Chang D, Dawson D C
Department of Physiology, University of Michigan Medical School, Ann Arbor 48109.
J Gen Physiol. 1988 Sep;92(3):281-306. doi: 10.1085/jgp.92.3.281.
Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of 86Rb, [3H]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K+ and Cl- currents were generated by imposing transmural ion gradients, and cytosolic free Ca2+ was manipulated by means of a Ca2+-EGTA buffer system in the mucosal bathing solution. Raising the cytosolic free Ca2+ concentration from the nanomolar to the micromolar range activated basolateral conductances for K+ and Cl-. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca2+-induced increases in the K+ and Cl- conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca2+-activated conductive pathways for K+ and Cl-. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca2+-activated channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.
将分离的龟结肠薄片在黏膜侧暴露于洋地黄皂苷，以化学方式去除顶端膜作为通透性屏障。黏膜对⁸⁶Rb、[³H]甘露醇和⁴⁵Ca-EGTA摄取的增加，以及黏膜浴中胞质标记酶乳酸脱氢酶的出现，证实了去污剂的通透作用。通过施加跨膜离子梯度产生基底外侧K⁺和Cl⁻电流，并借助黏膜浴溶液中的Ca²⁺-EGTA缓冲系统来调控胞质游离Ca²⁺。将胞质游离Ca²⁺浓度从纳摩尔范围提高到微摩尔范围，激活了基底外侧对K⁺和Cl⁻的电导。离子选择性、阻断剂特异性、钙激活动力学以及二价阳离子激活选择性方面的差异表明，Ca²⁺诱导的K⁺和Cl⁻电导增加是由于不同的通道群体所致。这些结果与以下观点一致：在保留基底外侧膜某些导电特性（特别是Ca²⁺激活的K⁺和Cl⁻导电途径）的条件下，龟结肠上皮细胞的顶端膜在功能上可以被去除。这种通透化制剂应能为鉴定假定的Ca²⁺激活通道所产生的宏观电流提供一种方法，并且还可能为通道调节机制的功能重建提供一个模型系统。
