Hogan E M, Davis B A, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
J Gen Physiol. 1997 Nov;110(5):629-39. doi: 10.1085/jgp.110.5.629.
We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl-. The purpose of the present study was to determine (a) whether Cl- plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl-]i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl-- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na+ and 0-194 mM Cl-. The artificial seawater bathing the BMF initially contained no Na+. After dialysis was halted, adding 50 mM Na+ to the artificial seawater caused an amiloride-sensitive pHi increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (JNa-H) increased with increasing [Cl-]i under both normo- and hypertonic conditions, with similar apparent Km values ( approximately 120 mM). However, the maximal JNa-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pHi requires Cl- under both normo- and hypertonic conditions, but at any given [Cl-]i, JNa-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl-]i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl--dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl--free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl-, dialyzing with GTPgammaS or AlF3, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl--depleted fibers, the Cl--dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF3 indicates that the G protein is a heterotrimeric G protein.
我们之前发现，在高渗溶液（1600 mosM/kg）中使藤壶肌纤维（BMF）收缩会刺激一种对氨氯吡咪敏感的钠氢交换体。这种激活由G蛋白介导，且需要细胞内的氯离子。本研究的目的是确定：（a）在等渗条件（975 mosM/kg）下，氯离子是否在钠氢交换的激活中发挥作用；（b）在等渗和高渗条件下，激活该交换体所需的细胞内氯离子浓度的剂量依赖性；（c）氯离子依赖性步骤和G蛋白依赖性步骤的相对顺序。我们通过用不含钠离子且氯离子浓度为0 - 194 mM的pH 6.5透析液对BMF进行内部透析来使其酸负荷增加。最初，浸泡BMF的人工海水中不含钠离子。透析停止后，向人工海水中添加50 mM钠离子会在等渗和高渗条件下引起对氨氯吡咪敏感的细胞内pH值升高。在等渗和高渗条件下，计算得出的钠氢交换通量（JNa-H）均随着细胞内氯离子浓度的增加而增加，表观米氏常数（Km）值相似（约120 mM）。然而，在高渗条件下，最大JNa-H增加了近90%。因此，在低细胞内pH值时，等渗和高渗条件下钠氢交换的激活都需要氯离子，但在任何给定的细胞内氯离子浓度下，高渗条件下的JNa-H都高于等渗条件。我们得出结论，细胞内氯离子浓度的增加不是主要的收缩信号，但可能作为辅助收缩信号。为了确定氯离子依赖性步骤是否在G蛋白依赖性步骤之后，我们将BMF预透析至无氯离子状态，然后试图通过激活G蛋白来刺激钠氢交换。我们发现，即使在没有氯离子的情况下，用GTPγS或AlF3透析，或注射霍乱毒素，都能刺激钠氢交换。由于在对照的氯离子缺失纤维中不存在钠氢交换活性，所以在收缩信号转导途径中，氯离子依赖性步骤在G蛋白处或之前。AlF3的刺激表明该G蛋白是一种异源三聚体G蛋白。
