Non-Uniform and Variable Arrangements of Ryanodine Receptors Within Mammalian Ventricular Couplons
Rationale: Single tilt tomograms of the dyads in rat ventricular myocytes indicated that type 2 ryanodine receptors (RYR2) were not positioned in a well-ordered array. Further, the orientation and packing strategy of purified type 1 ryanodine receptors (RYR1) in lipid bilayers is determined by the free Mg2+ concentration. These observations led us to test the hypothesis that RYR2 within the mammalian dyad have multiple and complex arrangements.
Objective: Determining 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 non-uniform containing a mix of checkerboard and side-by-side arrangements as well as isolated tetramers. Both phosphorylation and 0.1mmol/l Mg2+ moved the tetramers into a predominantly checkerboard configuration, while the 4 mmol/l Mg2+ induced a dense side-by-side arrangement. These changes occurred within ten 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.
- Received March 6, 2014.
- Revision received April 10, 2014.
- Accepted April 30, 2014.