Luminal regulation of the cardiac ryanodine receptor and its molecular mechanisms
Principal Investigator: Marta Gaburjáková
Duration: January 2017 – December 2019
Ryanodine receptor type 2 (RYR2) plays unchangeable role in the process of excitation-contraction coupling in the heart. The physiologically relevant Ca2+ regulation of the RYR2 channel is predominantly determined by its robust cytosolic domain that harbors binding sites for cytosolic Ca2+. Less characterized channel regulation by luminal Ca2+ could be mediated by two mechanisms namely luminal Ca2+ binding on the luminal RYR2 face and binding of luminal Ca2+ emanating the RYR2 pore to the cytosolic channel face. Our aim is to examine a mutual relationship between these two mechanisms of the luminal regulation. The recent findings about the structure of Ca2+ binding sites on the RYR2 channel allow us to employ non-standard experimental conditions. We expect that trivalent cations and their interaction with the RYR2 channel will contribute to the reconciling of current controversial results and bring a comprehensive picture of the RYR2 Ca2+ regulation that has been implicated in heart diseases.
RYR2 channel, luminal Ca2+, cytosolic Ca2+, Ca2+ binding sites, trivalent cations
The main aim of our project proposal is gaining new information about the RYR2 luminal regulation using trivalent cations that could fundamentally contribute fruitful findings to understanding details of molecular mechanism involved in this process. As the main experimental approach we will employ an ion channel reconstitution in a planar lipid membrane. This method enables us to examine detail functional properties of individual ion channel. First, we will determine an appropriate composition of experimental solutions because trivalent cations strongly interact with standardly used Ca2+ chelators; however, these interactions have not been quantitatively described at a sufficient level. It is essential to avoid undesired functional interference that could potentially result in incorrect data interpretation. The center of our work will be electrophysiological experiments with two types of trivalent cations with different ionic radius (Eu3+ and Yb3+). We will employ them to block Ca2+ binding sites first on the luminal and then on the cytosolic RYR2 faces that will provide a basis for studying effect of luminal Ca2+ on the channel activity over the broad spectrum of experimental conditions including the physiological relevant area. A detail and systematic comparison of obtained results with those published for the intact RYR2 regulation provides a best way how to determine which of proposed mechanisms of RYR2 luminal regulation is dominant or whether they interfere or compete. Our aim is not focused only on the pure biophysical approach; a part of our efforts to assess the physiological relevance of individual mechanisms will be devoted to experimental conditions that better simulate ionic conditions in the heart muscle cells.
|Nánási jr P, Gaburjakova M, Gaburjakova J , Almássy J (2017): Omecamtiv mecarbil activates ryanodine receptors from canine cardiac but not skeletal muscle. Eur J Pharmacol, 809:73-79.
|Gaburjakova J., Gaburjakova M.(2018):Reconstitution of Ion Channels in Planar Lipid Bilayers: New Approaches. V Advances in Biomembranes and Lipid Self-Assembly, Volume 27, Iglič A., Rappolt M. a Garcia Sáez A.J. (Eds.), Elsevier-Academic Press, str. 147-185, ISBN: 978-0-12-815772-5.