Structure-functional insights into the dantrolene binding site of the human cardiac ryanodine receptor: towards a deeper understanding of heart arrhythmias

Vladena Bauerova-Hlinkova¹, Konrad Beck², Ľubica Mačáková¹, Mário Benko¹, Dominka Hajdúchová¹, Eva Kutejová¹ and Jacob Bauer¹

¹Department of Biochemistry and Protein Structure, Institute of Molecular Biology SAS, Dúbravská cesta 21, 84551 Bratislava, Slovak republic;
²Cardiff University School of Dentistry, Heath Park, Cardiff CF14 4XY, Wales, UK

Ryanodine receptors (RyRs) are the largest known ion channels. Their main physiological role is to release calcium from the sarcoplasmic/endoplasmic reticulum into the cytosol of muscle cells. Three isoforms of this channel (1-3) have been identified in humans; isoforms 1 and 2 (RyR1 and 2) are predominantly expressed in skeletal and cardiac muscle, respectively.  Mutations identified in the genes of the human RyR1 and 2 isoforms are responsible for several severe dystrophies and cardiac arrhythmias, which often result in death at a young age [1].

Dantrolene is a postsynaptic muscle relaxant that decreases excitation-contraction coupling in muscle cells. It is currently used as an effective treatment for malignant hyperthermia, muscle spasticity, neuroleptic malignant syndrome and it is also used during resuscitation [2]. It has been shown that dantrolene binds to the skeletal (RyR1) and cardiac (RyR2) muscle ryanodine receptors that are responsible for Ca²⁺ release from the sarcoplasmic reticulum. Dantrolene binding sites are found in both RyR1 and RyR2 isoforms [3]. Although the structure of the RyR1 and RyR2 N-terminal domain has been solved, little is known about the molecular basis of its interaction with dantrolene. In the present work we have prepared several mutants of the hRyR2 N-terminal domain (hRyR2 NTD) in the dantrolene binding site (residues 600–620) by site-directed mutagenesis and investigated the effect of these mutations on the folding and thermal stability of this fragment. Our results show that these mutations have an impact on the thermal stability of the hRyR2 NTD. We also measured the binding affinities between dantrolene and the wild-type and mutant hRyR2 NTD and used molecular docking to model dantrolene binding in hRyR2. This work helps to clarify the role of the dantrolene binding site in hRyR2 and to characterize dantrolene binding on the molecular level.

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2.    S. Kobayashi, M. Yano, T. Suetomi, M.  Ono, H.  Tateishi, M. Mochizuki, X.  Xu, H. Uchinoumi, S. Okuda, T. Yamamoto, N. Koseki, H. Kyushiki, N. Ikemoto, M. Matsuzaki. Journal of the American College of Cardiology 53, (2009), p: 1993-2005.

3.    R. Wang, X. Zhong, X. Meng, A. Koop, X. Tian, P.P. Jones, B.R. Fruen, T. Wagenknecht, Z. Liu, S.R.W. Chen. Journal of Biological Chemistry 286, (2011), p: 12202-12212.

Acknowledgement: This work was financially supported by Slovak Grant Agency VEGA, grants no. 2/0140/16, 2/0131/20 and Interreg SK-AT StruBioMol ITMS: 305011X666.