Teng Jinfeng, Loukin Stephen H, Anishkin Andriy, Kung Ching
Laboratory of Molecular and Cell Biology and.
Department of Biology, University of Maryland-College Park, College Park, MD 20742.
Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3386-91. doi: 10.1073/pnas.1502366112. Epub 2015 Mar 3.
Unlike other cation channels, each subunit of most transient receptor potential (TRP) channels has an additional TRP-domain helix with an invariant tryptophan immediately trailing the gate-bearing S6. Recent cryo-electron microscopy of TRP vanilloid subfamily, member 1 structures revealed that this domain is a five-turn amphipathic helix, and the invariant tryptophan forms a bond with the beginning of the four-turn S4-S5 linker helix. By homology modeling, we identified the corresponding L596-W733 bond in TRP vanilloid subfamily, member 4 (TRPV4). The L596P mutation blocks bone development in Kozlowski-type spondylometaphyseal dysplasia in human. Our previous screen also isolated W733R as a strong gain-of-function (GOF) mutation that suppresses growth when the W733R channel is expressed in yeast. We show that, when expressed in Xenopus oocytes, TRPV4 with the L596P or W733R mutation displays normal depolarization-induced activation and outward rectification. However, these mutant channels have higher basal open probabilities and limited responses to the agonist GSK1016790A, explaining their biological GOF phenotypes. In addition, W733R current fails to inactivate during depolarization. Systematic replacement of W733 with amino acids of different properties produced similar electrophysiological and yeast phenotypes. The results can be interpreted consistently in the context of the homology model of TRPV4 molecule we have developed and refined using simulations in explicit medium. We propose that this bond maintains the orientation of the S4-S5 linker to keep the S6 gate closed. Further, the two partner helices, both amphipathic and located at the polar-nonpolar interface of the inner lipid monolayer, may receive and integrate various physiological stimuli.
与其他阳离子通道不同，大多数瞬时受体电位（TRP）通道的每个亚基都有一个额外的TRP结构域螺旋，紧跟在带有门控结构的S6之后，且有一个不变的色氨酸。最近对TRP香草酸亚家族成员1结构的冷冻电镜研究表明，该结构域是一个五圈两亲性螺旋，不变的色氨酸与四圈S4-S5连接螺旋的起始部分形成一个键。通过同源建模，我们在TRP香草酸亚家族成员4（TRPV4）中鉴定出了相应的L596-W733键。L596P突变会阻碍人类科兹洛夫斯基型脊椎干骺端发育不良中的骨骼发育。我们之前的筛选还分离出W733R作为一个强大的功能获得性（GOF）突变，当W733R通道在酵母中表达时会抑制生长。我们发现，当在非洲爪蟾卵母细胞中表达时，带有L596P或W733R突变的TRPV4表现出正常的去极化诱导激活和外向整流。然而，这些突变通道具有更高的基础开放概率，并且对激动剂GSK1016790A的反应有限，这解释了它们的生物学GOF表型。此外，W733R电流在去极化过程中不会失活。用不同性质的氨基酸对W733进行系统性替换产生了类似的电生理和酵母表型。在我们使用显式介质模拟开发和完善的TRPV4分子同源模型的背景下，可以对这些结果进行一致的解释。我们提出，这个键维持了S4-S5连接螺旋的方向，以保持S6门关闭。此外，这两个伙伴螺旋都是两亲性的，位于内部脂质单层的极性-非极性界面，可能接收并整合各种生理刺激。
