Figure 3: Residues in the linker of transmembrane domains 2 and 3 (L2–3) are critical for proton activation of TRPV3.
From: Selective potentiation of 2-APB-induced activation of TRPV1–3 channels by acid
(A) Representative inside-out recording showing that protons activated TRPV3 channel in a dose-dependent manner after sensitization by repeated applications of 100 μM 2-APB. (B) Summary data of current densities at −60 mV evoked by 2-APB [100 μM for wild type (WT), 500 μM for chimeras] obtained by whole-cell recordings. Only cells that expressed V3/V1(L2–3) showed response. Mouse TRPV3 mutants with the N terminus (Nt), linker of transmembrane domains 4 and 5 (L4–5), and the C terminus (Ct) swapped by the cognate segments of rat TRPV1, respectively, were not functional. (C) Intracellular protons failed to induce current in patches excised from cells that expressed mV3/V1(L2–3), a mouse TRPV3 mutant with the linker for transmembrane domains 2 and 3 replaced by the corresponding segment of rat TRPV1. The chimeric channel still responded to stimulation by 2-APB. (D) Top, putative membrane topology of a single TRPV3 subunit. Bottom, an amino acid alignment of the linker 2-3 between TRPV3 and TRPV1, with the different residues shaded in grey. (E–H) Representative inside-out recordings showing that protons failed to activate mV3(L508R) (E), mV3(D512N) (F), mV3(S518V) (G), and mV3(A520S) (H), even though the response to 2-APB was retained. (I) Summary of intracellular proton (pH 5.5)-activated currents, normalized to the maximum currents evoked by 2-APB after sensitization. Residues in the linker 2–3 of TRPV3 were mutated individually to the corresponding residues of TRPV1, except that D512 was substituted by N. Inside-out patches were excised from transiently transfected HEK293 cells and recorded while being held at +60 mV. *P < 0.05; **P < 0.001, different from WT. Error bars indicate SEM.
