(A) Overlay of the S1-S4 unit in the apo (orange) and toxin-bound (marine) structure; the root-mean-square difference (RMSD) of the C-α trace is only ~0.7 Å, indicating the internal structure of this unit is not altered upon toxin binding. (B) Overlay of the S1-S4 unit, plus the transmembrane portion of the neighboring S5, which belongs to the adjacent protomer (hence referred to as S5’); the RMSD is again ~0.7 Å. (C) Overlay of the pore helix (P') and the transmembrane portion of S6'; the RMSD is also ~0.7 Å. (D) Overlay of the S1-S4 unit, plus S5', the pore helix P' and S6’. Only the S1-S4 unit and S5' (in gray) are used for fitting, to highlight the relative displacement of S6' and the P' helix, which, as shown in panel (C), move as a largely rigid unit. (E) Overlay of the pore helix P’ and the transmembrane portion of S6 across the protomer interface, i.e. S6"; the RSMD is ~ 0.6 Å. (F) Overlay of two adjacent channel subunits, using the same fit as in panel (D). Owing to the quasi-rigid structure of the P-S6 interface across subunits (see panel E), the displacements induced upon toxin binding on the pore and S6 helices result in a change in the relative orientation of the S1-S4 units, which we propose leads to a change in the crossing-angle of S5 and S6 on the intracellular side, and thus the opening of the lower gate. (G,H) Close-up view of the changes induced upon toxin (yellow) binding on a hydrophobic cluster formed by side-chains from S5, the pore helix, the selectivity filter (SF) and S6. Mutation of the side-chains colored in gray has been shown to diminish toxin-induced TRPV1 activation (see text). (I) Evaluation of the compactness of the hydrophobic cluster shown in (G,H) during MD simulations of apo and toxin-bound TRPV1, in terms of the number of contacting atom pairs of a given side-chain with all others. Fewer contacts imply a less compact arrangement with greater exposure to the solvent, and potentially, an increased heat-capacity of the channel.