The role of R702 in LC-CoA modulation of TRPV1 channel function.

<p>(A) Representative current trace of WT TRPV1 during repeated exposure to 1 µM capsaicin. (B) Representative current traces of the R702A TRPV1 mutant in the presence or absence of 1 µM palmitoyl CoA following repeated exposure to acidic activating solution of pH 5.5. (C) Grouped data of the effect of the R702A mutation on TRPV1 channel kinetics in response to acidic solution of pH 5.5. (D) Grouped data of the effect of 1 µM palmitoyl CoA on WT and R702A TRPV1 channel kinetics in response to the pH 5.5 solution. *P<0.05, **P<0.01. Dashed line denotes zero current level.</p

The role of Ca²⁺ in PIP₂ and palmitoyl CoA regulation of TRPV1 currents.

<p>(A) Representative whole-cell recordings (n = 7) and (B) inside-out recording (n = 5) of currents elicited by 1 µM capsaicin in the absence of Ca²⁺. (C) Grouped data of the effect of Ca²⁺ on the desensitization of TRPV1 currents. (D) Representative inside-out recordings and histograms of the effect of 1 µM palmitoyl CoA (n = 7), 25 µM PIP₂ (n = 4) or 1 µM palmitoyl CoA combined with 25 µM PIP₂ (n = 9) on currents elicited by 1 µM capsaicin (n = 9) in the absence of Ca²⁺. (E) Representative inside-out recordings and grouped data of the application of 1 µM capsaicin (n = 6), 1 µM palmitoyl CoA (n = 8) or 25 µM PIP₂ (n = 8) in the presence of 2 mM Ca²⁺. (F) Representative inside-out recordings and grouped data of the effect of 1 µM capsaicin (n = 12), 1 µM palmitoyl CoA (n = 11) or 25 µM PIP₂ (n = 10) in the presence of 2 mMCa²⁺ and 2 µM U73122. *P<0.05. Dashed line denotes zero current level.</p

The role of K711 in LC-CoA modulation of TRPV1 channel function.

<p>(A) Amino acid sequence alignment of TRPV1. (B) Representative current trace of WT TRPV1 during repeated exposure to 1 µM capsaicin. (C) Representative current traces of the K711A TRPV1 mutant in the presence or absence of 1 µM palmitoyl CoA following repeated exposure to 1 µM capsaicin. (B,C) inset: Overlay of WT and K711A current traces normalized to maximum current illustrate the similar kinetics. (D) Grouped data of the effect of the K711A mutation on TRPV1 channel kinetics in response to 1 µM capsaicin. (E) Grouped data of the effect of 1 µM palmitoyl CoA on WT and K711A TRPV1 channel kinetics in response 1 µM capsaicin. *P<0.05, **P<0.01. Dashed line denotes zero current level.</p

The effect of palmitoyl CoA on TRPV1 currents.

<p>(A) Representative whole-cell recordings elicited by 1 µM capsaicin (n = 5) in the presence of (B) 25 µM PIP₂ (n = 12) or (C) 1 µM palmitoyl CoA (n = 9). (D) Representative whole-cell recordings elicited by acidic pH (pH = 5.5, n = 5) in the presence of (E) 1 µM palmitoyl CoA (n = 5). (F) Grouped data of the effects of PIP₂ and palmitoyl CoA on TRPV1 currents. *P<0.05. Dashed line denotes zero current level.</p

Chain-length, saturation and voltage-dependent effects of LC-CoA modulation of TRPV1 currents.

<p>(A) Representative inside-out recordings of the application various LC-CoAs (0.1 µM) in the presence, or (B) absence of 1 µM capsaicin. (C) Grouped data of the effect of 0.1 µM LC-CoAs in the presence or absence of 1 µM capsaicin. (D) I–V plot of currents elicited by 1 µM capsaicin in the presence of increasing concentration of palmitoyl CoA. (E) Concentration-effect curve of palmitoyl CoA modulation of currents elicited by 1 µM capsaicin. *P<0.05.</p

Kinetic parameters of WT TRPV1 and R702A mutant currents in the presence and absence of palmitoyl CoA.

<p>Currents were elicited by pH5.5. * Significantly different from values in the WT control (first pulse) (P<0.05). ** Significantly different from values in the WT control (second pulse) (P<0.05).</p

The effect of LC-CoAs on [Ca²⁺]_i.

<p>(A) Representative intracellular paired pulse Ca²⁺ recordings and grouped data of the effect of intracellular LC-CoAs on capsaicin-elicited (1 µM) Ca²⁺ levels in tsA201 cells expressing recombinant TRPV1 channel following repeated exposure to capsaicin, Ad-Scramble (n = 5) and Ad-ACSL-1 (n = 6) or (B) Jurkat 6.1 T-cells expressing endogenous channels, Ad-Scramble (n = 8) and Ad-ACSL-1 (n = 9). (C) Representative intracellular Ca²⁺ recordings and grouped data of the effect of intracellular LC-CoAs on Jurkat 6.1 T-cells, Ad-Scramble (n = 8) and Ad-ACSL-1 (n = 9) following application of 20 µg/ml PHA. Grouped data showing the effect of 1 µM capsazepine on intracellular calcium levels in Jurkat 6.1 T-cells, Ad-ACSL-1 (n = 9) and Ad-ACSL-1+CPZ (n = 10) following application of 20 µg/ml PHA. *P<0.05, **P<0.01.</p

Kinetic parameters of WT TRPV1 and K711A mutant currents in the presence and absence of palmitoyl CoA.

<p>Currents were induced by 1 mmol/l capsaicin.</p><p>* Significantly different from values in the WT control (first pulse) (P<0.05).</p><p>** Significantly different from values in the WT control (second pulse) (P<0.05).</p