Nonuniform and variable arrangements of ryanodine receptors within mammalian ventricular couplons

Abstract

RATIONALE:: Single-tilt tomograms of the dyads in rat ventricular myocytes indicated that type 2 ryanodine receptors (RYR2s) were not positioned in a well-ordered array. Furthermore, the orientation and packing strategy of purified type 1 ryanodine receptors in lipid bilayers is determined by the free Mg concentration. These observations led us to test the hypothesis that RYR2s within the mammalian dyad have multiple and complex arrangements. OBJECTIVES:: To determine the arrangement of RYR2 tetramers in the dyads of mammalian cardiomyocytes and the effects of physiologically and pathologically relevant factors on this arrangement. METHODS AND RESULTS:: We used dual-tilt electron tomography to produce en-face views of dyads, enabling a direct examination of RYR2 distribution and arrangement. Rat hearts fixed in situ; isolated rat cardiomyocytes permeabilized, incubated with 1 mmol/L Mg, and then fixed; and sections of human ventricle, all showed that the tetramer packing within a dyad was nonuniform containing a mix of checkerboard and side-by-side arrangements, as well as isolated tetramers. Both phosphorylation and 0.1 mmol/L Mg moved the tetramers into a predominantly checkerboard configuration, whereas the 4 mmol/L Mg induced a dense side-by-side arrangement. These changes occurred within 10 minutes of application of the stimuli. CONCLUSIONS:: The arrangement of RYR2 tetramers within the mammalian dyad is neither uniform nor static. We hypothesize that this is characteristic of the dyad in vivo and may provide a mechanism for modulating the open probabilities of the individual tetramers. © 2014 American Heart Association, Inc.