTR转染的细胞被酸化时，（用NH 4Cl脉冲），并在无Na+（N-甲基-D-葡糖胺取代）溶液（阻断Na+依赖性pH（i）调节机制），pH（i）保持酸性（pH约为6.5），直到用20 μ M毛喉素处理细胞（增加细胞[cc]）; pH（i）随后增加至在0.055 pH单位/分钟的速率下，毛喉素对pH（i）的速率没有影响，Delta F508和模拟转染细胞中的回收率。在不含HCO 3-/CO2的培养基中未观察到这种Na+非依赖性、毛喉素依赖性pH（i）恢复。毛喉素处理的WT-CFTR转染的（但不是Delta F508-CFTR或模拟转染的）细胞在含Cl-，无HCO 3-的溶液中显示Cl-通道具有线性I/V关系，在对称的150 mM Cl-中电导为10.4 +/- 0.5 pS。当通道在内部和外部用不同的[Cl-]和[HCO 3-]孵育时，Cl-/HCO 3-渗透率比（由I/V曲线的反转电位确定）为3.8 +/- 1.0（平均值+/- SEM; n = 9）;电导率比为3.9 +/- 0.5（在150 mM Cl-和127 mM HCO;）。我们的结论是，在酸化细胞的WT-CFTR的功能作为一个基地的加载器，允许cAMP依赖性的流入HCO 3-通过通道进行HCO 3-约四分之一的效率，因为它进行Cl-。在生理条件下，Cl-和HCO 3-的电化学梯度都是向外的，因此CFTR可能有助于上皮分泌这两种离子。HCO 3-分泌可能是重要的控制pH值的管腔，但可能不是细胞质，流体CFTR含有上皮细胞。在CF中，HCO 3-分泌减少可能导致腔液pH值降低。
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 [pH(i); 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 pH(i) regulatory mechanisms), pH(i) remained acidic (pH approximate to 6.5) until the cells were treated with 20 mu M forskolin (increases cellular [cc]); pH(i) then increased toward (but not completely to) control level (pH(i) 7.2) at a rate of 0.055 pH unit/min. Forskolin had no effect on rate of pH(i) recovery in Delta F508 and mock-transfected cells. This Na+-independent, forskolin-dependent pH(i) recovery was not observed in HCO3-/CO2-free medium. Forskolin-treated WT-CFTRtransfected (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 HCO;. 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.