Regulation of Ca2+-dependent Desensitization in the Vanilloid Receptor TRPV1 by Calcineurin and cAMP-dependent Protein Kinase

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 Ca 2؉ -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 Ser 116 and Thr 370 . In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca 2؉ -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 A⅐cyclophilin A complex (CsA⅐CyP), 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 CsA⅐CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA⅐CyP 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 CsA⅐CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca 2؉ -dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr 370 as a key amino acid residue.

Mechanism of Ca2+-dependent desensitization in TRP channels

“…38 In contrast, phosphorylation by protein kinase A and protein kinase C has been proposed to slow desensitization in TRPV1 channels, 39,40 and protein kinase C has also been implicated in reversing desensitization of TRPM4 channels. 41 Ca 2+ /CaM regulated phosphorylation/dephosphorylation has also been proposed: phosphorylation of TRPC5 by myosin light chain kinase is associated with slowing of desensitization 42 and dephosphorylation of TRPV1 by calcineurin is reported to promote desensitization.…”

Rapid, Opioid-sensitive Mechanisms Involved in Transient Receptor Potential Vanilloid 1 Sensitization

“…Notably, both PKA phosphorylation sites as well as putative SNARE interaction sites are located on the N-terminal of TRPV1 (8,47,48), such that phosphorylation of TRPV1 by PKA could lead to enhancement of these exocytosis pathways through to-date unknown mechanisms. Indeed, PKA-dependent trafficking pathways have been reported for a number of receptors and ion channels including the chloride-selective anion channel CFTR and AMPA receptor (49,50).…”

Ser/Thr-phosphoprotein phosphatases in chondrogenesis: neglected components of a two-player game

“…This hypothesis was confirmed with de facto electrophysiological data by Fomina and Levitan who demonstrated that in rat pituitary lactotrop cells inhibition of CaN activity by CsA has led to reduced ionic currents via voltage-gated Ca 2+ channels [91]. Since then, CaN has been shown to modulate a number of ion channels including inhibition of the arachidonate-regulated Ca 2+ channels (ARC channels that are closely related to the store-operated CRAC channel Orai proteins) [92]; the intracellular Ca 2+ release channels inositol trisphosphate receptor (IP 3 R) and ryanodine receptor (RyR) [61]; or the vanilloid receptor TRPV1 [93]. In smooth muscle cells, PP1 was described to increase the open probability of the large-conductance, voltage-and Ca 2+ -gated K + (BK Ca ) channel; in contrast, PP1 inhibited the A-type K + channel 19-pS in mouse colon cells through its fast inactivation mechanism.…”