Chalfant M L, Denton J S, Berdiev B K, Ismailov I I, Benos D J, Stanton B A
Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Am J Physiol. 1999 Feb;276(2):C477-86. doi: 10.1152/ajpcell.1999.276.2.C477.
Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Recently, three subunits (alpha, beta, gamma) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates Na+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to identifying the molecular mechanisms of proton-mediated regulation of apical membrane Na+ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinations of alpha-, beta-, and gamma-subunits of ENaC in Xenopus oocytes and studied ENaC currents by the two-electrode voltage-clamp and patch-clamp techniques. In addition, the effect of pH on the alpha-ENaC subunit was examined in planar lipid bilayers. We report that alpha,beta,gamma-ENaC currents were regulated by changes in intracellular pH (pHi) but not by changes in extracellular pH (pHo). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced single-channel open probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited alpha, beta-ENaC, alpha,gamma-ENaC, and alpha-ENaC currents. We conclude that pHi but not pHo regulates ENaC and that the alpha-ENaC subunit is regulated directly by pHi.
质子在包括皮质集合管、蛙皮和膀胱在内的多种上皮组织中调节电致钠吸收。最近，编码上皮钠通道（ENaC）的三个亚基（α、β、γ）被克隆出来。然而，尚不清楚pH是通过与三个ENaC亚基之一相互作用直接调节Na⁺通道，还是通过与一种调节蛋白相互作用间接调节。作为确定质子介导的上皮顶端膜Na⁺通透性调节分子机制的第一步，我们研究了pH对ENaC生物物理特性的影响。为此，我们在非洲爪蟾卵母细胞中表达了ENaC的α、β和γ亚基的各种组合，并通过双电极电压钳和膜片钳技术研究了ENaC电流。此外，还在平面脂质双分子层中研究了pH对α-ENaC亚基的影响。我们报告，α、β、γ-ENaC电流受细胞内pH（pHi）变化的调节，而不受细胞外pH（pHo）变化的调节。酸化通过电压非依赖机制降低通道活性，碱化则增加通道活性。此外，pHi降低会降低单通道开放概率，缩短单通道开放时间，并增加单通道关闭时间，而不改变单通道电导。细胞质溶液酸化也会抑制α、β-ENaC、α、γ-ENaC和α-ENaC电流。我们得出结论，是pHi而非pHo调节ENaC，并且α-ENaC亚基直接受pHi调节。
