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.

Department of Cellular Neurophysiology Institute of Physiology Academy of Sciences of the Czech Republic Videnska 1083 142 20 Prague 4 Czech Republic

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