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. 2016 Jul;157(7):1515-24.
doi: 10.1097/j.pain.0000000000000556.

Agonist-dependence of Functional Properties for Common Nonsynonymous Variants of Human Transient Receptor Potential Vanilloid 1

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Free PMC article

Agonist-dependence of Functional Properties for Common Nonsynonymous Variants of Human Transient Receptor Potential Vanilloid 1

Sen Wang et al. Pain. .
Free PMC article

Abstract

Transient receptor potential vanilloid 1 (TRPV1) is a polymodal receptor activated by capsaicin, heat, and acid, which plays critical roles in thermosensation and pain. In addition, TRPV1 also contributes to multiple pathophysiological states in respiratory, cardiovascular, metabolic, and renal systems. These contributions are further supported by evidence that variations in the human TRPV1 (hTRPV1) gene are associated with various physiological and pathological phenotypes. However, it is not well understood how the variations in hTRPV1 affect channel functions. In this study, we examined functional consequences of amino acid variations of hTRPV1 induced by 5 nonsynonymous single-nucleotide polymorphisms (SNPs) that most commonly exist in the human population. Using electrophysiological assays in HEK293 cells, we examined 9 parameters: activation, Ca permeation, and desensitization after activation by capsaicin, acid, and heat. Our results demonstrated that the 5 SNPs differentially affected functional properties of hTRPV1 in an agonist-dependent manner. Based upon the directionality of change of each phenotype and cumulative changes in each SNP, we classified the 5 SNPs into 3 presumptive functional categories: gain of function (hTRPV1 Q85R, P91S, and T469I), loss of function (I585V), and mixed (M315I). These results reveal a spectrum of functional variation among common hTRPV1 polymorphisms in humans and may aid mechanistic interpretation of phenotypes associated with nonsynonymous hTRPV1 SNPs under pathophysiological conditions.

Conflict of interest statement

Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Common nonsynonymous single-nucleotide polymorphism (SNPs) of human transient receptor potential vanilloid 1 (TRPV1) examined in this study. (A) RNA-sequencing of human DRG RNA samples pooled from 19 subjects revealed 10 splice variant forms of human TRPV1 (1–10). (B) FPKM (Fragments Per Kilobase of Exon Per Million Fragments Mapped) values of variants are plotted. (C) Identity and minor allele frequency (MAF) of 5 nonsynonymous TRPV1 SNPs. Minor allele frequency was obtained from the University of California Santa Cruz Genome browser (http://genome.ucsc.edu). (D) Schematics of TRPV1 structure and the location of amino acid variations induced by 5 SNPs. Human TRPV1 Q85, P91, M315I are located in amino terminal domain. Human TRPV1 T469I is located in extracellular linker between transmembrane domains 1 and 2. Human TRPV1 I585V is located in the fifth transmembrane domain.
Figure 2
Figure 2
Effects of amino acid variations of human transient receptor potential vanilloid 1 (hTRPV1) on capsaicin-induced and acid-induced activation (A) Representative current traces recorded from HEK293 cells transfected with TRPV1 WT. Currents were evoked by the consecutive application of 4 progressively increasing concentration of capsaicin. Bath solution did not contain Ca2+ (ext. B with 5 mM EGTA). Current densities were evaluated at −60 mV. Scale bar, 200 pA/pF and 1 min. (B and C) Capsaicin concentration-dependent relationships of hTRPV1 WT and variants. The 5 SNPs were arbitrarily divided into 2 groups and presented along with WT. Current amplitudes in each capsaicin concentration were normalized to the maximal response in each cell. Numbers in parenthesis within label represent the number of observations. The lines were derived from data fitting using sigmoidal logistics equation with fixed minimum and maximum value of 0 and 1, respectively. *P < 0.05 in extra sum-of-squares F test vs WT. Log(EC50) and Hill slope of WT, −6.87 ± 0.05 and 1.34 ± 0.15; Q85R, −6.79 ± 0.06 and 1.99 ± 0.38; P91S, −6.82 ± 0.05 and 1.3 ± 0.18; M315I, −6.73 ± 0.06 and 1.43 ± 0.27; T469I, −6.87 ± 0.06 and 1.27 ± 0.16; I585V, −6.67 ± 0.06 and 1.31 ± 0.27. (D) Representative current traces recorded from HEK293 cells transfected with TRPV1 WT (top) or TRPV1 A401T (bottom). Currents were evoked by the consecutive application of 4 progressively increasing concentrations of proton. Bath solution did not contain Ca2+ (ext. B with 5 mM EGTA). Vm = −60 mV. Scale bar, 200 pA/pF and 1 min. (E and F) Proton concentration-dependent relationships of hTRPV1 WT and variants. The 5 SNPs were arbitrarily divided into 3 groups and presented along with WT. Numbers in parenthesis within label represent the number of observations. *P <0.05 in extra sum-of-squares F test vs WT. EC50 and hill slope of WT, pH 5.91 ± 0.04 and 1.49 ± 0.33; Q85R, pH 6.03 ± 0.07 and 1.51 ± 0.60; P91S, pH 6.05 ± 0.05 and 1.55 ± 0.46; M315I, pH 5.97 ± 0.04 and 1.80 ± 0.69; A401T, pH 5.71 ± 0.08 and 2.44 ± 0.65; D412N, pH 5.94 ± 0.06 and 2.09 ± 1.65; T469I, pH 6.05 ± 0.05 and 1.51 ± 0.43; I585V, pH 5.80 ± 0.07 and 1.86 ± 0.55.
Figure 3
Figure 3
Effects of amino acid variations of human transient receptor potential vanilloid 1 (hTRPV1) on heat-induced activation. (A and B) Superimposed representative current–temperature relationships of hTRPV1 WT (black) and hTRPV1 SNPs in response to a heat ramp. Currents are relative to the respective maximum response of each cell. Vm =−60 mV. (C) Representative Arrhenius plot of hTRPV1 WT showing a threshold temperature. (D and E) Plots of threshold temperature (D) and ΔH° (E) of hTRPV1 WT and TRPV1 SNPs. Symbols represent scatter dot plot of raw data and the superimposed horizontal lines represent mean ± SEM. *P < 0.05; #P < 0.005 vs TRPV1 WT, Student t test. Numbers of observations are indicated within bars.
Figure 4
Figure 4
Effects of amino acid variations of human transient receptor potential vanilloid 1 (hTRPV1) on the relative permeability of Ca2+ to Na+ (PCa/PNa) after capsaicin-induced and heat-induced activation. (A and B) Representative current traces recorded from HEK293 cells transfected with TRPV1 WT (A) or TRPV1 M315I (B). Currents were evoked by 0.3 μM capsaicin in the presence of 10 mM extracellular Ca2+ (ext C) followed by 0 mM Ca2+ (ext B). The reversal potential (Erev) was measured at the potential where the capsaicin-evoked current traces intersected with the baseline in the presence (upward arrow) or absence (downward arrow) of Ca2+. (C) Relative permeability of Ca2+to Na+(PCa/PNa) upon activation by capsaicin calculated using the Equation described in Methods. *P < 0.05; #P < 0.0005, Student t test. (D and E) Representative current traces recorded from HEK293 cells transfected with TRPV1 WT (A) or TRPV1 I585V (B). Currents were evoked by the application of heat (~43–46°C) in the presence of 10 mM extracellular Ca2+(ext C) followed by 0 mM Ca2+(ext B). Erev was measured at the potential where the heat-evoked current traces intersected the baseline in the presence (upward arrow) or absence (downward arrow) of Ca2+. (F) PCa/PNa upon activation by heat. *P < 0.05; **P < 0.01, Student t test.
Figure 5
Figure 5
Effects of amino acid variations of human transient receptor potential vanilloid 1 (hTRPV1) on PCa/PNa following acid-induced activation of hTRPV1 SNPs and comparison of modality-dependence of PCa/PNa. (A and B) Representative current traces recorded from HEK293 cells transfected with TRPV1 WT (A) or TRPV1 P91S (B). Currents were evoked by the application of acid (pH 4.5) in the presence of 10 mM extracellular Ca2+ (ext C) followed by 0 mM Ca2+ (ext B). Erev was measured at the potential where the acid-evoked current traces intersected with the baseline in the presence (upward arrow) or absence (downward arrow) of Ca2+. (C) PCa/PNa ratio upon activation by acid. *P < 0.05; ***P < 0.005, Student t test. (D) Comparison of PCa/PNa ratio upon activation by capsaicin (black), acid (blue), or heat (red) in hTRPV1 WT and SNPs. Data (mean ± SEM) are derived from Figs. 4C, 4F, and 5C. Three groups of PCa/PNa values upon activation by capsaicin, acid, and heat were significantly different in hTRPV1 WT and each SNP (P < 0.005 in one-way analysis of variance) except A401T (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.005; #P < 0.001, Holm–Sidak post hoc analysis.
Figure 6
Figure 6
Effects of amino acid variations of hTRPV1 on capsaicin-induced and acid-induced desensitization (A) Representative current traces recorded from HEK293 cells transfected with TRPV1 WT (upper panel) or TRPV1 M315I (lower panel). Currents were evoked by 2 consecutive applications of capsaicin (1 μM) in the presence of 0.3 mM extracellular Ca2+(ext C with 0.3 mM Ca2+). Membrane potential (Vm) = −60 mV. Scale bar, 200 pA/pF and 1 min. (B) Ratios of second response/first response after 2 consecutive applications of capsaicin. *P < 0.05, Student t test. (C) Representative current traces recorded from HEK293 cells transfected with TRPV1 WT (upper panel) or TRPV1 Q85R (lower panel). Currents were evoked by 2 consecutive applications of acid (pH 4.5) in the presence of 2 mM extracellular Ca2+ (ext A with MES and 100 μM amiloride). Vm = −60 mV. Scale bar, 100 pA/pF and 1 min; *P < 0.05. (D) The ratio of second response/first response following 2 consecutive applications of acid. *P < 0.05, Student t test.

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