Copello J, Wehner F, Reuss L
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641.
Am J Physiol. 1993 May;264(5 Pt 1):C1128-36. doi: 10.1152/ajpcell.1993.264.5.C1128.
To patch clamp the basolateral cell membrane, sheets of Necturus gallbladder epithelium were stripped of the subepithelial tissue layers and affixed apical side down on cover slips coated with Cell-Tak [F. Wehner, L. Garretson, K. Dawson, Y. Segal, and L. Reuss. Am. J. Physiol. 258 (Cell Physiol. 27): C1159-C1164, 1990]. In 90% of the patches we observed K+ channels identical to the maxi-K+ channels previously demonstrated in the apical membrane (Y. Segal and L. Reuss. J. Gen. Physiol. 95: 791-818, 1990). To ascertain whether these channels were present in the native tissue, we carried out intracellular-microelectrode studies. We tested for activation of basolateral membrane K+ conductance by depolarization or by elevation of intracellular Ca2+ and for tetraethylammonium sensitivity of the basolateral membrane voltage and fractional resistance. The results were negative, indicating that maxi-K+ channels are not expressed in the basolateral membrane of the "intact" epithelium. Using the same intracellular-microelectrode protocol on the apical membrane, we demonstrated the presence of an apical K+ conductance attributable to maxi-K+ channels. Additional experiments revealed a Ba(2+)-sensitive basolateral K+ conductance in the native epithelium. We conclude that in the stripped preparation there is artifactual expression of maxi-K+ channels. In addition, the native basolateral membrane K+ channels either are not expressed in this preparation or have a low conductance and cannot be discerned from the background noise.
为了对基底外侧细胞膜进行膜片钳实验，将美西螈胆囊上皮片的上皮下组织层剥离，并将其顶端向下固定在涂有Cell-Tak的盖玻片上[F. 韦纳、L. 加勒森、K. 道森、Y. 西格尔和L. 罗伊斯。《美国生理学杂志》258(细胞生理学27):C1159 - C1164，1990]。在90%的膜片中，我们观察到与先前在顶端膜中证实的大电导钾通道相同的钾通道(Y. 西格尔和L. 罗伊斯。《普通生理学杂志》95:791 - 818，1990)。为了确定这些通道是否存在于天然组织中，我们进行了细胞内微电极研究。我们通过去极化或细胞内钙离子浓度升高来测试基底外侧膜钾离子电导的激活情况，以及基底外侧膜电压和电阻分数对四乙铵的敏感性。结果均为阴性，表明大电导钾通道在“完整”上皮的基底外侧膜中不表达。在顶端膜上使用相同的细胞内微电极方案，我们证实了归因于大电导钾通道的顶端钾离子电导的存在。进一步的实验揭示了天然上皮中一种对钡离子敏感的基底外侧钾离子电导。我们得出结论，在剥离的标本中存在大电导钾通道的人为表达。此外，天然的基底外侧膜钾离子通道要么在该标本中不表达，要么电导很低，无法从背景噪声中分辨出来。
