Ca 2 / Calmodulin Modulates TRPV 1 Activation by Capsaicin

TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159–212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487–517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature . 389:816–824). When activated, they produce depolarization through the influx of Na , but their high Ca 2 permeability is also important for mediating the response to pain. In particular, Ca 2 influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Neuroscience . 55:1015–1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525– 3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, Ca 2 /calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg 2 , reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by Ca 2 alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca 2 . Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca 2 -independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH 2 terminal domain demonstrated that a stretch of 30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca 2 -dependent manner. The physiological response to pain involves an influx of Ca 2 through TRPV1. Our results indicate that this Ca 2 influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin. key words: desensitization • capsaicin • TRPV1 • calcium • calmodulin I N T R O D U C T I O N The first TRP channel was identified in 1977 as a phototransduction mutant in Drosophila (Minke, 1977). Since that time the TRP channel family has grown to include at least 20 members (Clapham et al., 2001). The importance of TRP channels is demonstrated by the wide variety of their proposed functions: invertebrate phototransduction, responding to painful stimuli, responding to moderate temperature changes; repletion of intracellular calcium stores, receptor–mediated excitation, and modulation of the cell cycle (for reviews see Clapham et al., 2001; Montell, 2001; Montell et al., 2002). Despite the clear physiological importance of TRP channels, little is known about what regulates their function. One type of TRP channel, TRPV1, is a polymodal receptor that integrates a number of painful stimuli. These stimuli include: noxious heat, with a threshold of 42 C; extracellular acidification, with a pK a of 5.3 (Caterina et al., 1997; Jordt et al., 2000); anandamide and other arachidonic acid metabolites (Smart et al., 2000); and capsaicin, a pungent extract from plants in the Capsicum family (Szallasi and Blumberg, 1999; Caterina and Julius, 2001). Although all the details of the capsaicin binding site are not known, it has been shown to bind from the intracellular side (Jung et al., 1999), though this result is controversial (Vyklicky et al., 2003). With intracellular NH 2 and COOH-terminal domains, six putative membrane-spanning domains, and sequence similarity in the pore region, TRP channels fall within the superfamily of ion channels that includes voltage-gated Na , K , and Ca 2 channels and cyclic nucleotide–gated (CNG) channels (Jan and Jan, 1990, 1992). For those ion channels within this superfamily for which stoichiometry has been directly examined, all have been shown to be composed of four six transmembrane domain subunits or pseudosubunits, with auxiliary subunits sometimes present as well (Clapham et al., 2001). TRPV1 channels are nonselective cation channels that do not discriminate among monovalent cations and are Tamara Rosenbaum and Ariela Gordon-Shaag contributed equally to