Figure 5. Parallels between TRPV3_K169A and wild-type TRPV1.

(A) Representative whole-cell recording at +60 mV of WT (black), K169A (red), E751A (blue), W739A (orange) immediately following 2-APB sensitization protocol, with perfusion protocol of 5 s wash, followed by 15 s 30 μM 2-APB, prolonged 30 s wash and the residual current was blocked by 5 s application of 50 μM ruthenium red (RuR). The scale bars units are in current density (pA/pF). (B) Graphical representation of RuR sensitive residual current at the end of the recording in (A) (WT: n = 10 biologically independent experiments; K169A: n = 5 biologically independent experiments; E751A: n = 6 biologically independent experiments, W739A: n = 5 biologically independent experiments). (C) Basal current activity before application of ligand was determined by the blocking the current following a voltage step from 0 to +60 mV with 50 μM RuR. (D) Graphical representation of the mean blocked current density from the protocol in (C) (GFP control: n = 5 biologically independent experiments; WT: n = 6 biologically independent experiments; K169A: n = 5 biologically independent experiments; E751A: n = 5 biologically independent experiments, W739A: n = 6 biologically independent experiments). (E) Conductance voltage relation of WT, K169A, and E751A in the absence of ligand determined from peak tail current elicited from a −160 mV post step pulse following a voltage step between −120 to +200 mV (Δ 20 mV). The conductance of both mutants fail to saturate at +200 mV while WT channels are nonconductive at the tested voltages (WT: n = 3 biologically independent experiments; K169A: n = 3 biologically independent experiments; E751A: n = 3 biologically independent experiments). (F) Alignment of AR5 from TRPV3_WT (orange), TRPV3_{K169A 2APB} (magenta) and TRPV1 (light cyan). The AR5 loop of TRPV1 assumes a conformation similar to that of TRPV3_{K169A 2APB}. (G) The AR5 loop and the HLH_CD are within interaction distance in TRPV1.

Figure 5—figure supplement 1. Sequence alignment of rat TRPV1 (rTRPV), rabbit TRPV2 (rabTRPV2), human TRPV3 (hTRPV3) and xenopus TRPV4 (xTRPV4).

The alignment is colored by conservation and regions of interest are marked with purple lines. residues of interest are indicated with *. The distal CTD has a high helical propensity as calculated by PsiPred. Indicated by a helix cartoon. Abbreviations: ARD, ankyrin repeat domain; AR2, ankyrin repeat 2; AR5, ankyrin repeat 5; β_CD, β-sheet of the coupling domain (CD); HLH _CD, helix-loop-helix of the CD; Pre-S1 _CD, Pre-S1 of the CD; PH, pore helix; SF, selectivity filter; PL, pore loop.