Figure 1.
The effect of palmitoyl CoA on TRPV1 currents.
(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.
Figure 2.
The role of Ca2+ in PIP2 and palmitoyl CoA regulation of TRPV1 currents.
(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.
Figure 3.
Chain-length, saturation and voltage-dependent effects of LC-CoA modulation of TRPV1 currents.
(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.
Figure 4.
The effect of LC-CoAs on [Ca2+]i.
(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.
Figure 5.
The role of K711 in LC-CoA modulation of TRPV1 channel function.
(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.
Table 1.
Kinetic parameters of WT TRPV1 and K711A mutant currents in the presence and absence of palmitoyl CoA.
Figure 6.
The role of R702 in LC-CoA modulation of TRPV1 channel function.
(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.
Figure 7.
Model of LC-CoA interaction with conserved residues in human TRPV family.
(A) Amino acid sequence alignment of the proximal C-terminal residues in human TRPV1-TRPV6. (B) Whole protein transmembrane view of the apo-state TRPV1 channels helical TRP domain interacting with the 18 carbon LC-CoA stearoyl CoA. (C) Synaptic view of the helical TRP domains basic residues R702 and K711 interacting with the 18 carbon LC-CoA stearoyl CoA. All molecular modeling is based on the 3.4 Å resolution TRPV1 structure determined by electron cryo-microscopy (PDB# 3J5P, [37], [38]). Analysis was performed using Pymol software.
Table 2.
Kinetic parameters of WT TRPV1 and R702A mutant currents in the presence and absence of palmitoyl CoA.