Desensitization of Capsaicin-activated Currents in the Vanilloid Receptor TRPV1 Is Decreased by the Cyclic AMP-dependent Protein Kinase Pathway

Abstract

Proinflammatory prostaglandin E2 is known to sensitize sensory neurons to noxious stimuli. This sensitization is mediated by the cAMP-dependent protein kinase (PKA) signal pathway. The capsaicin receptor TRPV1, a non-selective cation channel of sensory neurons involved in the sensation of inflammatory pain, is a target of PKA-mediated phosphorylation. Our goal was to investigate the influence of PKA on Ca 2؉ -dependent desensitization of capsaicin-activated currents. By using site-directed mutagenesis, we created point mutations at PKA consensus sites and studied wild-type and mutant channels transiently expressed in HEK293t cells under whole-cell voltage clamp. We found that forskolin, a stimulator of adenylate cyclase, decreased desensitization of TRPV1. The selective PKA inhibitor H89 inhibited this effect. Mimicking phosphorylation at PKA consensus sites by replacing Ser-6, Ser-116, Thr-144, Thr-370, Ser-502, Ser-774, or Ser-820 with aspartate resulted in five mutations (S116D, T144D, T370D, S774D, and S820D) that exhibited decreased desensitization as well. However, disrupting phosphorylation by replacing respective sites with alanine resulted in four mutations (S6A, T144A, T370A, and S820A) with desensitization properties resembling those of the aspartate mutations. Significant changes in relative permeabilities for Ca 2؉ over Na ؉ or in capsaicin sensitivity could not explain changes in desensitization properties of mutant channels. In mutations S116A, S116D, T370A, and T370D, pretreatment of cells with forskolin did not reduce desensitization as compared with wild-type and other mutant channels. We conclude that Ser-116 and possibly Thr-370 are the most important residues involved in the mechanism of PKA-dependent reduction of desensitization of capsaicin-activated currents.

Agonist- and Ca2+-dependent Desensitization of TRPV1 Channel Targets the Receptor to Lysosomes for Degradation

“…6C, when applying our prolonged agonist exposure protocol (1 M capsaicin for 20 min), TRPV1-S116D-expressing cells displayed ϳ60% desensitization (R pH2/pH1 ϭ 0.37 Ϯ 0.01, n ϭ 506, n ϭ 5) as compared with 90% observed with TRPV1-WT (R pH2/pH1 ϭ 0.10 Ϯ 0.01%, n ϭ 409, n ϭ 8). We then asked whether the observed decrease in desensitization, which agrees with results reported by others (8,9), is because of decreased internalization of the S116D mutant. Using surface biotinylation/avidin pulldown assays, we found that in contrast to wild type channels, surface levels of TRPV1-S116D were not affected by capsaicin incubation, as revealed by the levels of the mutant protein, which remained similar to those of cells exposed to vehicle (Fig.…”

“…TRPV1 desensitization is a process markedly depending on Ca 2ϩ and involves various intracellular signaling pathways (6). Thus, dephosphorylation by the phosphatase calcineurin of TRPV1 previously phosphorylated by protein kinase A (PKA) or Ca 2ϩ -calmodulin-dependent kinase II leads to TRPV1 desensitization (7)(8)(9)(10). Several other mechanisms like binding of molecules such as calmodulin (11)(12)(13), ATP (13), or AKAP150 (14) or the depletion of phosphoinositol 4,5-diphosphate from the plasma membrane (15-18) also modulate receptor desensitization.…”

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.…”

TRP Channels and Pruritus

“…Besides direct activation mechanisms, several molecules are able to act on TRPV1 via first activating their specific receptors and then initiating downstream signaling pathways leading to the sensitization of TRPV1. Indeed, bradykinin [183,184], ATP [185], lipoxygenase products [182,183], prostaglandins [186,187], histamine [188], various neurotrophins (NGF, neurotrophin-3 and -4) [91,102,184], TNF- [189,190], pro-inflammatory chemokines [191], and PAR2 [192,193] were all reported to sensitize TRPV1 via various signal transduction pathways. TRPV1 was shown to be modulated by the protein kinase C (PKC) [183,[194][195][196], phospholipase C (PLC), and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) [184,[197][198][199][200] systems as well as by intracellular ATP [201].…”