ERG钾通道对远端肾单位闰细胞泌H+的调控

The intercalated cells (IC) in the distal nephron are responsible for maintaining acid-base homeostasis in our body through activating H+-ATPase in the apical membrane of the distal nephron thereby facilitating H+ secretion. Acidosis significantly stimulates H+-ATPase and enhances H+ secretion. However, the role of K channel in mediating proton secretion is not understood. In our preliminary experiments we have detected a hyperpolarization-activated K channel activity in IC. Based on electrophysiological properties and pharmacological profile, we confirmed that this K current is carried by ERG K channels. ERG family K channels are highly expressed in the excitable cells such as neurons and cardiac myocytes. They are responsible for maintaining the membrane potential in proper conditions thereby sustaining the cell excitability. Since the hyperpolarization-activated ERG K currents were only detected in IC but not in the principal cells, it is possible that the activation of ERG K channels will increase the inward K currents thereby decreasing the intracellular electronegativity and increasing the electronic driving force for H+ secretion. Thus, We propose the hypothesis that ERG K channels are involved in mediating H+ secretion in the IC and that the acidosis may stimulate H+ secretion by increasing the open probability and/or the protein expression of ERG K channel. To verify the hypothesis, we will examine the expression of ERG K channels and their activity in IC using molecular biology and patch-clamp approaches under control and acidosis conditions. In addition, we will study the phenotypes of transgenic mice to investigate the effect of the loss of ERG K channel function on the acid-base balance.

肾脏远端肾单位闰细胞的主要功能是调节机体的酸碱平衡。生理条件下闰细胞即通过泌H+作用参与维持机体pH值的相对稳定，这种作用在酸中毒时显著增强，但机制尚不清楚。ERG钾通道通常被认为只存在于心肌细胞和神经细胞等可兴奋性细胞，与自动节律性形成和兴奋性的维持有关。我们最近用膜片钳技术在肾脏远端肾单位的闰细胞记录到幅度极大的超极化激活钾电流，并根据电生理学和药理学特性确定介导这种电流的通道为ERG钾通道。由于这种超极化激活钾电流只存在于闰细胞，并未在主细胞观察到，因此，我们提出这样的假说：ERG钾通道与闰细胞的泌H+功能密切相关，酸中毒时，ERG钾通道活性增强和/或表达增加，使H+泵排酸能力增强。本项目拟用分子生物学手段和膜片钳技术研究ERG钾通道在闰细胞的特异性表达，以及酸中毒情况下ERG钾通道的表达及其活性的变化，并使用基因敲除动物研究ERG钾通道缺失对机体酸碱平衡的影响，以验证上述假说。

生理条件下，肾脏远端肾单位闰细胞通过泌H+参与机体酸碱平衡调节，这种作用在酸中毒时显著增强，但机制尚不清楚。本课题采用膜片钳技术在肾脏远端肾单位的闰细胞记录到幅度极大的超极化激活钾电流，并根据电生理学和药理学特性确定介导这种电流的通道为ERG钾通道。在肾小管酸化条件下，ERG钾通道电流显著增大，提示酸中毒引起的闰细胞泌H+增强可能与ERG钾通道激活有关。应用蛋白印迹技术和免疫荧光技术进一步证实ERG钾通道在远端肾单位肾小管广泛分布。这些结果表明，ERG钾通道可能与闰细胞的泌H+功能密切相关，酸中毒时，ERG钾通道活性增强和/或表达增加，使H+泵排酸能力增强。但这个结论还需要通过建立基因敲除动物研究肾小管ERG钾通道缺失对机体酸碱平衡影响来进一步证实。目前本课题组已与南京大学模式动物研究所签订协议，正在建立实验所需的肾小管上皮细胞ERG钾通道（基因型为KCNH2）特异性敲除小鼠，预计年内能完成这部分实验。本研究有助于进一步阐明闰细胞泌H+的内在机制，揭示酸中毒条件下闰细胞排酸能力增强的原因。