ANALYSIS OF BASOLATERAL ION CHANNELS IN SKIN

尽管有大量的研究，但对基底外侧的了解甚少。 钠转运上皮细胞的电导和因子 调节它以响应钠的变化率 运输 虽然很明显，大多数 青蛙皮肤的基底外侧传导，一种广泛用于 钠吸收上皮细胞，是由于钾，性质的 钾和其他离子通道及其可能的调节 细胞内因素是未知的。 最近的技术进展 使得记录单个离子通道电流成为可能 流经一小片细胞膜， 明确地描述复杂的成分 孤立细胞中的宏观电流。 初步实验 已经证明了应用这些膜片钳技术的可行性 酶促分离活上皮细胞的技术， 青蛙皮 实验建议在完整的隔离 上皮细胞和分散的细胞来表征基本的 特性（即单通道电导、电压和时间） 依赖性门控动力学、离子选择性 构成了基底外侧的传导通路 膜的 可能对通道活动的影响 细胞内调节剂（pH、游离Ca和Na 浓度，ATP）以及蛋白质的作用 磷酸化（通过cAMP依赖性蛋白激酶）将被 分析了 在完整上皮细胞中进行的其他实验， 离子载体将表征宏观K电流， 将有可能确定负责的渠道， 不同条件下的基底外侧膜K电导。 膜片钳技术也将用于研究 在分离的细胞和完整的基底外侧通道的反应 上皮细胞对Na转运调节剂（加压素，异丙肾上腺素， 等）。 这些实验的结果将有助于阐明 离子转运机制及其在 钠吸收上皮细胞的基底外侧膜，并将导致 更好地理解跨上皮细胞之间的相互作用 运输和细胞内稳态。

Despite much study little is known about the basolateral conductance in sodium transporting epithelia and of the factors regulating it in response to alterations in the rate of sodium transport. Although it is clearly established that most of the basolateral conductance in frog skin, a widely used model for sodium absorbing epithelia, is due to potassium, the nature of the potassium and other ion channels and their possible modulation by intracellular factors are unknown. Recent technical advances have made it possible to record single ion channel currents flowing through a small patch of cell membrane and to characterize unambiguously the components of complex macroscopic currents in isolated cells. Preliminary experiments have shown the feasibility of applying these "patch-clamp" techniques to enzymatically isolated viable epithelial cells from the frog skin. Experiments are proposed in intact isolated epithelia and dispersed cells to characterize the elementary properties (i.e. single-channels conductance, voltage- and time- dependent gating kinetics, ionic selectivity) of the ion channels that constitute the conductance pathways of the basolateral membrane. The effects on channel activity of possible intracellular regulating agents (pH, free Ca and Na concentrations, ATP) as well as the role of protein phosphorylation (by cAMP-dependent protein kinase) will be analyzed. Other experiments in intact epithelia treated with ionophores will characterize the macroscopic K current so that it will be possible to identify the channels responsible for the basolateral membrane K conductance under different conditions. Patch-clamp techniques will be used also to investigate the response of basolateral channels in isolated cells and intact epithelia to modifiers of Na transport (vasopressin, isoproterenol, etc.). The results of these experiments will help elucidate the mechanisms of ion transport and their regulation at the basolateral membrane of Na absorbing epithelia and will lead to a better understanding of the interactions between transepithelial transport and cell homeostasis.