Rosenbaum T et al. (2004), Ca2+/calmodulin modulates TRPV1 activation by c...

XB-ART-4200

J Gen Physiol 2004 Jan 01;1231:53-62. doi: 10.1085/jgp.200308906.

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Ca2+/calmodulin modulates TRPV1 activation by capsaicin.

Rosenbaum T , Gordon-Shaag A , Munari M , Gordon SE .

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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 Ca2+ permeability is also important for mediating the response to pain. In particular, Ca2+ 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, Ca2+/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg2+, 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 Ca2+ alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca2+. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca2+-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH2-terminal domain demonstrated that a stretch of approximately 30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca2+-dependent manner. The physiological response to pain involves an influx of Ca2+ through TRPV1. Our results indicate that this Ca2+ 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.

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Species referenced: Xenopus
Genes referenced: ank1 trpv1

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	Figure 1. . CaM mediates Ca2+ inhibition of TRPV1 channels expressed in Xenopus oocytes. (A) Current families obtained in the presence of 20 Î¼M capsaicin, 20 Î¼M capsaicin + 500 nM CaM + 50 Î¼M free Ca2+, 20 Î¼M capsaicin + 500 nM CaM, or 20 Î¼M capsaicin + 50 Î¼M free Ca2+, as indicated by the labels. (B) IV relation for currents shown in (A). Circles, capsaicin; squares, capsaicin + Ca2+; and triangles, capsaicin + Ca2+/CaM. (C) Time course of inhibition by and recovery from Ca2+/CaM. The intracellular (bath) solution contained 20 Î¼M capsaicin, 50 Î¼M free Ca2+, and/or 500 nM CaM, as indicated by the bars above the graph. The bar labels correspond to the bar immediately beneath each label.
	Figure 2. . Ca2+/CaM inhibits TRPV1 channels expressed in HEK 293 cells. (A) Current families activated by 20 Î¼M capsaicin, capsaicin + 50 Î¼M free Ca2+, or capsaicin + Ca2+ + 500 nM CaM, as indicated. The current family labeled âwashâ was recorded in capsaicin after Ca2+/CaM had been extensively washed from the patch. (B) IV relations from the current families shown in A. Circles, capsaicin; squares, capsaicin + Ca2+; and triangles, capsaicin + Ca2+/CaM. (C) Kinetics of onset and recovery from Ca2+/CaM regulation. The presence of capsaicin, Ca2+, and CaM were as indicated by the labeled bars.
	Figure 3. . Coexpression of wild-type and mutant CaM with TRPV1 alters the effects of Ca2+ on channel function in oocytes and HEK cells. (A) Box-plot of the percent decrease of capsaicin-activated (20 Î¼M) current in Xenopus oocytes produced by 50 Î¼M free Ca2+ and no added CaM (left, n = 8), 500 nM added CaM, (center, n = 8), or CaMwt coexpressed with TRPV1 and no added CaM (right, n = 4). For the boxes, the line indicates the median of the data, the box surrounds the 25th through 75th percentile of the data, and the whiskers reach the 10th and 90th percentiles. As indicated by the asterisks, both the center and right groups had statistically significant differences compared with Ca2+ and no added CaM. (B) Box-plot of the percent decrease of capsaicin-activated (20 Î¼M) current in HEK cells produced by 50 Î¼M free Ca2+ and either no added CaM (first box, n = 7), 500 nM added CaM (second box, n = 3), CaMwt coexpressed with TRPV1 and no added CaM (third box, n = 6), or CaM1,2,3,4v. coexpressed with TRPV1 and no added CaM (fourth box, n = 6). As indicated by the asterisks, both the center groups had statistically significant differences compared with Ca2+ and no added CaM. For the fourth condition, p exactly equalled 0.05, and therefore did not meet our criterion for significance. (C) Effects of CaMwt and CaM1,2,3,4 overexpression in HEK cells on the response to Ca2+ (50 Î¼M). Open circles represent the data from coexpression of TRPV1 and CaMwt and filled circles represent data from coexpression of TRPV1 and CaM1,2,3,4. Data were obtained from pulses to +60 mV every 3 s. Ca2+ was introduced at time t = 0. (D) Doseâresponse to different Ca2+ concentrations in the presence of exogenous CaM. Data were obtained at a voltage of +60 mV and are normalized to the current obtained in the presence of each Ca2+ concentration with CaM. Data are the mean values from three patches. The smooth curve is a fit with the Hill equation, giving an IC50 for inhibition by Ca2+ in the presence of CaM of 60 Î¼M and a Hill slope of 1.25.
	Figure 4. . Ca2+/CaM inhibition of TRPV1 reflects a decrease in channel open probability. (A) Currents from a patch containing a single TRPV1 channel in the absence of both capsaicin and Ca2+ (top), in the presence of 4 Î¼M capsaicin but no Ca2+ (middle), and in the presence of 4 Î¼M capsaicin and 300 nM free Ca2+ (bottom). Consecutive traces at +60 mV are shown. All-points histograms were made from all data collected under each condition (2 s without capsaicin, 12 s with capsaicin in the absence of Ca2+, and 6.1 s for capsaicin plus Ca2+). Open probabilities were taken from the area under the Gaussian representing open channels relative to the sum of the area under the Gaussians representing the closed and open channels, and were as follows: capsaicin alone = 0.99, capsaicin + Ca2+ = 0.72. For this experiment, the channels were expressed in Xenopus oocytes. (B) Currents in response to steps from a holding potential of â120 mV to between â150 mV and +110 mV, in steps of 20 mV. After the voltage steps the potential was returned to â120 mV. Currents activated by 20 Î¼M capsaicin (top), capsaicin + 50 Î¼M free Ca2+ (middle), or capsaicin + 50 Î¼M free Ca2+ and 500 nM CaM (bottom). (C) Instantaneous currents measured upon the return of the potential to â120 mV plotted versus the step potential preceding this repolarization. Currents activated by 20 Î¼M capsaicin (circles), capsaicin + 50 Î¼M free Ca2+ (squares), or capsaicin + 50 Î¼M free Ca2+ and 500 nM CaM (triangles). The currents were normalized to the maximum current activated by capsaicin (without Ca2+ or CaM). Solid curves are fits with the Boltzmann equation with the following parameters: capsaicin: z = 0.41e0 (elemental charge), V1/2 = â122 mV; Ca2+: z = 0.31e0, V1/2 = â109 mV; Ca2+/CaM: z = 0.2e0, V1/2 = +40 mV.
	Figure 5. . TRPV1 channels interact directly with CaM. (A) TRPV1 binds to CaM immobilized on agarose beads. Lanes 1 and 2 represent the input and lanes 3 and 4 represent the protein eluted from the CaM-agarose beads. Lanes 1 and 3 were from uninjected oocytes and lanes 2 and 4 were from oocytes injected with TRPV1 cRNA. (B) TRPV1 does not interact with beads alone. This control demonstrates that the interaction of TRPV1 and CaM is specific. Lanes 1â3 are from uninjected oocytes and lanes 4â6 are from oocytes injected with TRPV1 cRNA. The beads used in each case were as indicated in the figure. (C) The top panel represents inputs and the lower panel represents protein eluted from the CaM beads. Lanes 1â3 are from uninjected oocytes and lanes 4â6 are from oocytes injected with TRPV1 cRNA. Added Ca2+ and EGTA were as indicated.
	Figure 6. . The TRPV1 NH2-terminal region interacts directly with CaM in a Ca2+-dependent manner. (A) Cartoon diagram of TRPV1 NH2-terminal region (not to scale). The three Ankyrin repeats are shown in green (Ank1â3), the region required for interaction with CaM is shown in blue, and the first transmembrane domain (S1) is shown in white. GST-TRPV1 fusion proteins designated below, aligned in-scale to their corresponding sequence in the NH2 terminus. The numbers within each box refer to the amino acid numbers from TRPV1 encoded in each GST-fusion protein. (B) In vitro CaM binding assay shows that two overlapping GST-fusion proteins can bind CaM in a Ca2+-dependent manner. (Left) Western blot of proteins shown in A under three conditions: (1) Input. This represents the amount of each protein that was loaded on the CaM-agarose beads. For the subsequent two lanes, this amount of protein represents that maximum possible amount that can bind. (2) Proteins that bound to CaM agarose beads in the presence of 2 mM Ca2+. (3) Proteins that bound to CaM agarose beads in the presence of 2 mM EGTA with no added Ca2+. (Right) Bar graph showing the percent of each GST-fusion protein that bound CaM in the presence of Ca2+ (blue) or EGTA (white), as determine by densitometry of the experiment on the left. For each experimental condition (Ca2+ and EGTA), the values were normalized to that of the input lane.
	Figure 7. . NH2-terminal deletions reduce proteins levels as well as the fraction of the protein that binds to CaM. Lanes 1â4 represent the input and lanes 5â8 represent the protein eluted from the CaM beads. Each channel type is as indicated. The experiment was performed in the presence of 2 mM Ca2+.

References [+] :

Bhave, cAMP-dependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylation. 2002, Pubmed