Molecular mechanism underlying desensitization of the proton-activated chloride channel PAC
- Department of Physiology, Johns Hopkins University School of Medicine, United States
- Department of Structural Biology, Van Andel Institute, United States
- Department of Medicine, Johns Hopkins University School of Medicine, United States
- Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, United States
Figures
Figure 1 with 1 supplement
The proton-activated chloride (PAC) channel desensitizes at low pH.
(A) Representative whole-cell current traces of the endogenous PAC current (ICl, H) in wild-type (WT) HEK293T cells induced by extracellular acidification at pH 5.0, 4.6, 4.0, and 3.6. Cells were …
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Figure 1—source data 1
Raw data for Figure 1A–F.
- https://cdn.elifesciences.org/articles/82955/elife-82955-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
The proton-activated chloride (PAC) channel does not exhibit steady-state desensitization and shows fast recovery from desensitization.
(A) (Left) Protocol and (right) representative whole-cell trace for measuring the recovery from desensitization of the PAC current at pH 4.0. The cells were perfused with pH 4.0 solution to elicit …
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Figure 1—figure supplement 1—source data 1
Raw data for Figure 1—figure supplement 1A-D.
- https://cdn.elifesciences.org/articles/82955/elife-82955-fig1-figsupp1-data1-v1.xlsx
Figure 2
Association of H98 with E107/D109 in the extracellular domain–transmembrane domain (ECD–TMD) interface facilitates proton-activated chloride (PAC) channel desensitization.
(A) The structure of human PAC at pH 4. The acidic pocket residues are shown in yellow spheres. Histidine 98 is shown in a cyan sphere. Close-up views of the local interactions at the acidic pocket …
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Figure 2—source data 1
Raw data for Figure 2B-C, F-I.
- https://cdn.elifesciences.org/articles/82955/elife-82955-fig2-data1-v1.xlsx
Figure 3
E94 in the extracellular transmembrane helix 1 (TM1) extension is critical for proton-activated chloride (PAC) channel desensitization.
(A) The structure of human PAC at pH 4. The β10–β11 loop is shown in red. Glutamic acid (E) 94 and aspartic acid (D) 91 is shown in a cyan sphere. (B) Representative whole-cell current traces of …
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Figure 3—source data 1
Raw data for Figure 3B–D.
- https://cdn.elifesciences.org/articles/82955/elife-82955-fig3-data1-v1.xlsx
Figure 4
The E94R mutant promotes proton-activated chloride (PAC) channel desensitization by enhancing its interaction with the β10–β11 linker.
(A) A schematic figure shows the impact of E94/R94 on the β10–β11 linker of human PAC at pH 4. (B) The Cα atom root-mean-square-fluctuation (RMSF) of wild-type (WT) and E94R mutant simulations. As …
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Figure 4—source data 1
Raw data for Figure 4B–F.
- https://cdn.elifesciences.org/articles/82955/elife-82955-fig4-data1-v1.xlsx
Figure 5
D91 reduces proton-activated chloride (PAC) channel desensitization by destabilizing transmembrane helix 1 (TM1) and lipid interaction.
(A) Representative whole-cell current traces of wild-type (WT) PAC, and mutants D91R, and D91N induced by different extracellular acidic pH solutions (4.6, 5.0, 5.2, 5.4, 5.8, and 6.2) at +100 mV. …
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Figure 5—source data 1
Raw data for Figure 5A–C and E.
- https://cdn.elifesciences.org/articles/82955/elife-82955-fig5-data1-v1.xlsx
Figure 6
The β10–β11 linker regulates proton-activated chloride (PAC) channel desensitization via interactions with the transmembrane domain (TMD).
(A) Interactions between E250 in β10–β11 linker with TM1–β1 linker (R93) and β14–TM2 linker (D297). The left panel shows the desensitized state of PAC in which an arginine substitution at position …
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Figure 6—source data 1
Raw data for Figure 6B–E.
- https://cdn.elifesciences.org/articles/82955/elife-82955-fig6-data1-v1.xlsx
Figure 7
A schematic model showing proton-activated chloride (PAC) channel pH-dependent activation and desensitization.
For simplicity, PAC trimer is depicted as two subunits, in the closed, open, and desensitized states. Excess extracellular acidification leads to PAC desensitization as a result of remodeling of the …
Tables
Table 1
The predicted residue pKa value of relevant residues for the wild-type (WT) and mutants investigated.
| WT | H98R | E94R | D91R | |
|---|---|---|---|---|
| pKa(H98) | 6.10 | 12.42 (R) | 6.12 | 6.12 |
| pKa(E107) | 5.28 | 5.53 | 5.29 | 5.27 |
| pKa(D109) | 3.46 | 3.68 | 3.46 | 3.46 |
| pKa(E250) | 4.76 | 4.94 | 4.78 | 4.78 |
| pKa(E94) | 4.84 | 4.79 | 12.53 (R) | 4.52 |
| pKa(D91) | 3.70 | 3.69 | 3.59 | 12.45 (R) |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| cell line (Homo-sapiens) | HEK293T | ATCC | Cat#: CRL-3216 | |
| cell line PACC1 knockout (Homo-sapiens) | PAC KO HEK293T | doi:10.1126/science.aav9739 | ||
| recombinant DNA reagent | pIRES2-EGFP-hPAC | doi:10.1126/science.aav9739 | ||
| commercial assay or kit | Lipofectamine2000 | Invitrogen | Cat#: 11668019 | |
| commercial assay or kit | QuikChange II XL site-directed mutagenesis | Agilent Technologies | Cat#: 200522 | |
| software, algorithm | pCLAMP 10.7 | Molecular Devices | RRID:SCR_011323 | |
| software, algorithm | Clampfit 10.7 | Molecular Devices | ||
| software, algorithm | PyMOL 2.3.4 | Schrodinger, LLC | ||
| software, algorithm | Gromacs 2020.1 | doi: 10.1016/j.softx.2015.06.001 | ||
| software, algorithm | CHARMM-GUI | doi: 10.1002/jcc.23702 | ||
| software, algorithm | PPM Web Server | doi: 10.1093/nar/gkr703 | ||
| software, algorithm | Python | Python Software Foundation | ||
| software, algorithm | GraphPad Prism 8 | GraphPad |
