VEGA 2/0003/14

Principal Investigator: Marta Gaburjáková

Duration: January 2014 – December 2016
Coordinating Organization: Centre of Biosciences – Institute of Molecular Physiology and Genetics SAS, Bratislava

Annotation:

In cardiac muscle, the contraction is initiated by a massive Ca2+ release from the sarcoplasmic reticulum (SR) cisternae via activated ryanodine receptors (RYR2) functioning as Ca²⁺ channels. The major physiological activator of the RYR2 channel is Ca2+ that binds to its large cytosolic domain. However, there is growing evidence that the Ca2+ binding to a minor luminal region of the RYR2 channel can also be effectively involved in the channel regulation. Several studies indicate that calsequestrin (CSQ2) as the main Ca2+-binding protein in the SR lumen could play the role of a luminal Ca2+ sensor of the RYR2 channel. The aim of our work is to clarify the role of CSQ2 in the luminal regulation of the RYR2 channel by combination of electrophysiological and biochemical methods. This interdisciplinary approach is expected to contribute to the detail understanding of the functional communication between CSQ2 and the RYR2 channel from the biophysical as well as the physiological point of view.

Keywords:

RYR2 channel, calsequestrin, luminal Ca2+, planar lipid membrane

Objectives:

The main objective of our work is to systematically examine the role of CSQ2 in the luminal regulation of the RYR2 channel in the heart and define its physiological relevance. As the major experimental approach we will use the ion channel reconstitution in a planar lipid membrane. This electrophysiological method provides a unique way to investigate the functional profile of individual ion channels. At first, we will test the concentration and time dependence of effects exerted by Ca2+ and the high ionic strength of solutions on the physical interaction of CSQ2 and RYR2 channel employing the biochemical method – dot blotting. Our intention is to find an effective and powerful procedure for stripping endogenous CSQ2 from the channel complex under our experimental conditions in order to obtain the CSQ2 free microsomal sample. The luminal regulation of the RYR2 channel that is not controlled by CSQ2 will serve as a control for the next two sets of experiments where we will study the regulatory role of  endogenous and recombinant CSQ2 under the conditions that involve physiologically relevant range. Multiway data analysis and data comparison will provide qualitatively new information about the functional interaction between CSQ2 and RYR2 channel that could help us to clarify the role of RYR2 luminal regulation in the developing of serious cardiac arrhythmias. In addition, we will compare our results with that obtained in the mixture of luminal Ca2+ with Cs+. Considering several published studies we suspect that Cs+ exerts an inhibition effect on the RYR2 channel that led very likely to an incorrect data interpretation and the effect of Cs+ was mistakenly ascribed to the regulatory effect of CSQ2. Our results have also a potential to test this formulated hypothesis.

Publications: