Eskandari S, Snyder P M, Kreman M, Zampighi G A, Welsh M J, Wright E M
Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1751, USA.
J Biol Chem. 1999 Sep 17;274(38):27281-6. doi: 10.1074/jbc.274.38.27281.
The human epithelial sodium channel (hENaC) is a hetero-oligomeric complex composed of three subunits, alpha, beta, and gamma. Understanding the structure and function of this channel and its abnormal behavior in disease requires knowledge of the number of subunits that comprise the channel complex. We used freeze-fracture electron microscopy and electrophysiological methods to evaluate the number of subunits in the ENaC complex expressed in Xenopus laevis oocytes. In oocytes expressing wild-type hENaC (alpha, beta, and gamma subunits), clusters of particles appeared in the protoplasmic face of the plasma membrane. The total number of particles in the clusters was consistent with the whole-cell amiloride-sensitive current measured in the same cells. The size frequency histogram for the particles in the clusters suggested the presence of an integral membrane protein complex composed of 17 +/- 2 transmembrane alpha-helices. Because each ENaC subunit has two putative transmembrane helices, these data suggest that in the oocyte plasma membrane, the ENaC complex is composed of eight or nine subunits. At high magnification, individual ENaC particles exhibited a near-square geometry. Functional studies using wild-type alphabeta-hENaC coexpressed with gamma-hENaC mutants, which rendered the functional channel differentially sensitive to methanethiosulfonate reagents and cadmium, suggested that the functional channel complex contains more than one gamma subunit. These data suggest that functional ENaC consists of eight or nine subunits of which a minimum of two are gamma subunits.
人类上皮钠通道（hENaC）是一种由α、β和γ三个亚基组成的异源寡聚体复合物。了解该通道的结构和功能及其在疾病中的异常行为需要知晓构成通道复合物的亚基数量。我们使用冷冻断裂电子显微镜和电生理方法来评估非洲爪蟾卵母细胞中表达的ENaC复合物中的亚基数量。在表达野生型hENaC（α、β和γ亚基）的卵母细胞中，质膜原生质面上出现了颗粒簇。簇中颗粒的总数与在相同细胞中测量的全细胞氨氯地平敏感电流一致。簇中颗粒的大小频率直方图表明存在一种由17±2个跨膜α螺旋组成的整合膜蛋白复合物。由于每个ENaC亚基有两个假定的跨膜螺旋，这些数据表明在卵母细胞质膜中，ENaC复合物由八个或九个亚基组成。在高倍放大下，单个ENaC颗粒呈现出近似方形的几何形状。使用与γ-hENaC突变体共表达的野生型αβ-hENaC进行的功能研究表明，功能性通道复合物对甲硫代磺酸盐试剂和镉具有不同的敏感性，这表明功能性通道复合物包含不止一个γ亚基。这些数据表明，功能性ENaC由八个或九个亚基组成，其中至少两个是γ亚基。
