channelopathy mechanism revealed by direct calmodulin activation of TrpV4

• Published in: Proceedings of the National Academy of Sciences - PNAS 2015-07, Vol.112 (30), p.9400-9405
• Main Authors: Loukin, Stephen H, Jinfeng Teng, Ching Kung
• Format: Article
• Language: English
• Subjects: Alleles; Amino Acid Sequence; Animals; autoinhibition; Binding Sites; Biological Sciences; Bone Diseases - genetics; Bone Diseases - physiopathology; calcium; Calcium - chemistry; calmodulin; Calmodulin - chemistry; Channelopathies - physiopathology; Chelating Agents - chemistry; death; Gene Expression Profiling; Genes; Humans; ion channel; Ion Channel Gating; Molecular Sequence Data; mutants; Mutation; Oocytes - cytology; Peptides; Protein Binding - genetics; Protein Structure, Tertiary; Proteins; RNA, Complementary - metabolism; Sequence Homology, Amino Acid; skeletal dysplasia; transient receptor potential channels; TRPV Cation Channels - genetics; TRPV Cation Channels - physiology; TrpV1; Xenopus laevis
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1510602112

Over 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. Ca ²⁺ encourages TrpV4 opening through calmodulin (CaM). Here, we examined two channels mutated in close proximity to the Ca ²⁺-CaM–binding domain. They not only leak but also are greatly reduced in activation by Ca ²⁺-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 Ca ²⁺-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. Ca ²⁺-calmodulin (CaM) regulates varieties of ion channels, including Transient Receptor Potential vanilloid subtype 4 (TrpV4). It has previously been proposed that internal Ca ²⁺ increases TrpV4 activity through Ca ²⁺-CaM binding to a C-terminal Ca ²⁺-CaM binding domain (CBD). We confirmed this model by directly presenting Ca ²⁺-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 Ca ²⁺-CaM. Deleting a 10-residue upstream peptide (Δ795–804) that covers the two SD mutant sites resulted in strong constitutive activity and the complete lack of Ca ²⁺-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 Ca ²⁺-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.