Thiele I E, Hug M J, Hübner M, Greger R
Physiologisches Institut, Albert-Ludwigs-Universität, Freiburg, Deutschland.
Cell Physiol Biochem. 1998;8(1-2):61-74. doi: 10.1159/000016271.
The aim of this study was to examine whether the stable expression of wild-type cystic fibrosis transmembrane conductance regulator (CFTR) in Chinese hamster ovary (CHO) cells alters the properties of these cells towards hypotonic cell swelling and ATP. According to many previous studies this was not expected a priori, since overexpression of CFTR should not affect the conductive pathways upregulated by the purinergic agonist or cell swelling. Three types of CHO cells were examined: a control group of normal CHO cells; a group of CFTR-CHO cells stably expressing wild-type CFTR at high levels (CHO-CFTR), and a group delta F508-CFTR-CHO cells, stably expressing the frequent mutation delta F508 CFTR (CHO-delta F508). Whole cell patch-clamp studies were performed to measure the membrane voltage (Vm), the membrane conductance (Gm), and the membrane capacitance (C(m)). Hypotonic cell swelling (Hypo, 150 mosm/l) was used, because it activates Cl- and K+ channels and enables the cell to extrude KCl in many cells, and ATP because it is known to activate Ca(2+)-regulated channels in a large variety of cells. Hypo depolarized all three types of cells. This depolarization was accompanied by an increase in Cl- conductance. The selectivity of the conductance was I- > or = Br- > or = Cl- in CHO cells, but Cl- = Br- = I- in the CFTR cells. Even more surprising: ATP (100 mumol/l) hyperpolarized CHO and delta F508 cells and predominantly enhanced K+ conductance, whilst it depolarized and increased mostly a Cl- conductance in CFTR cells. The selectivity of this anion conductance was atypical for ATP: Br- > Cl- > I-. C(m) was increased by ATP and Hypo in all three types of cells. ATP enhanced cytosolic Ca2+ ([Ca2+]i) in all three types of cells but did not enhance cAMP. These data indicate that the expression of CFTR profoundly alters the properties of CHO cells. Agonists which stimulate characteristic Ca(2+)-regulated channels now enhance a Cl- conductance resembling the properties of CFTR-Cl- conductance.
本研究的目的是检测野生型囊性纤维化跨膜传导调节因子（CFTR）在中国仓鼠卵巢（CHO）细胞中的稳定表达是否会改变这些细胞对低渗性细胞肿胀和ATP的反应特性。根据之前的许多研究，事先并未预期会出现这种情况，因为CFTR的过表达不应影响由嘌呤能激动剂上调的传导途径或细胞肿胀。研究了三种类型的CHO细胞：正常CHO细胞的对照组；高水平稳定表达野生型CFTR的CFTR-CHO细胞组（CHO-CFTR），以及稳定表达常见突变型δF508 CFTR的δF508-CFTR-CHO细胞组（CHO-δF508）。进行全细胞膜片钳研究以测量膜电压（Vm）、膜电导（Gm）和膜电容（C(m)）。使用低渗性细胞肿胀（Hypo，150 mosm/l），因为它可激活Cl-和K+通道，并使许多细胞能够排出KCl，还使用ATP，因为已知它可激活多种细胞中的Ca(2+)调节通道。低渗使所有三种类型的细胞去极化。这种去极化伴随着Cl-电导的增加。CHO细胞中电导的选择性为I- >或= Br- >或= Cl-，但CFTR细胞中Cl- = Br- = I-。更令人惊讶的是：ATP（100 μmol/l）使CHO和δF508细胞超极化，并主要增强K+电导，而它使CFTR细胞去极化并主要增加Cl-电导。这种阴离子电导的选择性对ATP来说是非典型的：Br- > Cl- > I-。在所有三种类型的细胞中，ATP和低渗均使C(m)增加。ATP使所有三种类型的细胞中的胞质Ca2+（[Ca2+]i）增加，但不增加cAMP。这些数据表明CFTR的表达深刻改变了CHO细胞的特性。刺激特征性Ca(2+)调节通道的激动剂现在增强了一种类似于CFTR-Cl-电导特性的Cl-电导。
