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 Mg2+ 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 Mg2+, 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 Mg2+ moved the tetramers into a predominantly checkerboard configuration, whereas the 4 mmol/L Mg2+ 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.

Keywords: calcium; electron microscope tomography; excitation contraction coupling; phosphorylation; ryanodine receptor calcium release channel; sarcoplasmic reticulum.