Snyder P M, Bucher D B, Olson D R
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
J Gen Physiol. 2000 Dec;116(6):781-90. doi: 10.1085/jgp.116.6.781.
The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (alpha, beta, and gamma). The channel forms the pathway for Na(+) absorption in the kidney, and mutations cause disorders of Na(+) homeostasis. However, little is known about the mechanisms that control the gating of ENaC. We investigated the gating mechanism by introducing bulky side chains at a position adjacent to the extracellular end of the second membrane spanning segment (549, 520, and 529 in alpha, beta, and gammaENaC, respectively). Equivalent "DEG" mutations in related DEG/ENaC channels in Caenorhabditis elegans cause swelling neurodegeneration, presumably by increasing channel activity. We found that the Na(+) current was increased by mutagenesis or chemical modification of this residue and adjacent residues in alpha, beta, and gammaENaC. This resulted from a change in the gating of ENaC; modification of a cysteine at position 520 in betaENaC increased the open state probability from 0. 12 to 0.96. Accessibility to this side chain from the extracellular side was state-dependent; modification occurred only when the channel was in the open conformation. Single-channel conductance decreased when the side chain contained a positive, but not a negative charge. However, alterations in the side chain did not alter the selectivity of ENaC. This is consistent with a location for the DEG residue in the outer vestibule. The results suggest that channel gating involves a conformational change in the outer vestibule of ENaC. Disruption of this mechanism could be important clinically since one of the mutations that increased Na(+) current (gamma(N530K)) was identified in a patient with renal disease.
上皮钠离子通道（ENaC）由三个同源亚基（α、β和γ）组成。该通道构成了肾脏中钠离子重吸收的途径，其突变会导致钠稳态紊乱。然而，对于控制ENaC门控的机制却知之甚少。我们通过在与第二个跨膜片段细胞外末端相邻的位置引入大的侧链（α、β和γENaC中分别为549、520和529）来研究门控机制。秀丽隐杆线虫相关的DEG/ENaC通道中的等效“DEG”突变会导致肿胀性神经变性，推测是通过增加通道活性来实现的。我们发现，通过诱变或化学修饰α、β和γENaC中的该残基及相邻残基，钠离子电流会增加。这是由于ENaC门控的改变所致；修饰βENaC中520位的半胱氨酸会使开放状态概率从0.12增加到0.96。从细胞外侧对该侧链的可及性取决于通道状态；仅当通道处于开放构象时才会发生修饰。当侧链带正电荷而非负电荷时，单通道电导会降低。然而，侧链的改变并未改变ENaC的选择性。这与DEG残基位于外前庭的位置一致。结果表明，通道门控涉及ENaC外前庭的构象变化。由于在一名肾病患者中鉴定出了一种增加钠离子电流的突变（γ(N530K)），因此破坏这一机制在临床上可能很重要。
