Journal: Nature Communications

Article Title: Structural mechanism of TRPV3 channel inhibition by the anesthetic dyclonine

doi: 10.1038/s41467-022-30537-8

Figure Lengend Snippet: a Representative ratiometric Fura-2-based fluorescence measurements of changes in intracellular Ca 2+ for HEK 293 GnTI − cells expressing wild-type mouse TRPV3. The changes in the fluorescence intensity ratio at 340 and 380 nm ( F 340 / F 380 ) were monitored in response to the addition of 200 µM 2-APB (arrow) after pre-incubation of cells with various concentrations of dyclonine. The experiment was repeated independently seven times with similar results. b Dose–response curve for TRPV3 inhibition by dyclonine. The changes in the F 340 / F 380 ratio were normalized to its maximal value in the absence of dyclonine and fitted by the logistic equation (red line), with IC 50 = 29.8 ± 5.3 µM and n Hill = 1.61 ± 0.27 ( n = 7 independent experiments). The values are mean ± SEM. The inset shows the chemical structure of dyclonine. Source data are provided as a Source Data file. c , d 3D cryo-EM reconstruction of TRPV3 Dyc viewed from the side ( c ) or top ( d ), with subunits colored green, yellow, purple, and cyan. e , f The same cryo-EM density as in ( c , d ), but cut off along the dashed lines in ( d ) and ( c ), respectively. Putative densities for dyclonine and sodium ions are shown in red and green.

Article Snippet: To better understand the molecular mechanism of TRPV3 inhibition by dyclonine, we embarked on structural characterization of the TRPV3–dyclonine complex using cryo-electron microscopy (cryo-EM).

Techniques: Fluorescence, Expressing, Incubation, Inhibition, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Structural mechanism of TRPV3 channel inhibition by the anesthetic dyclonine

doi: 10.1038/s41467-022-30537-8

Figure Lengend Snippet: a , b TRPV3 Dyc structure viewed from the side ( a ) or top ( b ), with subunits colored green, yellow, purple, and cyan. Red mesh shows densities for dyclonine. Dyclonine molecules are shown as sticks. c , d Close-up views of all four dyclonine binding sites ( c ) and only one of them ( d ). e Representative ratiometric fluorescence measurements of changes in intracellular Ca 2+ for HEK 293 GnTI − cells expressing F666A mutant TRPV3 channels. The changes in the fluorescence intensity ratio F 340 / F 380 were monitored in response to the addition of 200 µM 2-APB (arrow) after pre-incubation of cells with various concentrations of dyclonine. The experiment was repeated independently three times with similar results. f Dose–response curves for inhibition of wild-type and mutant TRPV3 channels by dyclonine. The changes in the fluorescence intensity ratio F 340 / F 380 evoked by addition of 200 µM 2-APB after pre-incubation with various concentrations of dyclonine were normalized to their maximal values in the absence of dyclonine. Curves through the points are logistic equation fits, with IC 50 = 29.8 ± 5.3 µM and n Hill = 1.61 ± 0.27 ( n = 7 independent experiments) for wild-type TRPV3, IC 50 = 673 ± 37 µM and n Hill = 2.07 ± 0.08 ( n = 3 independent experiments) for F666A, IC 50 = 31.5 ± 1.6 µM and n Hill = 1.63 ± 0.07 ( n = 3 independent experiments) for I663W, IC 50 = 31.5 ± 1.0 µM and n Hill = 2.33 ± 0.11 ( n = 4 independent experiments) for Y564A and IC 50 = 9.8 ± 0.7 µM and n Hill = 1.68 ± 0.19 ( n = 6 independent experiments) for F633A. Source data are provided.

Article Snippet: To better understand the molecular mechanism of TRPV3 inhibition by dyclonine, we embarked on structural characterization of the TRPV3–dyclonine complex using cryo-electron microscopy (cryo-EM).

Techniques: Binding Assay, Fluorescence, Expressing, Mutagenesis, Incubation, Inhibition

Journal: Nature Communications

Article Title: Structural mechanism of TRPV3 channel inhibition by the anesthetic dyclonine

doi: 10.1038/s41467-022-30537-8

Figure Lengend Snippet: Pore domain in TRPV3 Dyc with dyclonine in the head towards the pore (blue, a , b ) or tail towards the pore (green, c , d ) orientations, viewed parallel to the membrane ( a , c ) or extracellularly ( b , d ). The F666 side chains and dyclonine molecules are shown in sticks. Only two of four TRPV3 subunits are shown in ( a ) and ( c ), with the front and back subunits omitted for clarity.

Article Snippet: To better understand the molecular mechanism of TRPV3 inhibition by dyclonine, we embarked on structural characterization of the TRPV3–dyclonine complex using cryo-electron microscopy (cryo-EM).

Techniques:

Journal: Nature Communications

Article Title: Structural mechanism of TRPV3 channel inhibition by the anesthetic dyclonine

doi: 10.1038/s41467-022-30537-8

Figure Lengend Snippet: a Dose–response curves for the F666A mutant and wild-type TRPV3 activation by 2-APB. The changes in F 340 /F 380 were normalized to their approximated maximal values at saturating concentrations of 2-APB. Curves through the points are fits with the logistic equation and EC 50 = 27.4 ± 4.5 µM and n Hill = 1.19 ± 0.09 ( n = 4 independent experiments) for wild-type TRPV3 and EC 50 = 20.7 ± 0.9 µM and n Hill = 0.80 ± 0.02 ( n = 3 independent experiments) for F666A. The data for wild-type TRPV3 have been published before . b Dose–response curves for inhibition of the F666A mutant and wild-type TRPV3 by osthole. The changes in F 340 /F 380 were normalized to their maximal values in the absence of osthole. Curves through the points are fits with the logistic equation and IC 50 = 20.5 ± 0.5 µM and n Hill = 1.84 ± 0.14 ( n = 4 independent experiments) for wild-type TRPV3 and IC 50 = 33.1 ± 3.8 µM and n Hill = 1.37 ± 0.19 ( n = 3 independent experiments) for F666A. The data for wild-type TRPV3 have been published before . c Double logarithmic Schild plot for 2-APB concentration dependencies of TRPV3 activation in the presence of 10 μM (pink circles, n = 3 independent experiments) and 60 μM (green circles, n = 3 independent experiments) of dyclonine. Lines through the points of the corresponding color are linear fits. For all panels, data points are presented as mean ± SEM and source data are provided.

Article Snippet: To better understand the molecular mechanism of TRPV3 inhibition by dyclonine, we embarked on structural characterization of the TRPV3–dyclonine complex using cryo-electron microscopy (cryo-EM).

Techniques: Mutagenesis, Activation Assay, Inhibition, Concentration Assay

Journal: Nature Communications

Article Title: Structural mechanism of TRPV3 channel inhibition by the anesthetic dyclonine

doi: 10.1038/s41467-022-30537-8

Figure Lengend Snippet: Superposition of the pore domains in TRPV3 Dyc (blue) and apo-state TRPV3 (PDB ID: 7MIN; orange) viewed parallel to the membrane ( a ) or extracellularly ( b ). The F666 side chains and dyclonine molecules are shown in sticks. Only two of four TRPV3 subunits are shown in ( a ), with the front and back subunits omitted for clarity. Blue arrows indicate flipping of the F666 side chain, movement of P-loop extracellularly and of dyclonine towards the pore center upon binding in the transmembrane portals.

Article Snippet: To better understand the molecular mechanism of TRPV3 inhibition by dyclonine, we embarked on structural characterization of the TRPV3–dyclonine complex using cryo-electron microscopy (cryo-EM).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Structural mechanism of TRPV3 channel inhibition by the anesthetic dyclonine

doi: 10.1038/s41467-022-30537-8

Figure Lengend Snippet: a Pore-forming domain in TRPV3 Dyc with the residues contributing to pore lining in the dyclonine-bound (TRPV3 Dyc ), closed (TRPV3 Closed ) and open (TRPV3 Open ) states shown as sticks. Only two of four subunits are shown, with the front and back subunits omitted for clarity. The pore profile is shown as a space-filling model (green). The region that undergoes α-to-π transition in S6 is highlighted in pink. b Pore radius for TRPV3 Dyc (blue), TRPV3 Closed (orange, PDB ID: 7MIN) and TRPV3 Open (green, PDB ID: 7MIO) calculated using HOLE. The vertical dashed line denotes the radius of a water molecule, 1.4 Å.

Article Snippet: To better understand the molecular mechanism of TRPV3 inhibition by dyclonine, we embarked on structural characterization of the TRPV3–dyclonine complex using cryo-electron microscopy (cryo-EM).

Techniques: