Abstract
We have previously demonstrated that type II ryanodine receptors (RyR2) tetramers can be rapidly rearranged in response to a phosphorylation cocktail. The cocktail modified downstream targets indiscriminately making it impossible to determine whether phosphorylation of RyR2 was an essential element of the response. We therefore used the β-agonist isoproterenol and mice with one of the homozygous mutations, S2030A +/+ , S2808A +/+ , S2814A +/+ , or S2814D +/+ , to address this question and to elucidate the role of these clinically relevant mutations. We measured the length of the dyad using transmission electron microscopy (TEM) and directly visualized RyR2 distribution using dual-tilt electron tomography. We found that: 1) The S2814D mutation, by itself, significantly expanded the dyad and reorganized the tetramers suggesting a direct link between the phosphorylation state of the tetramer and the microarchitecture. 2) All of the wild-type, as well as the S2808A and S2814A mice, had significant expansions of their dyads in response to ISO, while S2030A did not. 3) In agreement with functional data from the same mutants, S2030 and S2808 were necessary for a complete β-adrenergic response, whereas S2814 was not. 4) All the mutated residues had unique effects on the organization of their tetramer arrays. 5) The correlation of structure with function suggests that tetramer-tetramer contacts play an important functional role. We conclude that both the size of the dyad and the arrangement of the tetramers are linked to the state of the channel tetramer and can be dynamically altered by a β-adrenergic receptor agonist.
Summary
Analysis of RyR2 mutants suggests a direct link between the phosphorylation state of the channel tetramer and the microarchitecture of the dyad. All phosphorylation site mutations produced significant and unique effects on the structure of the dyad and its response to isoproterenol.
Full Text Availability
The license terms selected by the author(s) for this preprint version do not permit archiving in PMC. The full text is available from the preprint server.
