Schwab A, Westphale H J, Wojnowski L, Wünsch S, Oberleithner H
Department of Physiology, University of Würzburg, Germany.
J Clin Invest. 1993 Jul;92(1):218-23. doi: 10.1172/JCI116553.
Intracellular alkalinization is known to be associated with tumorigenic transformation. Besides phenotypical alterations alkali-transformed Madin-Darby canine kidney (MDCK) cells exhibit a spontaneously oscillating cell membrane potential (PD). Using single-channel patch clamp techniques, it was the aim of this study to identify the ion channel underlying the rhythmic hyperpolarizations of the PD. In the cell-attached patch configuration, we found that channel activity was oscillating. The frequency of channel oscillations is 1.1 +/- 0.1 min-1. At the peak of oscillatory channel activity, single-channel current was -2.7 +/- 0.05 pA, and in the resting state it was -1.95 +/- 0.05 pA. Given the single-channel conductance of 53 +/- 3 pS for inward (and of 27 +/- 5 pS for outward) current the difference of single-channel current amplitude corresponded to a hyperpolarization of approximately 14 mV. The channel is selective for K+ over Na+. Channel kinetics are characterized by one open and by three closed time constants. The channel is Ca2+ sensitive. Half maximal activation in the inside-out patch mode is achieved at a Ca2+ concentration of 10 mumol/liter. In addition, we also found a 13-pS K+ channel that shows no oscillatory activity in the cell-attached patch configuration and that was not Ca2+ sensitive. We conclude that the Ca(2+)-sensitive 53-pS K+ channel is underlying spontaneous oscillations of the PD. It has virtually identical biophysical properties as a Ca(2+)-sensitive K+ channel in nontransformed parent MDCK cells. Hence, alkali-induced transformation of MDCK cells did not affect the channel protein itself but its regulators thereby causing spontaneous fluctuations of the PD.
已知细胞内碱化与致瘤转化有关。除了表型改变外，碱转化的麦氏犬肾（MDCK）细胞还表现出自发振荡的细胞膜电位（PD）。本研究旨在使用单通道膜片钳技术鉴定PD节律性超极化背后的离子通道。在细胞贴附式膜片配置中，我们发现通道活性在振荡。通道振荡频率为1.1±0.1次/分钟。在振荡通道活性的峰值处，单通道电流为-2.7±0.05 pA，在静息状态下为-1.95±0.05 pA。考虑到内向电流的单通道电导为53±3 pS（外向电流为27±5 pS），单通道电流幅度的差异对应于约14 mV的超极化。该通道对K+的选择性高于Na+。通道动力学的特征是一个开放时间常数和三个关闭时间常数。该通道对Ca2+敏感。在膜内朝外模式下，在Ca2+浓度为10 μmol/升时实现半最大激活。此外，我们还发现了一个13-pS的K+通道，它在细胞贴附式膜片配置中没有振荡活性，并且对Ca2+不敏感。我们得出结论，Ca(2+)敏感的53-pS K+通道是PD自发振荡的基础。它与未转化的亲代MDCK细胞中的Ca(2+)敏感K+通道具有几乎相同的生物物理特性。因此，碱诱导的MDCK细胞转化并未影响通道蛋白本身，而是影响了其调节因子，从而导致PD的自发波动。
