Article Figures & Data
Figures
- Fig. 1.
A, Amino acid residues in the sixth transmembrane domain of the rat VR1 receptor are highly conserved. Rat SIC and rat VRL-1 are the recently cloned stretch-inhibitable nonselective cation channel and the vanilloid receptor like protein 1, respectively. The various TRP sequences are from the proteins in the transient receptor potential family. The sodium channel sequences are from the sixth transmembrane region of the different voltage-gated sodium channels. Shaded boxesindicate identical amino acids, and open boxes show the highly conserved amino acids. B, Putative membrane topology of the rat VR1 receptor. The locations of amino acids that were mutated in the rat VR1 receptor are indicated. Shaded circle P in the third outer loop represents the P613L mutation.Shaded circles C and P in the putative pore region in the membrane represent the CP621GL double mutant, andshaded circles N, M, and Lin the sixth transmembrane region represent the NML676FAP mutant.Solid circle H represents the H614T mutation. Mutated VR1 receptors were characterized by both restriction enzyme mapping and DNA sequencing. C, Western blot analysis of CHO cells transfected with wild-type rat VR1 receptor (WT), NML676FAP mutant (NML), or pUC19 plasmid (CON) using the N-terminal VR1 antibody (1:1000 dilution) and enhanced chemiluminescence (Amersham Pharmacia Biotech). The molecular weight marker is indicated.
- Fig. 2.
Comparison of wild-type and mutant VR1 receptor function in CHO cells. A, Wild-type VR1 responds with inward currents to 1 μm CAP, pH 5.0, and 50 nm RTX. B, In contrast, CHO cells expressing the NML676FAP mutant do not respond to 1 μm CAP or 50 nm RTX but respond partially to a pH of 5.0 (inset, proton response on a magnified scale; calibration: 200 pA, 200 msec). C, Control cells transfected with vector alone do not respond to any agonist. All experiments were performed in calcium-free external solution to prevent desensitization. D, Mean current density ± SEM of responses of wild-type VR1 and of NML676FAP, P613L, CP621GL, and H614T mutant receptors to capsaicin (1 μm; white bars), pH 5.0 (hatched bars), or resiniferatoxin (50 nm; gray bars). Note the break in the current density axis to emphasize the proton response of the NML676FAP mutant. *Significant difference (p < 0.005) between responses of the NML676FAP mutant and nontransfected CHO cells.
- Fig. 3.
The NML676FAP mutant is expressed and localized similarly to wild-type VR1 when transiently transfected in CHO cells. Shown are fluorescence images of EGFP (left) and a secondary antibody labeling a VR1 N-terminal antibody (right) in cells cotransfected with EGFP and either wild-type VR1 (top) or the NML676FAP mutant (bottom). Images were obtained as described in Materials and Methods.
- Fig. 4.
The NML676FAP mutant fails to respond to even high concentrations of capsaicin. Shown are Fluo3 fluorescence responses (arbitrary units) of CHO cells expressing wild-type VR1 (A) or the NML676FAP mutant (B) assessed in 96-well plates using an FLIPR spectrofluorimeter. Each symbol (identical for A, B) represents the average fluorescence of eight wells at the indicated concentration of capsaicin (micromolar).
- Fig. 5.
The vanilloid binding site is intact but altered in the NML676FAP mutant. The inward current elicited by protons (pH 5.0 solution) is inhibited by 10 μm capsazepine (Cpz) in CHO cells expressing either the wild-type VR1 receptor (A) or the NML mutant receptor (B). The inhibition is reversible, because the proton-induced inward currents recover after washout of Cpz. The experiment was performed in an external solution lacking calcium to reduce desensitization. Calibration, 50 sec. C, Specific binding of [3H]RTX to CHO cell membranes expressing wild-type VR1 (filled circles), the NML676FAP mutant (open circles), or pUC19 as a control (filled inverted triangles) normalized to theB max values in each case.Curves represent fits of the Hill equation to the data as described in Materials and Methods with the following parameters: wtVR1, B max = 139.4 ± 3.1 fmol/mg protein; K d, 43 pm; andn = 1.36; NML676FAP,B max = 260.2 ± 15.6 fmol/mg protein; K d, 1670 pm; andn = 1.51.
- Fig. 6.
Calcium-dependent desensitization of the inward currents induced by capsaicin and protons. CHO cells expressing the wild-type VR1 receptor (A–D) exhibit acute desensitization and tachyphylaxis when treated with 1 μmCAP (A) or a pH 5.0 solution (C) in the presence of external calcium. Removal of calcium from the external solution blocks desensitization (B, D). The proton response in CHO cells transfected with the NML676FAP mutant also exhibits desensitization in the presence (E) but not in the absence (F) of calcium in the external solution. Calibration, 50 sec.
- Fig. 7.
The NML676FAP mutant functions as a dominant-negative subunit. A, CHO cells were transiently transfected with either wild-type VR1 receptor (VR1) or equal amounts of wtVR1 and NML676FAP mutant receptors (VR1 + DN). Inward current density elicited by 1 μm capsaicin in CHO cells transfected with wild-type VR1 (n = 3) was significantly greater than in cells cotransfected with VR1 + DN (n = 12; *p < 0.001, Student's t test).B, Inward currents elicited by 1 μmcapsaicin or protons, pH 5.0, in a CHO cell clone stably expressing the rat VR1 receptor (n = 15) were significantly attenuated by the transiently transfected mutant receptor (capsaicin responses of VR1 + DN, n = 12; proton responses of VR1 + DN, n = 8; *p < 0.002, Student's t test). In contrast, transient transfection of wild-type VR1 receptor into the CHOVR1 clone (VR1 +WT) had no significant effect on capsaicin-induced inward current (n = 3). The current measurements were done in calcium-free external solution. All values of currents were divided by membrane capacitance (picoamperes per picofarads) to normalize for cell size differences.
- Fig. 8.
Assessment of capsaicin receptor functional stoichiometry. A, [3H]RTX binding was measured in CHO cells expressing different ratios of wild-type VR1 and a control plasmid, pUC19, transfected at a constant cDNA per sample of 4.0 μg. MeasuredB max values are plotted against predictedB max values normalized at the lowest wild-type VR1 concentration (0.23 μg). Numbers for each data point indicate concentrations of wild-type VR1 cDNA used, and the solid line represents identity (slope = 1.0). B, Current density values for capsaicin (1 μm)-induced currents in CHO cells transfected with different ratios of wild-type VR1 and NML676FAP plotted against the percentage of mutant cDNA. Data points represent mean ± SEM values of 8, 4, 8, 4, 9, and 13 measurements, respectively, for increasing percentage of mutant subunit.Lines represent predictions (scaled to the maximum current density) of a binomial distribution of assembled subunit combinations according to:
where C = n!/i! (n − i)!, n is the subunit stoichiometry, i is number of required subunits to block function − 1, and x is the fraction of wild-type subunits expressed of the total. Solid lines represent nvalues of 2, 4, and 8 for i = 1, and the dashed line corresponds to n = 4 and i = 2 (i.e., a tetrameric channel requiring two mutant subunits to block function). C, Specific binding of [3H]RTX to CHO cell membranes expressing ratios of wild-type VR1 to NML676FAP of 1:0 (filled circles), 0:1 (open circles), 4:1 (filled inverted triangles), or 1:4 (open triangles) normalized to the B
max values in each case.Curves represent fits of the Hill equation to each data set.D, K
d values derived from Hill equation fits to [3H]RTX binding curves for CHO cells expressing wild-type VR1, the NML676FAP mutant, or the indicated ratios of wild type to mutant. Bars are mean ± SEM values for two or three measurements in each case. Solid circles represent the predictions for a population of tetrameric receptors with binomially distributed assembly combinations assuming a single mutant subunit is sufficient to change the RTX affinity to that of the homomeric NML676FAP mutant receptor.
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