title: Functional changes in the vanilloid receptor subtype 1 channel during and after acute desensitization.
abstract: Agonist-induced desensitization of the transient receptor potential vanilloid receptor-1 (TRPV1) is one of the key strategies that offer a way to alleviate neuropathic and inflammatory pain. This process is initiated by TRPV1 receptor activation and the subsequent entry of extracellular Ca(2+) through the channel into sensory neurones. One of the prominent mechanisms responsible for TRPV1 desensitization is dephosphorylation of the TRPV1 protein by the Ca(2+)/calmodulin-dependent enzyme, phosphatase 2B (calcineurin). Of several consensus phosphorylation sites identified so far, the most notable are two sites for Ca(2+)/calmodulin dependent kinase II (CaMKII) at which the dynamic equilibrium between the phosphorylated and dephosphorylated states presumably regulates agonist binding. We examined the mechanisms of acute Ca(2+)-dependent desensitization using whole-cell patch-clamp techniques in human embryonic kidney (HEK) 293T cells expressing the wild type or CaMKII phosphorylation site mutants of rat TRPV1. The nonphosphorylatable mutant S502A/T704I was capsaicin-insensitive but the S502A/T704A construct was fully functional, indicating a requirement for a specific residue at position 704. A point mutation at the nearby conserved residue R701 strongly affected the heat, capsaicin and pH-evoked currents. As this residue constitutes a stringent CaMKII consensus site but is also predicted to be involved in the interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), these data suggest that in addition to dephosphorylation, or as its consequence, a short C-terminal juxtamembrane segment adjacent to the transient receptor potential box composed of R701 and T704 might be involved in the decelerated gating kinetics of the desensitized TRPV1 channel.
authors.affiliation: Department of Cellular Neurophysiology, Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic.
authors.forename: K
authors.initials: K
authors.lastname: Novakova-Tousova
authors.forename: L
authors.initials: L
authors.lastname: Vyklicky
authors.forename: K
authors.initials: K
authors.lastname: Susankova
authors.forename: J
authors.initials: J
authors.lastname: Benedikt
authors.forename: A
authors.initials: A
authors.lastname: Samad
authors.forename: J
authors.initials: J
authors.lastname: Teisinger
authors.forename: V
authors.initials: V
authors.lastname: Vlachova
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D002211:Capsaicin / Q000494:pharmacology; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D009154:Mutation / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D051381:Rats; D013329:Structure-Activity Relationship; D050916:TRPV Cation Channels / Q000235:genetics / Q000502:physiology*; D013696:Temperature; D014162:Transfection
chemical_list: D019269:Phosphatidylinositol 4,5-Diphosphate; D050916:TRPV Cation Channels; C493922:Trpv1 protein, rat; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002211:Capsaicin; D002118:Calcium