VEGA 2/0006/15

Principal Investigator: Jana Gaburjáková

Duration: January 2015 – December 2017
Coordinating Organization: Centre of Biosciences – Institute of Molecular Physiology and Genetics SAS, Bratislava

Annotation:

The key determinant of cardiac contractility is Ca2+ released from the sarcoplasmic reticulum via the ryanodine receptor (RYR2). The major regulation domain of the RYR2 channel is a huge cytosolic head. A smaller luminal domain participates in the channel regulation as well and its dysfunction leads to generation of severe ventricular arrhythmias. The main luminal regulator of the RYR2 channel is Ca2+. Other metal cations such as Mg2+ and Zn2+ can also contribute to the luminal regulation by binding to luminal RYR2 sites. These binding sites are localized either on the channel protein or on the associated protein – calsequestrin (CSQ2). Our aim is to examine the luminal regulation of the RYR2 channel in more comprehensive manner in respect to cationic composition of the luminal space. On the single channel level we will examine the role of Mg2+ and Zn2+ in the luminal RYR2 regulation at either diastolic or systolic luminal Ca2+. Outcomes of experiments will be correlated to the CSQ2 presence in the RYR2 complex.

Keywords:

RYR2 channel, planar lipid membrane, luminal regulation, Mg2+, Zn2+

Objectives:

The major scientific goal of the project is to obtain a more comprehensive view on the luminal regulation of the cardiac ryanodine receptor (RYR2) focusing on a cationic composition of the luminal channel space. The main luminal regulator of the RYR2 channel is unbound Ca2+. However, in the SR lumen there are also present other metal cations such as Mg2+ and Zn2+. Both cations are able to occupy  luminal RYR2 sites, and thus, they can actively contribute to the luminal regulation of the channel. Many clinical trials have documented cardioprotective effect of both Mg2+ and Zn2+, but the molecular mechanism underlying Zn2+ action has not been revealed. We would like to contribute to deeper understanding of the Zn2+ protective role by examining its regulatory impact directly on the RYR2 channel as this channel is a fundamental player in the cardiac excitation-contraction coupling. Comparable ionic radius of Mg2+ and Zn2+ leads us to an idea that there might be a similar competition between Zn2+ and Ca2+ for luminal RYR2 sites as it was reported for Mg2+. So, competitive displacement of Ca2+ from the luminal sites by other cations could suppress the potentiating effect of luminal Ca2+. Investigation of luminal Ca2+/Mg2+ and Ca2+/Zn2+ competition could reveal which cation is a dominant physiological competitor of luminal Ca2+, and thus, plays more important role in the regulation of luminal Ca2+ effect. Protein calsequestrin (CSQ2) interacting with the luminal domain of the RYR2 channel is important in the channel regulation. However, CSQ2 dissociates from the channel complex at higher Ca2+ that could be one of its physiological roles. Currently, it is missing any systematic study about the CSQ2 stripping process from theRYR2 channel. Therefore, we will test the concentration dependence of stripping power of Ca2+, Mg2+, Zn2+ and their mixtures. Additionally, these experiments should help us to unravel whether CSQ2 aggregation is essential for its dissociation from the RYR2 complex employing distinct aggregation ability of tested metal cations.

Publications: