These data suggest that the R702 residue may stabilize the region where LC-CoAs interact with the channel, as mutation of this residue results in gating instability and altered channel kinetics. We propose that LC-CoAs interact with the same C-terminal basic residues as PIP2, likely via the negatively charged phosphate groups on the CoA moiety. It has been previously been shown that PIP2 interacts with a hydrophobic pocket formed by S4–S5 linker voltage-sensing region. However, we did not observe any alterations to the current-voltage relationship in the presence of palmitoyl CoA, suggesting, unlike PIP2, LC-CoAs are not interacting with voltage-sensing domains of the TRPV1 channel. Our data also show that LC-CoAs modulate TRPV1 channel activity in a saturation and side-chain length dependent manner. Increasing unsaturation decreases the magnitude of efficacy of LC-CoAs. We propose that the acyl side chain can partition into the membrane, leading to allosteric alterations in TRPV1 protein structure that result in changes in channel activity. Increasing side-chain length may strengthen the membrane partitioning of the LC-acyl tail resulting in increases in channel activity. Similarly, increasing unsaturation by the addition of double bonds would increase mobility and decrease lipophilicity of the acyl tail that may reduce acyl tail/membrane interactions and the magnitude of TRPV1 activation. This mechanism is similar to that proposed to play a role in LC-CoA activation of the KATP channel. In support of this notion, it has been shown that LCCoAs associate with membranes through insertion of the acyl side chain into the bilayer, with the interaction increasing with longer side chains. Furthermore, LC-CoAs may aggregate near areas of membrane curvature, such as membrane proteins, resulting higher local concentrations of LC-CoAs in the vicinity of TRPV1 channels. The combination of the increased membrane interaction and decreased lateral diffusion rate of saturated and longer chain LC-CoAs may increase the longevity of TRPV1 channel opening by maintaining the CoA head group in closer contact to the basic residues identified in this study. Intracellular LC-CoA levels are highly buffered by LC-CoA binding proteins, sterol carrier proteins and fatty acid bindings proteins. These binding proteins are thought to be essential for correct cellular function by keeping unbound LC-CoA levels in the nanomolar range. Interestingly, our finding that the palmitoyl CoA EC50 for the TRPV1 channel is 91 nM, suggests that the observed LC-CoA modulation of the TRPV1 channel is physiologically relevant. Furthermore, LC-CoA levels fluctuate in response to alterations in metabolic status, transmembrane fatty acid transport and activity/expression of acyl CoA synthetases such as ACSL-1. Indeed, overexpression of ACSL-1 in either 1) cells expressing recombinant TRPV1 channels.