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Structural basis for inhibition of a voltage-gated Ca2+ channel by Ca2+ antagonist drugs

Nature volume 537, pages 117–121 (2016)Cite this article

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Abstract

Ca2+ antagonist drugs are widely used in therapy of cardiovascular disorders1,2. Three chemical classes of drugs bind to three separate, but allosterically interacting, receptor sites on CaV1.2 channels, the most prominent voltage-gated Ca2+ (CaV) channel type in myocytes in cardiac and vascular smooth muscle3,4,5,6,7,8,9. The 1,4-dihydropyridines are used primarily for treatment of hypertension and angina pectoris and are thought to act as allosteric modulators of voltage-dependent Ca2+ channel activation, whereas phenylalkylamines and benzothiazepines are used primarily for treatment of cardiac arrhythmias and are thought to physically block the pore1,2. The structural basis for the different binding, action, and therapeutic uses of these drugs remains unknown. Here we present crystallographic and functional analyses of drug binding to the bacterial homotetrameric model CaV channel CaVAb, which is inhibited by dihydropyridines and phenylalkylamines with nanomolar affinity in a state-dependent manner. The binding site for amlodipine and other dihydropyridines is located on the external, lipid-facing surface of the pore module, positioned at the interface of two subunits. Dihydropyridine binding allosterically induces an asymmetric conformation of the selectivity filter, in which partially dehydrated Ca2+ interacts directly with one subunit and blocks the pore. In contrast, the phenylalkylamine Br-verapamil binds in the central cavity of the pore on the intracellular side of the selectivity filter, physically blocking the ion-conducting pathway. Structure-based mutations of key amino-acid residues confirm drug binding at both sites. Our results define the structural basis for binding of dihydropyridines and phenylalkylamines at their distinct receptor sites on CaV channels and offer key insights into their fundamental mechanisms of action and differential therapeutic uses in cardiovascular diseases.

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Figure 1: Structural basis for inhibition of CaVAb by amlodipine.
Figure 2: Inhibition of CaVAb by dihydropyridine binding at a lipid site.
Figure 3: Dihydropyridine binding allosterically modifies Ca2+ binding in the selectivity filter.
Figure 4: State-dependent inhibition by pore block with Br-verapamil and UK-59811.

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Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank with the following accession codes 5KLB (CavAb, 5 mM Ca2+ 2.7Å; 5KLG (CavAb-W195Y-UK-59811, 5 mM Ca2+); 5KLS (CavAb-UK-59811, 5 mM Ca2+); 5KMD (CavAb-W195Y-amlodipine, 5 mM Ca2+); 5KMF (CavAb-W195Y nimodipine, 5 mM Ca2+); and 5KMH (CavAb-Br-verapamil, 5 mM Ca2+).

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Acknowledgements

We are grateful to the beamline staff at the Advanced Light Source (BL8.2.1 and BL8.2.2) for their assistance during data collection. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health under award number R01 HL112808 (W.A.C. and N.Z.), and a National Research Service Award from training grant T32 GM008268 (T.M.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by research grants from the Howard Hughes Medical Institute (N.Z.) and by the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health under award number R01 NS26254 (W.A.C.). D.C.P. acknowledges support from Neusentis, Pfizer Inc., Cambridge, UK during the course of this work.

Author information

Author notes

  1. Ning Zheng and William A. Catterall: These authors jointly supervised this work.

Authors and Affiliations

  1. Department of Pharmacology, University of Washington, Seattle, 98195-7280, Washington, USA

    Lin Tang, Tamer M. Gamal El-Din, Teresa M. Swanson, Todd Scheuer, Ning Zheng & William A. Catterall

  2. Howard Hughes Medical Institute, University of Washington, Seattle, 98195-7280, Washington, USA

    Lin Tang & Ning Zheng

  3. Curadev Pharma, Discovery Park, Sandwich, CT14 9FF, Kent, UK

    David C. Pryde

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Contributions

L.T., T.M.G., T.M.S., T.S., D.C.P., N.Z., and W.A.C. designed the experiments. D.C.P. provided compound UK-59811. L.T. conducted protein purification, crystallization, and X-ray diffraction experiments for dihydropyridines. L.T. and T.M.S. conducted protein purification, crystallization, and X-ray diffraction experiments for Br-verapamil. L.T. and T.M.S. determined the structures and analysed the structural results with input from T.M.G. and N.Z. T.M.G. designed and analysed mutants that block drug binding and performed all of the electrophysiological studies. T.M.G., T.S., and W.A.C. analysed the electrophysiological results. All authors contributed to the interpretation of the structures in light of the physiological data. L.T., N.Z., and W.A.C. wrote the manuscript with input from all co-authors.

Corresponding authors

Correspondence to Ning Zheng or William A. Catterall.

Extended data figures and tables

Extended Data Figure 1 Biophysical characterization of CaVAb I199S.

a, Ba2+ currents recorded from a holding potential of −120 mV to test potentials from −60 mV to 20 mV in 10 mV steps for I199S. b, G–V curves of CaVAb and CaVAb I199S derived from peak I–V relationships. The voltages for half-maximal activation and slopes are: CaVAb: V1/2 = −18.8 ± 0.3, k = 3.68 ± 0.43, n = 7; CaVAb I199S: V1/2 = −18.8 ± 0.3, k = 3.88 ± 0.47 (n = 5). c, Repetitive depolarization to 0 mV at 1 Hz from a holding potential of −120 mV (n = 5). d, Steady-state inactivation of CaVAb and CaVAb I199S. Two pulses were applied: a 300-ms conditioning pulse to the indicated potentials followed by 50-ms test pulse to 0 mV (n = 3). e, State-dependent block of CaVAb I199S by 10 nM (green), 100 nM (blue), or 1.5 μM (red) amlodipine during repetitive depolarizations to 0 mV (left, n = 3–5 cells). Ba2+ currents in 100 nM amlodipine for CaVAb I199S (right). f, Concentration-dependent block of CaVAb I199S by nimodipine at 100 nM (blue), 1 μM (red), 5 μM (brown), 10 μM (grey) and 50 μM (black) (left, n = 4–5 cells for each curve). Ba2+ currents in the presence of 5 μM nimodipine for CaVAb I199S (right).

Extended Data Figure 2 Structural comparison of the binding modes of amlodipine, nimodipine, and UK-59811.

a, Superposition of CaVAb in complexes with amlodipine (cyan), nimodipine (yellow), and UK-59811 (magenta) at the dihydropyridine binding site viewed from the side of the pore module. The side chains of dihydropyridine-interacting residues are shown in sticks. b, An Fo–Fc simulated annealing omit map contoured at 2.5σ for nimodipine. c, An Fo–Fc simulated annealing omit map contoured at 2.5σ for UK-59811. d, An Fo–Fc simulated annealing omit map contoured at 2.5σ for DMPC.

Extended Data Figure 3 Biophysical characterization and drug block of CaVAb W195 and CaVAb Y195.

a, Sequence alignment of CaVAb S6 segment and CaV1.1 DIV S6. W195 in CaVAb is equivalent to Y1358 in CaV1.1. b, Ba2+ currents recorded from a holding potential of −120 mV to test potentials from −60 mV to 20 mV in 10 mV steps for CaVAb W195Y. c, G–V curves for CaVAb W195 and CaVAb Y195 derived from peak I–V relationships. The voltages for half-maximal activation and slopes are: CaVAb W195 V1/2 = −18.8 ± 0.3, k = 3.7 ± 0.43, n = 7; CaVAb Y195, V1/2 = −9 ± 0.3, k = 7.4 ± 0.1, n = 5. d, Steady-state inactivation of CaVAb W195 and CaVAb Y195 (n = 3). Two pulses were applied: a 300-ms conditioning pulse followed by 50-ms test pulse to 0 mV. e, State-dependent block of CaVAb W195Y by nimodipine at 100 nM (white), 500 nM (blue), 1 μM (green), 5 μM (red), and control (grey). f, Concentration-dependent block of CaVAb W195Y by nimodipine. IC50 = 508 ± 93 nM (n = 4–5 cells for each point).

Extended Data Figure 4 Biophysical characterization of block by UK-59811.

a, Ba2+ currents for state-dependent block by different concentrations of UK-59811. b, State-dependent block of CaVAb by UK-59811 at 0 nM (black), 100 nM (green), 500 nM (red), 1 μM (blue), and 5 μM (brown). For each curve, n = 4–5 cells. c, Concentration-response curve for UK-59811. Data were fit with a Hill equation assuming a 1:1 binding. IC50 = 194 ± 22 nM, n = 4–5.

Extended Data Figure 5 Evidence for the partially dehydrated Ca2+ binding and carboxyl-carboxylate pairs at the selectivity filter entryway.

a, Top view of an Fo–Fc simulated annealing omit map contoured at 3σ for residues 178 and 181 for the wild-type channel without drug. b, Top view of an Fo–Fc simulated annealing omit map contoured at 3σ for residues 178 and 181 for CaVAb–amlodipine. c, Top view of an Fo–Fc simulated annealing omit map contoured at 2.5σ for residues 178 and 181 for CaVAb–nimodipine. d, Top view of an Fo–Fc simulated annealing omit map contoured at 3σ for residues 178 and 181 for CaVAb–UK-59811. e, Top view of Site 1 with the anomalous difference Fourier map density (red mesh, contoured at 3σ) calculated with diffraction data of crystals collected at 1.75 Å wavelength. Ca2+ is shown as a green sphere. Site 1 residues are shown in sticks. Hydrogen bonds are indicated with dashed lines. f, Top view of Site 2 with the anomalous difference Fourier map density (magenta mesh, contoured at 3σ).

Extended Data Figure 6 Biophysical characterization of verapamil block of CaVAb and functional properties of CaVAb T206S.

a, Chemical structure of verapamil. b, Concentration dependence of verapamil inhibition of CaVAb. The amplitude of the peak Ba2+ current was recorded after applying 20 pulses at a frequency of 1 Hz, where the block reaches steady state. The data were fit by a Hill equation assuming a 1:1 binding ratio. n = 4–7 cells. IC50 = 475 ± 25 nM. c, Ba2+ currents of CaVAb T206S. d, G–V curves. CaVAb (black): V1/2 = 18.8 ± 0.3 mV, k = 3.7 ± 0.43 (n = 5); CaVAb T206S (blue): V1/2 = −15 ± 1.8 mV, k = 6.6 ± 0.4 (n = 5). e, Current traces of CaVAb (black) and CaVAb T206S (blue) during a 1-s depolarizing pulse from a holding potential of –120 mV to −10 mV. f, State-dependent inhibition of CaVAb T206S by Br-verapamil at 10 μM (black), 25 μM (green), 50 μM (red), and 100 μM (blue). For each curve, n = 4–5 cells.

Extended Data Figure 7 Comparison of dihydropyridine binding site in CaVAb and CaV1.2.

The pore domain of CaVAb is illustrated with two subunits in view, one in tan corresponding to domain III of CaV1.2 and one in blue corresponding to domain IV of CaV1.2. The amino acid residues in CaVAb corresponding to those that are important for dihydropyridine binding to CaV1.2 channels are highlighted in red. Bound amlodipine is illustrated with green sticks.

Extended Data Figure 8 Amlodipine binding breaks symmetry.

a, The overall structure of CaVAb in complex with amlodipine (shown in ribbon representation). Measuring the Cα distances of V196 (nearing the amlodipine binding pocket) from the 4 subunits shows the channel is asymmetrical. b, Binding of amlodipine (sticks in red) induces asymmetry and causes rearrangement of the lipid in the central cavity. c–f, The amlodipine binding pocket showing the Cα–Cα distance at two layers (Y195–G164 and I199–F167) horizontally. At layer 1 (Y195–G164), the Cα–Cα distance of its neighbouring sites (11.0 Å in d and 11.0 Å in f) matches the drug binding site (10.9 Å in c), but the diagonal site (e) is too narrow (10.6 Å). At layer 2 (I199–F167), the pocket width of the diagonal site (11.1 Å in e) matches the drug-binding site (11.0 Å in c), but the two diagonal sites are too wide (11.4 Å in d and 11.3 Å in f).

Extended Data Figure 9 Br-verapamil binding breaks symmetry.

a, Alignment of the 4 subunits of CaVAb in complex with Br-verapamil showing the voltage sensor module (VSD) and the ends of S6 are different. b, Measuring the Cα distances between T206 residues in adjacent subunits shows that the channel is indeed asymmetrical with Br-verapamil in the pore.

Extended Data Table 1 Data collection, phasing and refinement statistics
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Tang, L., Gamal El-Din, T., Swanson, T. et al. Structural basis for inhibition of a voltage-gated Ca2+ channel by Ca2+ antagonist drugs. Nature 537, 117–121 (2016). https://doi.org/10.1038/nature19102

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