Determinants of 4α-phorbol sensitivity in transmembrane domains 3 and 4 of the cation channel TRPV4

Abstract

TRPV4, a Ca2+-permeable member of the vanilloid subgroup of the transient receptor potential (TRP) channels, is activated by cell swelling and moderate heat (>27 °C) as well as by diverse chemical compounds including synthetic 4α-phorbol esters, the plant extract bisandrographolide A, and endogenous epoxyeicosatrienoic acids (EETs; 5,6-EET and 8,9-EET). Previous work identified a tyrosine residue located in the first half of putative transmembrane segment 3 (TM3) as a crucial determinant for the activation of TRPV4 by its most specific agonist 4α-phorbol 12,13-didecanoate (4α-PDD), suggesting that 4α-PDD interacts with the channel through its transmembrane segments. To obtain insight in the 4α-PDD-binding site and in the mechanism of ligand-dependent TRPV4 activation, we investigated the consequences of specific point mutations in TM4 on the sensitivity of the channel to different chemical and physical stimuli. Mutations of two hydrophobic residues in the central part of TM4 (Leu584 and Trp586) caused a severe reduction of the sensitivity of the channel to 4α-PDD, bisandrographolide A, and heat, whereas responses to cell swelling, arachidonic acid, and 5,6-EET remained unaffected. In contrast, mutations of two residues in the C-terminal part of TM4 (Tyr591 and Arg594) affected channel activation of TRPV4 by all stimuli, suggesting an involvement in channel gating rather than in interaction with agonists. Based on a comparison of the responses of WT and mutant TRPV4 to 4α-PDD and different 4α-phorbol esters, we conclude that the length of the fatty acid moiety determines the ligand binding affinity and propose a model for the interaction between 4α-phorbol esters and the TM3/4 region of TRPV4. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.