LETTER RESEARCH 18Z at al 9 e505. of the pheny 21 stat-dependent 22 IS5-S6 li l pore-omin 23 nistdru 24 25 2A 28 46.660-868 394 y of voltag ary Inforn vailable in the online version of the pape he staff at the Advance es of Health und e Award from m2222 ind doe 33-7ne the How by 0990 C T ages supp 0zhoro cl o Cher200 Auth and xar USA84.5478 821987 11 calcium channel blocker NZ.TM.G 328313 3181987 n 13. 11 14. 15. 1.5 )5KMD( re no comp ting finan 17.Letter RESEARCH 1 september 2016 | VOL 537 | NA T U RE | 121 pore4–6,24,25. Our structural results correlate precisely with this expec￾tation and reveal the exact structure of the drug–receptor complex. Br-verapamil is stretched between two subunits of CaVAb, consistent with drug binding at the interface of domains III and IV in CaV1.2 channels4–6,24,25. As for dihydropyridines, phenylalkylamine bind￾ing at this site disrupts the fourfold symmetry of the pore (Extended Data Fig. 9). Location of the phenylalkylamine receptor site deep in the central cavity in the pore reveals why binding of these drugs is state-dependent. Access of phenylalkylamines to their receptor is greatly enhanced by opening the intracellular activation gate, which allows diffusion to the drug receptor site. Drug binding is therefore frequency-dependent, allowing selective block of CaV1.2 channels in rapidly firing cardiac myocytes2 . This mechanism is the basis for use of verapamil for cardiac arrhythmias. Overall, our structural studies illuminate the complex pharmacology and therapeutic uses of Ca2+ antagonist drugs in treatment of differ￾ent cardiovascular disorders at the atomic level (see Supplementary Discussion). These structural models will be important for design and development of next-generation Ca2+ antagonist drugs to provide safer and more effective treatment of hypertension, angina pectoris, cardiac arrhythmia, and other medical conditions. Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper. received 11 March; accepted 12 July 2016. Published online 24 August 2016. 1. Zamponi, G. W., Striessnig, J., Koschak, A. & Dolphin, A. C. The physiology, pathology, and pharmacology of voltage-gated calcium channels and their future therapeutic potential. Pharmacol. Rev. 67, 821–870 (2015). 2. Hondeghem, L. M. & Katzung, B. G. Antiarrhythmic agents: the modulated receptor mechanism of action of sodium and calcium channel-blocking drugs. Annu. Rev. Pharmacol. Toxicol. 24, 387–423 (1984). 3. Murphy, K. M., Gould, R. J., Largent, B. L. & Snyder, S. H. A unitary mechanism of calcium antagonist drug action. Proc. Natl Acad. Sci. USA 80, 860–864 (1983). 4. Catterall, W. A. & Striessnig, J. Receptor sites for Ca2+ channel antagonists. Trends Pharmacol. Sci. 13, 256–262 (1992). 5. Hockerman, G. H., Peterson, B. Z., Johnson, B. D. & Catterall, W. A. Molecular determinants of drug binding and action on L-type calcium channels. Annu. Rev. Pharmacol. Toxicol. 37, 361–396 (1997). 6. Striessnig, J. Pharmacology, structure and function of cardiac L-type Ca2+ channels. Cell. Physiol. Biochem. 9, 242–269 (1999). 7. Hofmann, F., Lacinová, L. & Klugbauer, N. Voltage-dependent calcium channels: from structure to function. Rev. Physiol. Biochem. Pharmacol. 139, 33–87 (1999). 8. Cheng, R. C., Tikhonov, D. B. & Zhorov, B. S. Structural model for phenylalkylamine binding to L-type calcium channels. J. Biol. Chem. 284, 28332–28342 (2009). 9. Tikhonov, D. B. & Zhorov, B. S. Structural model for dihydropyridine binding to L-type calcium channels. J. Biol. Chem. 284, 19006–19017 (2009). 10. Takahashi, M., Seagar, M. J., Jones, J. F., Reber, B. F. & Catterall, W. A. Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle. Proc. Natl Acad. Sci. USA 84, 5478–5482 (1987). 11. Tanabe, T. et al. Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328, 313–318 (1987). 12. Mikami, A. et al. Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature 340, 230–233 (1989). 13. Wu, J. et al. Structure of the voltage-gated calcium channel Cav1.1 complex. Science 350, aad2395 (2015). 14. Ren, D. et al. A prokaryotic voltage-gated sodium channel. Science 294, 2372–2375 (2001). 15. Catterall, W. A. & Zheng, N. Deciphering voltage-gated Na+ and Ca2+ channels by studying prokaryotic ancestors. Trends Biochem. Sci. 40, 526–534 (2015). 16. Payandeh, J., Scheuer, T., Zheng, N. & Catterall, W. A. The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011). 17. Payandeh, J., Gamal El-Din, T. M., Scheuer, T., Zheng, N. & Catterall, W. A. Crystal structure of a voltage-gated sodium channel in two potentially inactivated states. Nature 486, 135–139 (2012). 18. Zhang, X. et al. Crystal structure of an orthologue of the NaChBac voltage￾gated sodium channel. Nature 486, 130–134 (2012). 19. Tang, L. et al. Structural basis for Ca2+ selectivity of a voltage-gated calcium channel. Nature 505, 56–61 (2014). 20. Catterall, W. A., Perez-Reyes, E., Snutch, T. P. & Striessnig, J. Voltage-gated calcium channels: introduction. IUPHAR/BPS Guide to Pharmacology (http:// guidetopharmacology.org/GRAC/FamilyIntroductionForward?familyId=80) (2011). 21. Striessnig, J., Murphy, B. J. & Catterall, W. A. Dihydropyridine receptor of L-type Ca2+ channels: identification of binding domains for [3H](+)-PN200-110 and [3H]azidopine within the α1 subunit. Proc. Natl Acad. Sci. USA 88, 10769–10773 (1991). 22. Yamaguchi, S. et al. Key roles of Phe1112 and Ser1115 in the pore-forming IIIS5-S6 linker of L-type Ca2+ channel α1C subunit (CaV 1.2) in binding of dihydropyridines and action of Ca2+ channel agonists. Mol. Pharmacol. 64, 235–248 (2003). 23. Hescheler, J., Pelzer, D., Trube, G. & Trautwein, W. Does the organic calcium channel blocker D600 act from inside or outside on the cardiac cell membrane? Pflügers Archiv 393, 287–291 (1982). 24. Striessnig, J., Glossmann, H. & Catterall, W. A. Identification of a phenylalkylamine binding region within the α1 subunit of skeletal muscle Ca2+ channels. Proc. Natl Acad. Sci. USA 87, 9108–9112 (1990). 25. Hockerman, G. H., Johnson, B. D., Scheuer, T. & Catterall, W. A. Molecular determinants of high affinity phenylalkylamine block of L-type calcium channels. J. Biol. Chem. 270, 22119–22122 (1995). 26. Yatani, A. & Brown, A. M. The calcium channel blocker nitrendipine blocks sodium channels in neonatal rat cardiac myocytes. Circ. Res. 56, 868–875 (1985). 27. Kass, R. S., Arena, J. P. & Chin, S. Block of L-type calcium channels by charged dihydropyridines. Sensitivity to side of application and calcium. J. Gen. Physiol. 98, 63–75 (1991). 28. Bangalore, R., Baindur, N., Rutledge, A., Triggle, D. J. & Kass, R. S. L-type calcium channels: asymmetrical intramembrane binding domain revealed by variable length, permanently charged 1,4-dihydropyridines. Mol. Pharmacol. 46, 660–666 (1994). 29. Peterson, B. Z. & Catterall, W. A. Calcium binding in the pore of L-type calcium channels modulates high affinity dihydropyridine binding. J. Biol. Chem. 270, 18201–18204 (1995). 30. Glossmann, H., Ferry, D. R., Goll, A., Striessnig, J. & Zernig, G. Calcium channels and calcium channel drugs: recent biochemical and biophysical findings. Arzneimittelforschung 35, 1917–1935 (1985). Supplementary Information is available in the online version of the paper. 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 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. Author Information The coordinates and structure factors have been deposited in the Protein Data Bank with the following accession codes 5KLB (CavAb, 5mM Ca2+ 2.7Å; 5KLG (CavAb-W195Y-UK-59811, 5mM Ca2+); 5KLS (CavAb-UK-59811, 5mM Ca2+); 5KMD (CavAb-W195Y-amlodipine, 5mM Ca2+); 5KMF (CavAb-W195Y nimodipine, 5mM Ca2+); and 5KMH (CavAb￾Br-verapamil, 5mM Ca2+). Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to N.Z. (nzheng@uw.edu) or W.A.C. (wcatt@uw.edu). © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved