The Cardiac Ryanodine Receptor (RyR2): Investigating Mechanisms of Gating at the Selectivity Filter

Abstract

Ryanodine receptors (RyR) provide the pathway for release of intracellular calcium ions (Ca2+) that initiate muscle contraction. Mutations in cardiac RyRs, (RyR2) underlie arrhythmia and sudden cardiac death. RyRs are therefore an attractive therapeutic target, however more information regarding structure and function is required. The enormous size of RyRs precludes detailed structural analysis. Models, based on potassium channel templates, exist for the RyR pore-forming region (PFR) providing an invaluable framework in which predictions of specific processes of ion translocation and gating mechanisms can be tested.Potassium channels have a conserved gating mechanism involving a distinct hydrogen-bonding network of residues at the selectivity filter that is responsible for holding the filter in an inactive, non-conducting conformation. This study examines interactions of equivalent residues in RyR2 to ascertain whether a similar gating mechanism exists.Three alanine (D4829A, Y4813A, Y4839A) and one conserved tryptophan (Y4839W) RyR2 mutations were constructed to assess a proposed hydrogen-bonding network. Mutated channels formed functional homotetramers in vivo whereby they released Ca2+ upon caffeine addition. Differences in [3H]-ryanodine binding on isolated WT and mutant mix membrane populations revealed 1) altered ryanodine binding site and/or 2) altered calcium sensitivity for mutant channels. Preliminary single-channel experiments assessing ion handling and gating properties under steady state conditions suggest that the selectivity filer has a role in channel gating. Conductance and open probability (Po) for Y4839A was reduced by 15 % and 76 % respectively compared to WT RyR2. No single-channel experiments were performed for Y4813A due to inherent protein instability when purified. Four unique gating modes including subconductance states for D4829A were observed. Further experiments are required to assess the role of the selectivity filter in gating.Study supported by the British Heart Foundation.