Two amino acid residues determine 2-APB sensitivity of the ion channels TRPV3 and TRPV4

Abstract

Temperature-activated transient receptor potential ion channels (thermoTRPs) are polymodal detectors of various stimuli including temperature, voltage, and chemicals. To date, it is not known how TRP channels integrate the action of such disparate stimuli. Identifying specific residues required for channel-activation by distinct stimuli is necessary for understanding overall TRP channel function. TRPV3 is activated by warm temperatures and various chemicals, and is modulated by voltage. One potent activator of TRPV3 is 2-aminoethyl diphenylborinate (2-APB), a synthetic chemical that modulates many TRP channels. In a high-throughput mutagenesis screen of approximately 14,000 mutated mouse TRPV3 clones, we found 2 residues (H426 and R696) specifically required for sensitivity of TRPV3 to 2-APB, but not to camphor or voltage. The cytoplasmic N-terminal mutation H426N in human, dog, and frog TRPV3 also effectively abolished 2-APB activation without affecting camphor responses. Interestingly, chicken TRPV3 is weakly sensitive to 2-APB, and the equivalent residue at 426 is an asparagine (N). Mutating this residue to histidine induced 2-APB sensitivity of chicken TRPV3 to levels comparable for other TRPV3 orthologs. The cytoplasmic C-terminal mutation R696K in the TRP box displayed 2-APB specific deficits only in the presence of extracellular calcium, suggesting involvement in gating. TRPV4, a related thermoTRP, is 2-APB insensitive and has variant sequences at both residues identified here. Remarkably, mutating these 2 residues in TRPV4 to TRPV3 sequences (N426H and W737R) was sufficient to induce TRPV3-like 2-APB sensitivity. Therefore, 2-APB activation of TRPV3 is separable from other activation mechanisms, and depends on 2 cytoplasmic residues.

Fig. 1.

Electrophysiological characterization of TRPV3-H426N and TRPV3-R696K. (A) Concentration-response curves of 2-APB- and camphor-activated responses in wild-type and H426N mutant reveal specific loss of 2-APB response, but not camphor-evoked response. (B) In R696K mutant, 2-APB-activated response was also specifically affected, whereas camphor-activated responses were similar between wild-type TRPV3 and R696K mutant. For R696K mutant, the acute peak response was used to calculate the EC₅₀ value. Error bars are SEs. For 2-APB responses: n = 17 for wild-type TRPV3; n = 9 for H426N mutant; and n = 6 for R696K mutant. For camphor responses: n = 10 for wild-type TRPV3; n = 5 for H426N mutant; and n = 10 for R696K mutant.

Fig. 2.

A conserved requirement of H426 for 2-APB responses among mammalian and amphibian TRPV3 orthologs. (A) Sequence alignment of mouse, rat, human, dog, frog, and chicken TRPV3. Position 426 is indicated. (B) FLIPR EC₅₀ values of 2-APB (B) and camphor (C) in H426N equivalent mutants of frog, human, and dog TRPV3; fTRPV3 refers to frog TRPV3; hTRPV3 refers to human TRPV3; and dTRPV3 refers to dog TRPV3. Error bars are SEs; n = 5 for each clone.

Fig. 3.

N427H mutation restores 2-APB sensitivity of chicken TRPV3. (A) Concentration-response curves of 2-APB and camphor in chicken wild-type TRPV3, cTRPV3-N427H, and pcDNA on FLIPR. Error bars are SEs; n = 6 for each clone. (B) Whole-cell patch-clamp current-density concentration-response curves of 2-APB-activated currents in HEK293 cells transfected with wild-type or N427H mutant chicken TRPV3 at +100 and −100 mV. Error bars are SEs; n = 4 for wild-type chicken TRPV3; and n = 5 for cTRPV3-N427H mutant. (C) Inside-out single channel recordings of wild-type chicken TRPV3 treated with 25 μM 2-APB or 3 mM camphor. (D) Single channel current traces (inside-out configuration) of cTRPV3-N427H channel treated with 25 μM 2-APB or 6 mM camphor. (E) Average fold increase of single channel activities over basal levels evoked by 2-APB or camphor in inside-out patches expressing wild-type cTRP3 or cTRPV3-N427H mutant (*, P < 0.05, Student's t test; n = 4 for wild-type cTRPV3; and n = 5 for cTRPV3-N427H mutant).

Fig. 4.

Mutations of TRPV4 at 2 cytoplasmic residues identified render TRPV4 sensitive to 2-APB. (A) Sequence alignment of the N-terminal region and C-terminal TRP box of mouse TRPV3 and rat TRPV4. Residues required for mouse TRPV3 responses to 2-APB and equivalent residues in TRPV4 are indicated. Diagrams illustrate the positions of the 2 intracellular residues and show a schematic representation of the double mutations in TRPV4. Error bars are SEs; n = 5 for each clone. (B) Concentration-dependent responses to 2-APB and 4-α-PDD in HEK293 cells transfected with wild-type or mutated TRPV4; n = 5 for each clone.