Abstract

TRPV4, a Ca(2+)-permeable member of the vanilloid subgroup of the transient receptor potential (TRP) channels, is activated by cell swelling and moderate heat (>27 degrees C) as well as by diverse chemical compounds including synthetic 4 alpha-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 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), suggesting that 4 alpha-PDD interacts with the channel through its transmembrane segments. To obtain insight in the 4 alpha-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 (Leu(584) and Trp(586)) caused a severe reduction of the sensitivity of the channel to 4 alpha-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 (Tyr(591) and Arg(594)) 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 4 alpha-PDD and different 4 alpha-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 4 alpha-phorbol esters and the TM3/4 region of TRPV4.

Highlights

  • In the present study, we identify residues in TM4 of TRPV4 that are critically involved in ligand binding

  • Sequence alignment of the putative transmembrane segment 3 (TM3) and TM4 of TRPV4 with the corresponding region of TRPV1 displayed a high level of similarity (60% homology/40% identity) (Fig. 1)

  • TRPV4 activation, we constructed a series of point mutations within TM4 and tested the response of the mutant channelexpressed HEK293 cells to the different known TRPV4 stimuli using intracellular Ca2ϩ ([Ca2ϩ]i) measurements and wholecell patch clamp recordings

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Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—Human embryonic kidney cells, HEK293, were grown in Dulbecco’s modified Eagle’s medium containing 10% (v/v) human serum, 2 mM L-glutamine, 2 units/ml penicillin, and 2 mg/ml streptomycin at 37 °C in a humidity-controlled incubator with 10% CO2. Solutions—For electrophysiological measurements, the standard extracellular solution contained (in mM): 150 NaCl, 6 CsCl, 1 MgCl2, 5 CaCl2, 10 glucose, 10 HEPES, buffered at pH 7.4 with NaOH. The osmolarity of this solution, as measured with a vapor pressure osmometer (5 Wescor 5500, Schlag, Gladbach, Germany), was 320 Ϯ 5 mosM. When measuring swelling-activated currents, we used an isotonic solution containing (in mM): 105 NaCl, 6 CsCl, 5 CaCl2, 1 MgCl2, 10 HEPES, 90 D-mannitol, 10 glucose, buffered pH 7.4 with NaOH (320 Ϯ 5 mosM). Data are expressed as mean Ϯ S.E., and significance (p Ͻ 0.01) between individual groups was tested using unpaired Student’s t test

RESULTS
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DISCUSSION
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