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Abstract
The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A.cyclophilin A complex (CsA.CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA.CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA.CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA.CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue.
Highlights
EXPERIMENTAL PROCEDURESSite-directed Mutagenesis and Transient Transfection—Mutagenesis of rat TRPV1-cDNA was performed with rTRPV1-pcDNA3 by means of the transformer site-directed mutagenesis kit (BD Biosciences Clontech, Palo Alto, CA) as described previously [19]
Heat (Ͼ42 °C), capsaicin, protons, [1, 2], the endogenous cannabinoid anandamide [3], lipoxygenase products, and other lipids related to arachidonic acid [4] and ethanol [5]
We found that intracellular application of the cyclosporin A1⁄7cyclophilin A complex (CsA1⁄7CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-wild type (WT) currents
Summary
Site-directed Mutagenesis and Transient Transfection—Mutagenesis of rat TRPV1-cDNA was performed with rTRPV1-pcDNA3 by means of the transformer site-directed mutagenesis kit (BD Biosciences Clontech, Palo Alto, CA) as described previously [19]. Chemicals and Solutions—Capsaicin (8-methyl-N-vanillyl-6-nonenamide) and cyclosporin A (CsA; both Sigma-Aldrich) were dissolved in absolute ethanol to give stock solutions of 10 mM. Cyclophilin A (CyP; Sigma-Aldrich) was dissolved in Tris-Cl, pH 7.4, containing HEPES, 1,4-dithio-DL-threitol, phenylmethanesulfonyl fluoride, and sodium azide to give a stock solution of 20 M. Standard bath solutions contained 70 mM NaCl, 70 mM choline Cl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose (adjusted to pH 7.4 with tetramethylammonium hydroxide). Pipette solution contained 140 mM KCl, 2 mM MgCl2, 5 mM EGTA, and 10 mM HEPES (adjusted to pH 7.4 with KOH). Electrophysiological Technique and Data Analysis—Currents were recorded at room temperature with the whole cell configuration of the patch-clamp method. An unpaired Student’s t test (SigmaStat; SSPS Science, Chicago, IL) was used to evaluate the significance of changes in mean values. An unpaired Student’s t test (SigmaStat; SSPS Science, Chicago, IL) was used to evaluate the significance of changes in mean values. p values Ͻ0.05 were considered statistically significant
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