Pore mutation N617D in the skeletal muscle DHPR blocks Ca2+ influx due to atypical high-affinity Ca2+ binding
- Department of Pharmacology, Medical University of Innsbruck, Austria
- Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Austria
Figures
Figure 1
Mutant DHPR(N617D) remains Ca2+ impermeant despite strong or long depolarizations in the presence of DHP agonist Bay K.
(a) Representative whole-cell Ca2+ current recordings elicited by 200 ms depolarizations from −50 to +80 mV from ncDHPR (top) and wt (center) myotubes before (left) and after (right) perfusion with …
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Figure 1—source data 1
Data for IV graph.
- https://cdn.elifesciences.org/articles/63435/elife-63435-fig1-data1-v2.xlsx
Figure 2
Mutant DHPR(N617D) does not conduct inward Na+ currents in the presence of near physiological [Na+].
(a) Plots of current-voltage relationship for DHPR-mediated Na+ currents recorded from ncDHPR myotubes indicate the absence of slow-activating, non-inactivating inward Na+ currents in the presence …
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Figure 2—source data 1
Data for IV graphs.
- https://cdn.elifesciences.org/articles/63435/elife-63435-fig2-data1-v2.xlsx
Figure 3
Binding of Ca2+ ions with nanomolar affinity within the pore of mutant DHPR(N617D) precludes Ca2+ permeation.
Representative whole-cell Li+ current recordings from wt and ncDHPR myotubes in response to 200 ms depolarizations from −50 to +40 mV in the presence of 100 mM external Li+ and either 0 (a), 1 µM (b)…
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Figure 3—source data 1
Data for dose-response graph.
- https://cdn.elifesciences.org/articles/63435/elife-63435-fig3-data1-v2.xlsx
Figure 4
Inward Li+ currents conducted by DHPR(N617D) are sensitive to nifedipine block.
(a) Plots of current-voltage relationship for DHPR-mediated Li+ currents recorded from ncDHPR myotubes in the presence (Imax = −0.35 ± 0.13 pA/pF; n = 16) and absence (Imax = −2.41 ± 0.27 pA/pF; n = …
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Figure 4—source data 1
Data for IV graph.
- https://cdn.elifesciences.org/articles/63435/elife-63435-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
Ca2+ selectivity and conductance mechanisms in the wt and mutant DHPR(N617D) channel pore.
(a, b) De novo conformation prediction of peptide F600 - I624 constituting the selectivity filter and adjacent pore helices P1 and P2 of DHPRα1S repeat II (P1II, P2II) (left) and of peptide F1309 - …
Figure 5—figure supplement 1
Additional blocking strategies of DHPR Ca2+conductance in the evolution of skeletal muscle EC coupling.
Symbols and nomenclature are identical to Figure 5. (a) Zebrafish slow-muscle specific DHPRα1S carries a distorted EEEE locus, due to substitution of E292 of repeat I by Q. Exchange of E292 with Q292…
Figure 6 with 2 supplements
Structure models of selectivity filter regions of wt DHPR (left panels) and mutant DHPR(N617D) channel pores (right panels) showing the movements of Ca2+ ions in simulation studies.
(a) Top view of the pore illustrating the EEEE and DCS loci. The residues of the EEEE locus are displayed in red and the DCS locus is indicated by the position of the residues N617 or D617. (b) Side …
Figure 6—video 1
Movement of Ca2+ ions through the EEEE locus of the wt DHPR pore toward the cytosolic side.
The dark blue spheres represent van der Waals radii of Ca2+ ions. The residues of the EEEE locus are displayed in red.
Figure 6—video 2
Movement of Ca2+ ions through the DCS and EEEE loci of the DHPR(N617D) pore.
Ionic interactions between D617 of the DCS locus and the upper Ca2+ ion are reflected by the slow detachment and thus slow migration of the Ca2+ ion toward the cytosolic side, suggesting occlusion …
Tables
Table 1
Effect of varying free external Ca2+ concentrations on peak inward Li+ currents (Imax) in wt and ncDHPR myotubes.
Imax values of inward ILi+ are represented as mean ± SEM with corresponding number of recordings (n) from wt and ncDHPR myotubes. *p<0.05; ***p<0.001, unpaired Student’s t-test.
| Free [Ca2+] | wt | ncDHPR | ||
|---|---|---|---|---|
| Imax (pA/pF) | n | Imax (pA/pF) | n | |
| 0 | −2.07 ± 0.47 | 9 | −2.32 ± 0.35 | 16 |
| 10 nM | −2.08 ± 0.19 | 6 | −2.30 ± 0.19 | 12 |
| 30 nM | ‒ | ‒ | −2.17 ± 0.27 | 12 |
| 100 nM | −1.94 ± 0.23 | 5 | −1.98 ± 0.30 | 9 |
| 300 nM | −2.08 ± 0.16 | 8 | −1.33 ± 0.29 * | 8 |
| 1 µM | −1.68 ± 0.25 | 6 | −0.47 ± 0.10 *** | 8 |
| 3 µM | −0.24 ± 0.04 | 6 | −0.06 ± 0.02 *** | 7 |
| 10 µM | −0.09 ± 0.05 | 6 | ‒ | ‒ |
| 30 µM | −0.01 ± 0.02 | 5 | −0.02 ± 0.02 | 8 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | ncDHPR | doi:10.1038/s41467-017-00629-x Dayal et al., 2017 | ||
| Chemical compound, drug | (±)Bay K 8644 | Sigma-Aldrich | Cat#: B112 | 10 µM |
| Chemical compound, drug | Nifedipine | Sigma-Aldrich | Cat#: N7634 | 10 µM |
| Chemical compound, drug | Tetraethylammonium chloride (TEA-Cl) | Sigma-Aldrich | Cat#: T2265 | 145 mM |
| Chemical compound, drug | N-benzyl-p-toluene sulphonamide (BTS) | Santa Cruz Biotechnology, Inc | Cat#: sc-202087 | 100 µM |
| Software, algorithm | MaxChelator simulation program | https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/ | RRID:SCR_018807 | |
| Software, algorithm | ClampFit | Axon Instruments | version 10.7 | |
| Software, algorithm | SigmaPlot | Systat Software, Inc. | RRID:SCR_010285 | version 11.0 |
| Software, algorithm | GraphPad Prism | GraphPad Software, LLC | RRID:SCR_002798 | version 8 |
| Software, algorithm | PEP-FOLD 3.5 | RPBS web portal | Version 3.5 | |
| Software, algorithm | GROMACS | University of Stockholm, University of Upsala | RRID:SCR_014565 | version 2019.2 |
| Software, algorithm | MOE | Chemical Computing Group ULC | RRID:SCR_014882 | version 2020.01 |
| Software, algorithm | AMBER | University of California, San Francisco. | RRID:SCR_014230 | Version 2020 |
| Software, algorithm | PyMOL | Schrödinger, LLC | RRID:SCR_000305 | Version 2.4.0 |
Additional files
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Supplementary file 1
Composition of external solutions with different free Ca2+concentrations used for recording Ca2+ block of inward ILi+.
The indicated free Ca2+ concentrations in the bath solutions were achieved by adjusting the concentrations of CaCl2 and TEA-Cl, calculated using the MaxChelator simulation program (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/).
- https://cdn.elifesciences.org/articles/63435/elife-63435-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/63435/elife-63435-transrepform-v2.pdf
