Morris Ryan G, Schafer James A
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
J Gen Physiol. 2002 Jul;120(1):71-85. doi: 10.1085/jgp.20018547.
Antidiuretic hormone and/or cAMP increase Na(+) transport in the rat renal collecting duct and similar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was undertaken to determine if that increment in Na(+) transport could be explained quantitatively by an increased density of ENaC Na(+) channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retrovirally transfected with rat alpha-, beta-, and gammaENaC subunits, each of which were labeled with the FLAG epitope in their extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and B.C. Rossier. 1996. PROC: Natl. Acad. Sci. USA. 93:15370-15375). The density of ENaC subunits was quantified by specific binding of (125)I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4-8 nM. Transepithelial Na(+) transport was measured as the amiloride-sensitive short-circuit current (AS-I(sc)) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS-I(sc) measured in the same experiments. Stimulation with cAMP (20 microM 8-p-chlorothio-cAMP plus 200 microM IBMX) significantly increased AS-I(sc) from 11.2 +/- 1.3 to 18.1 +/- 1.3 microA/cm(2). M2 binding (at 1.7 nM M2) increased in direct proportion to AS-I(sc) from 0.62 +/- 0.13 to 1.16 +/- 0.18 fmol/cm(2). Based on the concentration dependence of M2 binding, the quantity of Na(+) channels per unit of AS-I(sc) was calculated to be the same in the presence and absence of cAMP, 0.23 +/- 0.04 and 0.21 +/-0.05 fmol/microA, respectively. These values would be consistent with a single channel conductance of approximately 5 pS (typically reported for ENaC channels) only if the open probability is <0.02, i.e., less than one-tenth of the typical value. We interpret the proportional increases in binding and AS-I(sc) to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the I(sc) increase produced by cAMP.
抗利尿激素和/或环磷酸腺苷（cAMP）可增加大鼠肾集合管及类似上皮组织（包括培养的马-达二氏犬肾（MDCK）细胞单层）中的钠离子转运。本研究旨在确定钠离子转运的增加是否可以通过顶端膜上上皮钠通道（ENaC）密度的增加来定量解释。将无内源性ENaC表达的MDCK细胞用大鼠α、β和γ ENaC亚基进行逆转录病毒转染，如前所述，每个亚基在其细胞外环均用FLAG表位标记（菲尔索夫，D.，L. 席尔德，I. 高施，A.-M. 梅里拉特，E. 施内贝格尔，和B.C. 罗西耶。1996年。《美国国家科学院院刊》。93:15370 - 15375）。通过将（125）I标记的抗FLAG抗体（M2）与顶端膜的特异性结合来定量ENaC亚基的密度，发现其是M2浓度的饱和函数，在4 - 8 nM时达到半数最大结合。跨上皮钠离子转运通过测量生长在可渗透支持物上的MDCK细胞的氨氯地平敏感短路电流（AS - I(sc)）来测定。在相同实验中，特异性M2结合与AS - I(sc)呈正相关。用cAMP（20 μM 8 - 对氯硫代 - cAMP加200 μM异丁基甲基黄嘌呤）刺激后，AS - I(sc)从11.2±1.3显著增加至18.1±1.3 μA/cm²。M2结合（在1.7 nM M2时）与AS - I(sc)成正比增加，从0.62±0.13增加至1.16±0.18 fmol/cm²。根据M2结合的浓度依赖性，计算出在有和无cAMP时每单位AS - I(sc)的钠离子通道数量相同，分别为0.23±0.04和0.21±0.05 fmol/μA。仅当开放概率<0.02，即小于典型值的十分之一时，这些值才与约5 pS的单通道电导（ENaC通道通常报道的值）一致。我们将结合和AS - I(sc)的成比例增加解释为表明顶端膜中ENaC亚基密度的增加可以完全解释cAMP产生的I(sc)增加。
