Institute of Molecular Physiology and Genetics
Luminal regulation of the cardiac ryanodine receptor and its molecular mechanisms
Principal Investigator: Jana Gaburjáková
Coordinating Organization: Centre of Biosciences – Institute of Molecular Physiology and Genetics SAS, Bratislava
Duration: January 2018 – December 2020
Annotation:
The cardiac ryanodine receptor (RYR2) is an ion channel that mediates huge Ca2+ release from intracellular stores; this Ca2+ is essential for cardiac muscle contraction. During physical exercise or emotional stress beta-adrenoceptors on the plasma membrane are stimulated to trigger phosphorylation cascades targeting also
the RYR2 channel. Phoshorylation of the RYR2 channel within domain harboring phosphosites Ser-2808 and Ser-2814 is also mediated by protein kinase A (PKA). We will study effects of RYR2 phosphorylation solely induced by endogenous PKA in vitro on RYR2 regulation by physiologically-relevant ligands (Ca2+, Mg2+, ATP)
or antiarrhythmic agent flecainide. The time course of PKA action within RYR2 phosphorylation domain and importance of basal phosphorylation for the channel function will be evaluated. Experimental approach will be extended by bioinformatics to localize other potential sites and domains relevant for phosphorylation in the RYR2
channel protein.
Objectives:
In cardiac muscle, the key process in contraction is Ca2+ release from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RYR2) that gets phosphorylated during beta-adrenergic stimulation. The main goal of the project is a complex evaluation of RYR2 phosphorylation at the functional level using a technique of ion channel incorporation into a planar lipid membrane. Phosphorylation of the RYR2 channel will be solely induced by endogenous PKA and we will focus on cytoplasmic and luminal RYR2 regulation by physiological agents (Ca2+, Mg2+, ATP). Since beta-adrenergic stimulation affects also the SR lumen we will study if phosphorylation influences interactions between the RYR2 channel and its associated protein calsequestrin (CSQ2) that mainly regulates Ca2+ kinetics in the SR lumen. Impaired interactions between these two proteins caused by single mutation within one of them could be a culprit of a genetic disorder that manifests by specific ventricular arrhythmias solely triggered by beta-adrenergic stimulation (CPVT). In this regard, we will test effects of flecainide, a promising drug in treatment of CPVT, on RYR2 regulation under conditions that mimic beta-adrenergic stimulation (the RYR2 channel phosphorylated by PKA+elevated luminal Ca2+ level). Phosphosites in proteins have a tendency to be localized close to each other in order to be managed by the same kinase protein, however with some delay. Ser-2808 is mostly phosphorylated by PKA and Ser-2814 is closed enough to be phosphorylated by the same PKA protein. We will investigate time dependence of the phoshorylation process occurring at these phosphosites using available phospho-antibodies and consequently we will examine how it is translated to RYR2 regulation. We will evaluate the importance of basal phoshorylation status of the RYR2 channel that is not related to the beta-adrenergic pathway employing dephosphorylation assay. In addition, bioinformatics will help us to identify new potential PKA phosphosites and domains in the RYR2 channel.
Keywords:
ryanodine receptor, phosphorylation, PKA, calsequestrin, flecainide
Publications:
- 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.