The HCN domain couples voltage gating and cAMP response in hyperpolarization-activated cyclic nucleotide-gated channels
- University of Milan, Italy
- TU-Darmstadt, Germany
- Columbia University, United States
Figures
Figure 1
Hydrophobic and hydrophilic interactions established by the HCN domain with the voltage sensor domain and the C-linker.
(A) Side view of two opposite subunits of HCN1 (PDB_ID: 5U6O) with the HCN domain (HCND) shown in orange, the voltage sensor domain VSD (TM S1-S4) in violet and C-linker ‘elbow’ (helices A’ and B’) …
Figure 2 with 3 supplements
Interaction of HCND with the voltage sensor domain: mutational analysis in the first hydrophobic pocket.
(A) Detailed view of one HCN1 subunit (PDB_ID: 5U6O) color coded as in Figure 1 showing I135 side chain inserted in the hydrophobic pocket formed by F141 in the TM S1 and M192 in the TM S2 of the …
Figure 2—figure supplement 1
Cellular localization of EGFP-HCN2 I177D/G and I176D mutant channels.
HEK293T cells were transfected with the constructs indicated on the left of each panel and analysed by confocal microscopy. From left: the first column shows the Bright Field (BF) image, scale bar …
Figure 2—figure supplement 2
Analysis of currents recorded from HCN1 I134, I135 and F109 mutants.
Representative whole-cell currents recorded, at the indicated voltages, from HEK293T cells transiently expressing wt, I135A, I135G, I134V, I134A, F109A and F109E mutants. The graphs on the right …
Figure 2—figure supplement 3
Cellular localization of EGFP-HCN1 I135G, I134A and F109E mutant channels.
HEK293T cells were transfected with the constructs indicated on the left of each panel and analysed by confocal microscopy. From left: the first column shows the Bright Field (BF) image, scale bar …
Figure 3
Interaction of HCND with the voltage sensor domain: MD simulation on the second hydrophobic pocket.
(A) Enlarged view of HCN1 subunit (PDB_ID: 5U6O) color coded as in Figure 1 showing the hydrophobic interactions of F109 with I284 and M287 from TM S4 and Y138 from the loop connecting HCND to TM …
Figure 4 with 2 supplements
Interaction of HCND with the voltage sensor domain: mutational analysis of HCN2 residue F151.
(A) Representative whole-cell currents recorded, at the indicated voltages, from HCN2 wt, F151V, F151A and F151E channels in the absence and in the presence of 15 µM cAMP. Graphs to the right show …
Figure 4—figure supplement 1
Analysis of currents recorded from HCN2 F151 mutants.
Representative whole-cell currents recorded, at the indicated voltages, from HEK293T cells transiently expressing wt (A), F151W (B) and F151M (C) HCN2 channel both in the absence and in the presence …
Figure 4—figure supplement 2
Patch-clamp analysis and MD simulation of HCN2 F151R, F151K and F151D channels.
(A) Representative whole-cell currents recorded, at the indicated voltages, from HEK293T cells transiently expressing wt, F151R, F151K and F151D HCN2 channels. (B) Mean tail current activation …
Figure 5 with 1 supplement
Linear response theory simulations demonstrate the mechanical coupling of C-linker, HCN domain and S4 helix in HCN1.
(A) One HCN1 subunit (PDB_ID: 5U6O) color coded as in Figure 1. Positions and directions of force-displacements on residue A425 of the C-linker ‘elbow’, residue K108 of the HCN domain, and residue …
Figure 5—figure supplement 1
Linear response theory null model of HCN1 channel and clustering of different perturbation directions.
The HCN1 protein (PDB_ID: 5U6O) was perturbed simultaneously on all four monomers at defined residues, by using 1000 external force vectors (shown as colored beads in spheres) with strength of 1600 …
Figure 6 with 2 supplements
Salt bridge interactions between the HCN domain and the C-linker control cAMP effect in HCN2.
(A) In HCN1 subunit (PDB_ID: 5U6O) (color coded as in Figure 1), the main chain carbonyl group of M113 in the HCN domain contacts the side chain of K422 residue on the C-linker of the opposite …
Figure 6—figure supplement 1
Kinetics analysis of C-linker mutants in HCN isoforms.
Mean activation and deactivation time constants (before and after the x-axis break, respectively) of (A) HCN2, (B) HCN4 and (C) HCN1, wt (black symbols) and mutant (colored symbols) channels in the …
Figure 6—figure supplement 2
Mutant cycle analysis of coupling between HCN4 R154A and E478A mutations.
(A) Representative whole-cell currents recorded, at the indicated voltages, from HEK293T cells transiently expressing wt, the single mutants R154A, E478A and the double mutant R154A-E478A. (B) Mean …
Figure 7 with 1 supplement
C-linker mutations preventing cAMP effect in all HCN isoforms.
(A) Half activation voltage (V1/2 ± SEM) values of HCN1 wt, K422A-E436A, R549E and K422A-E436A-R549E (blue symbols), HCN2 wt and K464A-E478A (green symbols), HCN4 wt and K543A-E557A (orange …
Figure 7—figure supplement 1
Properties of the C-linker double mutants in HCN1 and HCN4.
(A) Representative whole-cell currents (left and center) and mean activation curves (right) of: HCN1 wt in control solution (black filled squares) and in the presence of 15 µM cAMP (red squares); …
Author response image 1
Patch clamp analysis of HCN2 I176A mutant.
(A) Representative whole-cell currents of HCN2 wt and I176A channels recorded from +50 to -120 mV with -15mV increment. A wt trace with a low current amplitude similar to those of I176A was selected …
Videos
Video 1
Mechanical continuum between C-linker, HCND and VSD.
Morphing video of HCN1 showing the concerted movement of C-linker, HCND and VSD. The structural models used for the morphing were derived from computational data obtained with the linear response …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (human) | HCN1 | Xention Ltd. (Cambridge,UK) | NM_021072.3 | |
| Gene (mouse) | HCN2 | PMID: 11331358 | NM_008226.2 | |
| Gene (rabbit) | HCN4 | PMID: 10212270 | NM_001082707 | |
| Strain, strain background (E. coli) | Stbl2 | Thermo Fisher Scientific | ||
| Cell line (human) | HEK 293T | ATCC (Authenticated by STR profiling) | RRID:CVCL_0063 | Tested for mycoplasma: negative result |
| Recombinant DNA reagent | pcDNA 3.1 (plasmid) | Clontech Laboratories | ||
| Recombinant DNA reagent | pCI (plasmid) | Promega | ||
| Recombinant DNA reagent | pEGFP (plasmid) | Clontech Laboratories | ||
| Transfected construct (human) | HCN1 (cDNA) | Xention Ltd (Cambridge,UK) | All HCN1 mutants transfected in the paper were obtained starting from this wt cDNA. For further details please see Materials and methods | |
| Transfected construct (mouse) | HCN2 (cDNA) | Laboratory of Steven A. Siegelbaum | All HCN2 mutants transfected in the paper were obtained starting from this wt cDNA. For further details please see Materials and methods | |
| Transfected construct (rabbit) | HCN4 (cDNA) | PMID: 10212270 | All HCN4 mutants transfected in the paper were obtained starting from this wt cDNA. For further details please see Materials and methods | |
| Transfected construct (human) | GFP-HCN1 (cDNA) | This paper | All GFP-HCN1 mutants transfected in the paper were obtained starting from this wt cDNA. For further details please see Materials and methods | |
| Transfected construct (mouse) | GFP-HCN2 (cDNA) | PMID: 15564593 | All GFP-HCN2 mutants transfected in the paper were obtained starting from this wt cDNA. For further details please see Materials and methods | |
| Commercial assay or kit | QuickChange Lightning Site- Directed Mutagenesis Kit | Agilent | ||
| Commercial assay or kit | Exprep Plasmid SV kit | GeneAll | ||
| Commercial assay or kit | Thermo Scientific TurboFect Transfection Reagent | Thermo Fisher Scientific | ||
| Chemical compound, drug | Adenosine 3', 5'-cyclic monophosphate (cAMP) | SIGMA | ||
| Chemical compound, drug | CellMask Deep Red Plasma membrane Stain | Thermo Fisher Scientific | ||
| Software, algorithm | pClamp - Clampfit | Molecular Devices | RRID:SCR_011323 | Version 10.7 |
| Software, algorithm | pClamp - Clampex | Molecular Devices | RRID:SCR_011323 | Version 10.7 |
| Software, algorithm | EZ-Patch | Elements srl https://elements-ic.com/ | Version 1.0.0 | |
| Software, algorithm | Gromacs | http://www.gromacs.org/ | RRID:SCR_008395 | Version 9.22 |
| Software, algorithm | Modeller | http://salilab.org/modeller/modeller.html | RRID:SCR_014565 | Version 2018.x |
| Software, algorithm | Python | http://www.python.org | RRID:SCR_008394 | Version 3.7.3 |
| Software, algorithm | Pymol | https://pymol.org | RRID:SCR_000305 | Version 2.2.0 |
| Software, algorithm | Fiji ImageJ | http://fiji.sc/ |
Additional files
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Supplementary file 1
(A) Fitting parameters of the activation curves in HCN2 (Figures 2,4,6,7). From left to right: half-activation voltage (V1/2), inverse slope factor (k) obtained by fitting data to a Boltzmann function (Material and methods) in absence or presence of cAMP; n = number of cell tested in each condition; cAMP-induced shift in V1/2; number of cells that expressed a measurable HCN current. *p<0.05 by One-way ANOVA with Fisher’s test compared to wt HCN2; §p<0.05 by Student’s T-test compared to control condition (without cAMP); n.s. not statistically different; n.t. not tested; n.d. not detectable. cAMP concentration was 15 µM in all cases, except for last row (HCN2 K464-E478A#, 100 µM cAMP). (B) Fitting parameters of the activation curves in HCN1 and HCN4 (Figures 2,7). From left to right: half-activation voltage (V1/2), inverse slope factor (k) obtained by fitting data to a Boltzmann function (Material and methods) in absence or presence of cAMP; n = number of cell tested in each condition; cAMP-induced shift in V1/2; number of cells that expressed a measurable HCN current. *p<0.05 by One-way ANOVA with Fisher’s test compared to wild-type HCN1 or HCN4; §p<0.05 by Student’s T-test compared to control condition (without cAMP); n.s. not statistically different; n.t. not tested; n.d. not detectable. cAMP concentration used for HCN1 and HCN4 was 15 µM and 30 µM respectively. (C) Number of molecules for each simulation of molecular dynamics performed on HCN1. POPC: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; TIP3P: water model; K: K+ ion; CL: Cl- ion
- https://cdn.elifesciences.org/articles/49672/elife-49672-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/49672/elife-49672-transrepform-v2.docx
