Conservation of the cooling agent binding pocket within the TRPM subfamily
- Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
Figures
Figure 1
Structures of vanilloid bound to TRPV1 and cooling agent bound to TRPM8.
Side views of (A) TRPM8 bound to icilin (CPK) and Ca2+ (green sphere) and (B) TRPV1 bound to RTx (CPK). Intracellular views of (C) TRPM8 bound to icilin and Ca2+ and (D) TRPV1 bound to RTx.
Figure 2 with 1 supplement
Sequence and structure conservation of the icilin binding pocket in TRPM and TRPA channels.
(A) Structure-based sequence alignment of S1–S4 peripheral domains and transient receptor potential (TRP) helix of selected TRP channel structures, with residues contributing to the icilin binding …
Figure 2—figure supplement 1
S1–S4 residues contributing to the icilin binding pocket in TRPM8 structures 7wre and 6nr3 are shown as blue licorice, viewed from the intracellular side of the membrane, with the transient receptor potential (TRP) box omitted for clarity.
Cooling agent binding pocket mutations used in the present study are shown with carbon atoms colored gold and labeled in TRPM8 and TRPM4, and the equivalent residues in other channels are colored …
Figure 3
WT TRPM4 is sensitive to intracellular Ca2+, voltage, and icilin.
(A) Sample current families obtained using a holding voltage of −60 mV with 200 ms steps to voltages between −100 and +160 mV (Δ 20 mV) before returning to −60 mV. Control traces in the left column …
-
Figure 3—source data 1
Excel file with electrophysiology data for Figure 3B.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig3-data1-v1.xlsx
-
Figure 3—source data 2
Excel file with electrophysiology data for Figure 3C.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig3-data2-v1.xlsx
Figure 4 with 1 supplement
Icilin modulates voltage-dependent activation and closure kinetics of TRPM4.
(A) Sample current traces illustrating the fraction of current that activates rapidly (Iinst) compared to the steady-state current at the end of the pulse (ISS). The pulse protocols used a holding …
-
Figure 4—source data 1
Excel file with electrophysiology data for Figure 4B–D.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Icilin does not modulate voltage-dependent activation of TRPM3α2.
(A) Sample current families obtained using a holding voltage of −60 mV with 50 ms prepulses to −150 mV before 100 ms steps to voltages between −160 and +160 mV (Δ 20 mV), and then returning to −60 …
-
Figure 4—figure supplement 1—source data 1
Excel file with electrophysiology data for Figure 4—figure supplement 1A.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig4-figsupp1-data1-v1.xlsx
-
Figure 4—figure supplement 1—source data 2
Excel file with electrophysiology data for Figure 4—figure supplement 1B, C.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig4-figsupp1-data2-v1.xlsx
Figure 5
A867G mutant TRPM4 retains sensitivity to Ca2+ and voltage, but has enhanced sensitivity to icilin.
(A) Sample current families obtained using a holding voltage of −60 mV with 200 ms steps to voltages between −100 and +160 mV (Δ 20 mV) before returning to −60 mV. Control traces in the left column …
-
Figure 5—source data 1
Excel file with electrophysiology data for Figure 5B.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig5-data1-v1.xlsx
-
Figure 5—source data 2
Excel file with electrophysiology data for Figure 5C.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig5-data2-v1.xlsx
Figure 6
Icilin modulation of TRPM4 is enhanced in the A867G mutant.
(A) Sample current traces illustrating the fraction of current that activates rapidly (Iinst) compared to the steady-state current at the end of the pulse (ISS). The pulse protocols used a holding …
-
Figure 6—source data 1
Excel file with electrophysiology data for Figure 6B–D.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig6-data1-v1.xlsx
Figure 7
R901H mutant TRPM4 is sensitive to intracellular Ca2+ and voltage, but icilin does not promote opening.
(A) Sample current families obtained using a holding voltage of −60 mV with 200 ms steps to voltages between −100 and +160 mV (Δ 20 mV) before returning to −60 mV. Control traces in the left column …
-
Figure 7—source data 1
Excel file with electrophysiology data for Figure 7B.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig7-data1-v1.xlsx
-
Figure 7—source data 2
Excel file with electrophysiology data for Figure 7C.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig7-data2-v1.xlsx
Figure 8
Icilin modulation of the voltage-dependent activation of TRPM4 is disrupted in the R901H mutant.
(A) Sample current traces illustrating the fraction of current that activates rapidly (Iinst) compared to the steady-state current at the end of the pulse (ISS). The pulse protocols used a holding …
-
Figure 8—source data 1
Excel file with electrophysiology data for Figure 8B, C.
- https://cdn.elifesciences.org/articles/99643/elife-99643-fig8-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293 | ATCC | CRL-1573 ATCC Cat# PTA-4488, RRID:CVCL_0045 | |
| Transfected construct (Mus musculus) | mTRPM4b (plasmid) | Received from Youxing Jiang | pEGFP-N1 vector | |
| Transfected construct (Mus musculus) | mTRPM3α2 (plasmid) | Received from Thomas Voets | pCAGGS/IRES-GFP vector | |
| Commercial assay or kit | FuGENE6 Transfection Reagent | Promega | E269 | |
| Commercial assay or kit | QuikChange Lightning | Agilent Technologies | 21051 | |
| Chemical compound, drug | Icilin | Sigma | CAS 36945-98-9 | Source # 0000088284 Source # 0000141718 |
| Chemical compound, drug | Pregnenolone sulfate | Sigma | P162-25MG | Lot # MKCQ8813 |
| Software, algorithm | IgorPro | WaveMetrics | RRID:SCR_000325 | Data analysis |
| Software, algorithm | Python programming language | RRID:SCR_008394 | Data analysis | |
| Software, algorithm | SciPy | RRID:SCR_008058 | Data analysis | |
| Software, algorithm | Matplotlib | RRID:SCR_008624 | Data visualization | |
| Software, algorithm | seaborn | RRID:SCR_018132 | Data visualization | |
| Software, algorithm | PyMOL | Schrödinger | RRID:SCR_000305 | Structure visualization |
| Software, algorithm | PoseFilter | Subha Kalyaanamoorthy; https://doi.org/10.1093/bioinformatics/btab188 | Data analysis PyMOL Plugin | |
| Software, algorithm | pClamp 11 | Molecular Devices | RRID:SCR_011323 | Acquisition patch clamp data |
| Software, algorithm | JalView | RRID:SCR_006459 | Sequence alignment visualization | |
| Software, algorithm | Fr-TM-Align | Pandit & Skolnick; https://doi.org/10.1186/1471-2105-9-531 | Data analysis |
