ANALYSIS OF BASOLATERAL ION CHANNELS IN SKIN

尽管有很多研究，但对基侧向知之甚少。 钠转运上皮细胞的电导及其影响因素 调节它以响应钠速率的变化 运输。尽管很明显，大多数人 青蛙皮肤的基侧电导，一个被广泛使用的模型 钠吸收上皮细胞，是由于钾的性质 钾离子通道和其他离子通道及其可能的调节 细胞内的因素是未知的。最新技术进步 使记录单个离子通道电流成为可能 流经一小块细胞膜，然后 毫不含糊地描述复合体的成分 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”的可行性 酶法分离活体上皮细胞的技术 青蛙皮。建议在原封不动的隔离环境中进行实验 上皮细胞和分散的细胞来表征基本的 属性(即单通道电导、电压和时间) 依赖于门控动力学、离子选择性)的离子通道 它们构成了基底侧向的传导路径 薄膜。可能对通道活动的影响 细胞内调节剂(pH、游离钙和钠 浓度、ATP)以及蛋白质的作用 磷酸化(通过cAMP依赖的蛋白激酶)将是 分析过了。在完整的上皮细胞上进行的其他实验 离子载体将表征宏观K电流，从而使其 将可能确定负责该事件的渠道 基侧膜在不同条件下的K电导。 膜片钳技术也将被用来研究 基底外侧通道在分离细胞和完整细胞中的反应 上皮细胞对钠转运调节剂(加压素、异丙肾上腺素、 等)。这些实验的结果将有助于阐明 离子输运机制及其调控 钠吸收上皮细胞的基侧膜，并将导致 更好地理解跨上皮细胞之间的相互作用 运输和细胞动态平衡。

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.