THE STUDY OF HUMAN RYANODINE RECEPTOR 2 N-TERMINAL REGION RESPONSIBLE FOR HEART DYSFUNCTION

Ľ. BORKO^{1, 2}, V. BAUEROVÁ-HLINKOVÁ², E. HOSTINOVÁ², J. GAŠPERÍK², J. KOŠŤAN³, K. BECK⁴, A. ZÁHRADNÍKOVÁ² AND J. ŠEVČÍK²

¹Department of Molecular Biology FNS Comenius University, Bratislava

²Institute of Molecular Biology SAS, Bratislava

³ Department of Structural and Computational Biology, Max F. Perutz Laboratories, Vienna

⁴Cardiff University School of Dentistry, Cardiff, UK

The ryanodine receptor 2 (RyR2) is the calcium release channel responsible for contraction in mammalian myocardium cells. It is a large homotetramer, composed of four subunits with a molecular weight of ~560 kDa [1] and it is localized in the membrane of sarcoplasmatic reticulum [2]. The main RyR2 opening/closing regulation mechanism is considered to be the interaction between the N – terminal (aa ~ 1-600) and central region (aa ~ 2100-2500) [3] in the cytoplasmatic part. A hypothesis was proposed that mutations in these interacting regions cause regulation failure and thus lead to nonspecific Ca²⁺ release which results in several heart diseases [4].

Prediction performed by PFAM database revealed the presence of 14 domain in the RyR2 molecule [5]. In our work we have predominantly focused on the RyR2_1-606 and RyR2_1-655 fragments which involve three putative PFAM domains: Ins145_P3_rec, MIR, RIH [5,6] as well as several mutantions of RyR2_1-606 which may cause heart failure. The purity and solubility of RyR2_1-606 and RyR2_1-655 were verified by gel filtration and SDS PAGE. Monodispersity and stability in time was confirmed by dynamic light scattering (DLS). Thermofluor shift assay was used to find the conditions of highest thermal stability. Circular dichroism spectra [5,6] as well as preliminary thermofluor shift assay results indicate that the protein is folded and starts melting at around 40°C. Obtained purity, solubility and stability of RyR2_1-606 and RyR2_1-655 allow us to apply structure determining approaches: small angle scattering (SAXS) and X-ray crystallography, which could contribute to the RyR2 structure-function relation study.

Acknowledgement: This work was supported by the research grants from the Slovak Grant Agency VEGA No. 2/0131/10 and Slovak Research and development agency APVV-0628-10.

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4. Yano M, Yamamoto T, Ikeda Y, Matsuzaki M. 2006. Mechanisms of Disease: ryanodine receptor defects in heart failure and fatal arrhythmia. Nat Clin Pract Cardiovasc Med 3: 43-52.

Web site containing full list of RyR2 mutations: http://www.fsm.it/cardmoc/

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6. Bauerova-Hlinkova V, Hostinova E, Gasperik J, Beck K, Borko L, Lai FA, Zahradnikova A, Sevcik J. 2010. Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2. Protein Expr Purif 71: 33-41.