Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels
- Section of Neurobiology, Department of Biological Sciences, University of Southern California, United States
- Program in Neuroscience, University of Southern California, United States
- Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, United States
Figures
Figure 1
Three vertebrate OTOP channels vary in their I-pH response profile across a broad pH range.
(A) Proton currents elicited in response to acidic stimuli (pH 6–4.5) in the absence of extracellular Na+ measured from HEK-293 cells expressing each of the three OTOP channels as labeled (Vm = -80 …
-
Figure 1—source data 1
Source data for Figure 1C.
- https://cdn.elifesciences.org/articles/77946/elife-77946-fig1-data1-v1.xlsx
Figure 2
OTOP2 is a proton selective ion channel that is open at neutral pHo.
(A) Representative OTOP2 current elicited in response to a pH 5.5 solution with 160 mM Na+ replacing NMDG+ (top) or 140 mM Methane sulfonate-/ 20 mM Cl- replacing 160 mM Cl- (bottom) in the …
-
Figure 2—source data 1
Source data for Figure 2B and C.
- https://cdn.elifesciences.org/articles/77946/elife-77946-fig2-data1-v1.xlsx
Figure 3
OTOP channels mediate the influx and efflux of protons as measured with intracellular pH imaging.
(A–D) Changes in intracellular fluorescence emission upon exposure to changing extracellular pH, as indicated, were measured from HEK-293 cells co-expressing OTOP channels and the pH-sensitive …
-
Figure 3—source data 1
Source data for Figure 3A–D.
- https://cdn.elifesciences.org/articles/77946/elife-77946-fig3-data1-v1.xlsx
Figure 4 with 1 supplement
Activation kinetics vary dramatically between OTOP channels.
(A) Representative current traces (black) from cells expressing each of the three OTOP channels in response to the application and the removal of pH 5.5 solutions. The activation and the decay …
-
Figure 4—source data 1
Source data for Figure 4C and D.
- https://cdn.elifesciences.org/articles/77946/elife-77946-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Fit to activation kinetics of OTOP currents.
(A-C) Representative traces showing the activation of OTOP1 (A), OTOP2 (B), and OTOP3 (C) currents in response to solutions with extracellular pH titrated to 6.0 (left panel), 5.5 (middle panel), …
Figure 5 with 1 supplement
Changes in the slope conductance of OTOP channels as a function of extracellular pH.
(A) Voltage and solution exchange protocol used to measure the slope conductance in response to changing extracellular pH. Vm was held at –80 mV and ramped to +80 mV (1 V/s at 1 Hz). The first ramp …
-
Figure 5—source data 1
Source data for Figure 5C.
- https://cdn.elifesciences.org/articles/77946/elife-77946-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Changes in reversal potentials of OTOP currents during prolonged exposure to acid stimuli.
(A) I-V curves from experiments in which OTOP1 currents were evoked in response to a pH 5.0 stimulus with Vm = –80 mV and ramp depolarizations (–80 mV to +80 mV; 1 V/s) applied every second as in Fig…
-
Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1B, C.
- https://cdn.elifesciences.org/articles/77946/elife-77946-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 1 supplement
Chimeric channels with external linkers swapped reveal potential gating modules.
(A-F) Representative current traces in response to varying pH from 10 to 5 measured from HEK-293 cells expressing OTOP2 channels and chimeras with an OTOP2 backbone (A) or OTOP3 channels and …
-
Figure 6—source data 1
Source data for Figure 6B–E and G–J.
- https://cdn.elifesciences.org/articles/77946/elife-77946-fig6-data1-v1.xlsx
-
Figure 6—source data 2
Statistical tests comparing chimeric channels with wildtype channels with Mann-Whitney U test.
P values and sample size are indicated.
- https://cdn.elifesciences.org/articles/77946/elife-77946-fig6-data2-v1.docx
Figure 6—figure supplement 1
Topology and sequence alignment of OTOP channels.
Mouse OTOP1, 2, and 3 channels are aligned. The transmembrane helices are depicted in cylinders above the sequence. Residuals conserved in all three channels are shaded pink. External loops that …
Figure 7
Increase in activation kinetics of mOTOP3 chimeric channels.
(A–D) Time constants for activation of chimeric as compared with wildtype channels, measured from traces as in Figure 6A and F, using methods as in Figure 4. The τon of OTOP2 and its chimeras (A, B) …
-
Figure 7—source data 1
Source data for Figure 7A–D.
- https://cdn.elifesciences.org/articles/77946/elife-77946-fig7-data1-v1.xlsx
Figure 8 with 1 supplement
Predicted structures of mOTOP1.
mOTOP2 and mOTOP3. (A) Top views (top) and side views (bottom) of AlphaFold predicted structural models of mOTOP1, mOTOP2, and mOTOP3. The N- and C- domain halves of mOTOP1, mOTOP2, and mOTOP3 are …
Figure 8—figure supplement 1
Comparison between AlphaFold predictions and cryoEM structures.
(A) Superimposed structural alignments of the AlphaFold2 predicted model for zebrafish OTOP1 (DrOTOP11; cyan) and DrOTOP1 cryoEM structure PDB:6NF4 (purple) using single subunits, N domain, or C …
Figure 9
pH tuning of OTOP channels.
Lowering extracellular pH increases the slope conductance of OTOP1 and OTOP3 channels while it lowers the slope conductance of OTOP2 channels. OTOP1 also has a peak of activity at mildly alkaline pH.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (M. musculus) | Otop1, Otop2 and Otop3 | Tu et al., 2018. PMID:29371428 | ||
| Cell line (Homo-sapiens) | HEK293 | ATCC | CRL-1573 | |
| Cell line (Homo-sapiens) | PAC-KO HEK293 cells | Yang et al., 2019a. PMID:31023925 | ||
| Recombinant DNA reagent | Otop1, Otop2, and Otop3 in pcDNA3.1 | Tu et al., 2018. PMID:29371428 | ||
| Recombinant DNA reagent | Otop1, Otop2, and Otop3 – GFP | Saotome et al., 2019. PMID:31160780 | ||
| Recombinant DNA reagent | mO2_O3 loop swap mutations | This paper | cDNAs encode chimeric channels (see methods and Figure 6—figure supplement 1). Available upon request | |
| Recombinant DNA reagent | mO3_O2 loop swap mutations | This paper | cDNAs encode chimeric channels (see methods and Figure 6—figure supplement 1). Available upon request | |
| Recombinant DNA reagent | pHluorin in pcDNA3 | Miesenböck et al., 1998. PMID:9671304 | ||
| Chemical compound, drug | CHES | Sigma | C2885 | |
| Chemical compound, drug | PIPES | Sigma | P6757 | |
| Chemical compound, drug | Homopiperazine-1,4-bis(2-ethanesulfonic acid) | Sigma | 53,588 | |
| Software, algorithm | GraphPad Prism 8 and 9 | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | pClamp and clampfit | Molecular Devices | RRID:SCR_011323 | |
| Software, algorithm | Origin | OriginLab corporation | RRID:SCR_002815 | |
| Software, algorithm | CorelDraw | Corel | RRID:SCR_014235 | |
| Software, algorithm | SimplePCI | HCImage | https://hcimage.com/simple-pci-legacy/ |
