期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：11
页码：3386-3391
DOI：10.1073/pnas.1502366112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceTransient receptor potential (TRP) families are front-line channels that transduce various physical or chemical stimuli into ion fluxes. TRP subunits have architecture similar to that of voltage-gated K+ (Kv) channels, but most have an additional TRP-domain helix of unknown function. An invariant tryptophan in this helix forms a hydrogen bond with a residue that begins another helix, the S4-S5 linker, which is the lever that operates the channel gate in Kv channels. By electrophysiological and other examinations of TRP vanilloid subfamily, member 4 (TRPV4) channels mutated at the two bonding partners, we show that this bond is needed to stabilize the closed conformation. Destabilized channels leak ions, explaining the harmful phenotypes of mutant TRPV4 in budding yeast (growth stoppage) and in human (bone-development blockage). 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.
关键词：TRP channel ; TRP domain ; gating ; open mechanism
