Inagaki A, Yamaguchi S, Ishikawa T
Laboratory of Physiology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Am J Physiol Cell Physiol. 2004 Feb;286(2):C380-90. doi: 10.1152/ajpcell.00373.2003. Epub 2003 Oct 22.
Surface cells of the mammalian distal colon are shown to molecularly express the amiloride-sensitive epithelial Na+ channel composed of three homologous subunits (alpha-, beta-, and gamma-ENaC). However, because basic electrophysiological properties of amiloride-sensitive Na+ channels expressed in these cells are largely unknown at the cellular level, functional evidence for the involvement of the subunits in the native channels is incomplete. Using electrophysiological techniques, we have now characterized functional properties of native ENaC in surface cells of rectal colon (RC) of rats fed a normal Na+ diet. Ussing chamber experiments showed that apical amiloride inhibited a basal short-circuit current in mucosal preparation of RC with an apparent half-inhibition constant (Ki) value of 0.20 microM. RT-PCR analysis confirmed the presence of transcripts of alpha-, beta-, and gamma-rENaC in rectal mucosa. Whole cell patch-clamp experiments in surface cells of intact crypts acutely isolated from rectal mucosa identified an inward cationic current, which was inhibited by amiloride with a Ki value of 0.12 microM at a membrane potential of -64 mV, the inhibition being weakly voltage dependent. Conductance ratios of the currents were Li+ (1.8) > Na+ (1) >> K+ ( approximately 0), respectively. Amiloride-sensitive current amplitude was almost the same at 15 or 150 mM extracellular Na+, suggesting a high Na+ affinity for current activation. These results are consistent with the hypothesis that a heterooligomer composed of alpha-, beta-, and gamma-ENaC may be the molecular basis of the native channels, which are responsible for amiloride-sensitive electrogenic Na+ absorption in rat rectal colon.
哺乳动物远端结肠的表面细胞在分子水平上表达由三个同源亚基（α-、β-和γ-ENaC）组成的amiloride敏感上皮钠通道。然而，由于这些细胞中表达的amiloride敏感钠通道的基本电生理特性在细胞水平上很大程度上未知，亚基参与天然通道的功能证据并不完整。我们现在使用电生理技术，对喂食正常钠饮食的大鼠直肠结肠（RC）表面细胞中天然ENaC的功能特性进行了表征。Ussing室实验表明，顶端amiloride抑制了RC黏膜制剂中的基础短路电流，表观半抑制常数（Ki）值为0.20μM。RT-PCR分析证实直肠黏膜中存在α-、β-和γ-rENaC的转录本。从直肠黏膜急性分离的完整隐窝表面细胞的全细胞膜片钳实验确定了一种内向阳离子电流，在膜电位为-64 mV时，该电流被amiloride抑制，Ki值为0.12μM，抑制作用弱电压依赖性。电流的电导比分别为Li+（1.8）>Na+（1）>>K+（约0）。在15或150 mM细胞外Na+时，amiloride敏感电流幅度几乎相同，表明电流激活对Na+具有高亲和力。这些结果与以下假设一致，即由α-、β-和γ-ENaC组成的异源寡聚体可能是天然通道的分子基础，这些通道负责大鼠直肠结肠中amiloride敏感的电生性Na+吸收。
