Departments of Physiology and Biophysics, Weill-Cornell Medical College, New York, NY.
Department of Medicine, Weill-Cornell Medical College, New York, NY.
J Gen Physiol. 2022 Oct 3;154(10). doi: 10.1085/jgp.202213124. Epub 2022 Aug 8.
The epithelial Na+ channel (ENaC) is a heterotrimeric protein whose assembly, trafficking, and function are highly regulated. To better understand the biogenesis and activation of the channel, we quantified the expression of individual subunits of ENaC in rat kidneys and colon using calibrated Western blots. The estimated abundance for the three subunits differed by an order of magnitude with the order γENaC ∼ βENaC ≫ αENaC in both organs. Transcript abundance in the kidney, measured with digital-drop PCR and RNAseq, was similar for the three subunits. In both organs, the calculated protein expression of all subunits was much larger than that required to account for maximal Na+ currents measured in these cells, implying a large excess of subunit protein. Whole-kidney biotinylation indicated that at least 5% of β and γ subunits in the kidney and 3% in the colon were expressed on the surface under conditions of salt restriction, which maximizes ENaC-dependent Na+ transport. This indicates a 10- to 100-fold excess of βENaC and γENaC subunits at the surface relative to the requirement for channel activity. We conclude that these epithelia make much more ENaC protein than is required for the physiological function of the channel. This could facilitate rapid regulation of the channels at the cell surface by insuring a large population of inactive, recruitable subunits.
上皮钠离子通道（ENaC）是一种三聚体蛋白，其组装、运输和功能受到高度调控。为了更好地理解通道的生物发生和激活，我们使用校准的 Western blot 定量测定了大鼠肾脏和结肠中 ENaC 各亚基的表达。在这两个器官中，三个亚基的估计丰度相差一个数量级，γENaC ∼ βENaC ≫ αENaC。用数字滴 PCR 和 RNAseq 测量的肾脏中的转录丰度，三个亚基相似。在这两个器官中，所有亚基的计算蛋白表达都远大于这些细胞中测量到的最大 Na+电流所需的表达量，这意味着亚基蛋白存在大量过剩。全肾生物素化表明，在盐限制条件下，肾脏中至少 5%的β和γ亚基和结肠中 3%的β和γ亚基在表面表达，这最大限度地增加了 ENaC 依赖的 Na+转运。这表明相对于通道活性的要求，βENaC 和 γENaC 亚基在表面的过剩程度为 10 到 100 倍。我们得出的结论是，这些上皮组织产生的 ENaC 蛋白远远超过了通道生理功能所需的蛋白。这可以通过确保大量无活性、可募集的亚基来促进细胞表面通道的快速调节。
