The transient receptor potential vanilloid 1 (TRPV1) channel is activated by capsaicin, heat, and protons. Residues in the outer pore loop (E600 and E648) have been shown to be involved in proton gating, but the conformational landscape underlying this process near this site remains unclear. Here, we used single-molecule fluorescence resonance energy transfer (smFRET) to map conformational states near the protonation site and to examine proton-driven dynamics. Measurements between subunits across the pore revealed a shift from a high-FRET state (shorter distance) at pH 7.4 to a low-FRET state (longer distance) at pH 5.5. At low pH, the channel also displayed frequent transitions between these states, consistent with conformational switching. The E600D mutant, which is insensitive to proton activation in the pH 7.4 to pH 5.5 range showed primarily the high-FRET state at both pH values. While the E600K mutant, which shifts proton sensitivity to higher pH, showed primarily low-FRET state at pH 5.5 with minimal transitions. Capsaicin binding did not show any significant changes at this site, indicating that this region is primarily sensitive to proton binding. These studies show that proton-binding at the outer pore loop alters the conformational landscape near this site and also highlight the role of dynamics in proton sensitivity and its disruption by pore mutations.
Krishnamoorti et al. (Sun,) studied this question.