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标题：A channelopathy mechanism revealed by direct calmodulin activation of TrpV4
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作者：Stephen H. Loukin ; Jinfeng Teng ; Ching Kung 等
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：30
页码：9400-9405
DOI：10.1073/pnas.1510602112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceOver 50 mutations in the ion channel Transient Receptor Potential vanilloid subtype 4 (TrpV4) cause diseases ranging from dwarfism to prenatal death. We previously examined 14 mutant channels and found them to leak. Ca2+ encourages TrpV4 opening through calmodulin (CaM). Here, we examined two channels mutated in close proximity to the Ca2+-CaM-binding domain. They not only leak but also are greatly reduced in activation by Ca2+-CaM compared with the wild-type or other mutant channels. These mutations likely define an autoinhibitory domain that keeps the channel closed, to which adjacent detachable Ca2+-CaM binding interferes with this inhibition. The scattered disease alleles may all make the channel leak but apparently by different means, including the loss of an autoinhibition shown here. Ca2+-calmodulin (CaM) regulates varieties of ion channels, including Transient Receptor Potential vanilloid subtype 4 (TrpV4). It has previously been proposed that internal Ca2+ increases TrpV4 activity through Ca2+-CaM binding to a C-terminal Ca2+-CaM binding domain (CBD). We confirmed this model by directly presenting Ca2+-CaM protein to membrane patches excised from TrpV4-expressing oocytes. Over 50 TRPV4 mutations are now known to cause heritable skeletal dysplasia (SD) and other diseases in human. We have previously examined 14 SD alleles and found them to all have gain-of-function effects, with the gain of constitutive open probability paralleling disease severity. Among the 14 SD alleles examined, E797K and P799L are located immediate upstream of the CBD. They not only have increase basal activity, but, unlike the wild-type or other SD-mutant channels examined, they were greatly reduced in their response to Ca2+-CaM. Deleting a 10-residue upstream peptide ({Delta}795-804) that covers the two SD mutant sites resulted in strong constitutive activity and the complete lack of Ca2+-CaM response. We propose that the region immediately upstream of CBD is an autoinhibitory domain that maintains the closed state through electrostatic interactions, and adjacent detachable Ca2+-CaM binding to CBD sterically interferes with this autoinhibition. This work further supports the notion that TrpV4 mutations cause SD by constitutive leakage. However, the closed conformation is likely destabilized by various mutations by different mechanisms, including the permanent removal of an autoinhibition documented here.
关键词：skeletal dysplasia ; calcium ; ion channel ; autoinhibition ; TrpV1