Valverde M A, Mintenig G M, Sepúlveda F V
AFRC Babraham Institute, Cambridge, United Kingdom.
J Membr Biol. 1994 Feb;137(3):237-47. doi: 10.1007/BF00232592.
The ionic currents spontaneously present in T84 intestinal epithelial cells, a line of colonic carcinoma origin, have been studied using the whole-cell recording mode of the patch-clamp technique and the single-electrode voltage-clamp method. Patch-clamp experiments showed that nonstimulated T84 cells already possess large currents but that these tend to disappear during the course of the experiments, presumably through the dialysis of some essential cytoplasmic component against the micropipette solution. The main charge carrier in these experiments appears to be Cl- as judged from ion replacement. Microelectrode impalement of T84 cells gave a membrane potential of around -30 mV, similar to the equilibrium potential for Cl- estimated from previously published values for intracellular Cl- concentration. Voltage-clamp experiments with a single microelectrode revealed three kinetically distinguishable current patterns; currents decaying during hyperpolarizing voltage pulses, currents slowly activating during hyperpolarizing pulses and time-independent currents. The appearance of these distinct kinetic patterns was not predictable from cell to cell, and was not dependent on extracellular Ca2+. Ionic replacement experiments suggest that the charge carrier was always Cl-, regardless of the kinetic pattern observed. No K+ currents appear to be present in the non-stimulated T84 cells. Exposure of T84 cells to the muscarinic agonist carbachol induced a shift in the membrane potential towards more negative values, consistent with an activation of a K+ conductance. Thus, we suggest that the resting membrane potential in T84 cells is determined by the distribution of Cl-. This might imply that activation of K+ conductance could by itself support secretion by T84 monolayers through tonically active Cl- channels.
利用膜片钳技术的全细胞记录模式和单电极电压钳方法，对源自结肠癌细胞系的T84肠上皮细胞中自发存在的离子电流进行了研究。膜片钳实验表明，未受刺激的T84细胞已经具有大电流，但这些电流在实验过程中往往会消失，推测是由于一些必需的细胞质成分与微吸管溶液发生了透析作用。从离子置换判断，这些实验中的主要电荷载体似乎是Cl-。对T84细胞进行微电极刺入，测得膜电位约为 -30 mV，与根据先前发表的细胞内Cl-浓度值估算的Cl-平衡电位相似。用单个微电极进行的电压钳实验揭示了三种动力学上可区分的电流模式：在超极化电压脉冲期间电流衰减、在超极化脉冲期间电流缓慢激活以及与时间无关的电流。这些不同动力学模式的出现无法在细胞之间进行预测，并且不依赖于细胞外Ca2+。离子置换实验表明，无论观察到的动力学模式如何，电荷载体始终是Cl-。未受刺激的T84细胞中似乎不存在K+电流。将T84细胞暴露于毒蕈碱激动剂卡巴胆碱会导致膜电位向更负值偏移，这与K+电导的激活一致。因此，我们认为T84细胞的静息膜电位由Cl-的分布决定。这可能意味着K+电导的激活本身可以通过持续活跃的Cl-通道支持T84单层细胞的分泌。
