Goodwin P A, Campbell K E, Evans B A, Wann K T
Welsh School of Pharmacy, Cardiff University of Wales, United Kingdom.
J Pharmacol Toxicol Methods. 1998 Jun;39(4):229-33. doi: 10.1016/s1056-8719(98)00029-x.
We have conducted single-channel patch-clamp experiments in skin fibroblasts maintained in culture. Two different cell lines, a mouse 3T3-L1 cell line and a human B17 cell line, were selected for these pilot studies. Recordings were made from both cell-attached and excised inside-out patches at room temperature. In the case of the 3T3-L1 cells, the success rate in obtaining good seals (> 1Gomega) was low, and channel openings in either cell-attached or excised patches were rare. We have, however, identified a channel in a cell-attached configuration with a slope conductance of 39 pS in symmetrical K+ solutions. In the case of the human B17 cells, good quality seals were more readily obtained. One principal type of channel opening was identified. In cell-attached patches, the prevalent type of channel in symmetrical K+ solutions had a conductance of 187 pS. This channel was activated by strong depolarization, and there was usually more than one active channel in the patch. It was blocked by extracellular tetraethylammonium (20 mM), and persisted when external Cl- was replaced by aspartate. In excised inside-out patches bathed in symmetrical K+, this channel was activated by an increase in Ca+ applied to the intracellular face. A large conductance channel (175 pS) was also observed in excised inside-out patches, with a reverse physiological K+ gradient. This channel had a reversal potential > 40 mV and appeared not to be voltage-dependent under these recording conditions (2 mM Ca(2+)i). We conclude that the channel we have identified in these cells belongs to the maxi-K+ channel class.
我们在培养的皮肤成纤维细胞中进行了单通道膜片钳实验。为这些初步研究选择了两种不同的细胞系，一种小鼠3T3-L1细胞系和一种人B17细胞系。在室温下从细胞贴附型和内面向外膜片进行记录。对于3T3-L1细胞，获得良好封接（>1GΩ）的成功率较低，细胞贴附型或内面向外膜片中的通道开放很少见。然而，我们在对称K+溶液中鉴定出一种细胞贴附型配置的通道，其斜率电导为39 pS。对于人B17细胞，更容易获得高质量的封接。鉴定出一种主要的通道开放类型。在细胞贴附型膜片中，对称K+溶液中普遍存在的通道类型电导为187 pS。该通道由强去极化激活，膜片中通常有不止一个活性通道。它被细胞外四乙铵（20 mM）阻断，当外部Cl-被天冬氨酸替代时仍然存在。在浸浴于对称K+中的内面向外膜片中，该通道通过施加到细胞内表面的Ca+增加而被激活。在内面向外膜片中还观察到一个大电导通道（175 pS），具有反向生理K+梯度。该通道的反转电位>40 mV，在这些记录条件下（2 mM Ca(2+)i）似乎不依赖电压。我们得出结论，我们在这些细胞中鉴定出的通道属于大电导K+通道类别。
