From the Department of Cellular and Physiological Sciences (P.A., D.R.L.S., E.D.W.M.), Department of Pediatrics (S.S.), and Department of Surgery (S.K.G., A.I.M.C.), University of British Columbia, Vancouver, British Columbia, Canada.
Circ Res. 2014 Jul 7;115(2):252-62. doi: 10.1161/CIRCRESAHA.115.303897. Epub 2014 Apr 30.
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.
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.
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.
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.
大鼠心室肌细胞二联体的单倾斜断层扫描表明，2 型兰尼碱受体（RYR2）并未排列有序。此外，纯化的 1 型兰尼碱受体在脂质双层中的取向和包装策略由游离 Mg2+浓度决定。这些观察结果促使我们检验这样一个假设，即在哺乳动物二联体中的 RYR2 具有多种复杂的排列方式。
确定哺乳动物心肌细胞二联体中 RYR2 四聚体的排列方式，以及生理和病理相关因素对这种排列方式的影响。
我们使用双倾斜电子断层扫描产生二联体的面视图，从而可以直接检查 RYR2 的分布和排列。原位固定的大鼠心脏；分离的大鼠心肌细胞通透化，在 1mmol/L Mg2+孵育后固定；以及人的心室切片，均显示二联体中四聚体的包装不均匀，包含棋盘和并排排列以及孤立的四聚体的混合物。磷酸化和 0.1mmol/L Mg2+都将四聚体移动到主要的棋盘式构象，而 4mmol/L Mg2+则诱导密集的并排排列。这些变化在施加刺激后的 10 分钟内发生。
哺乳动物二联体中 RYR2 四聚体的排列既不均匀也不固定。我们假设这是体内二联体的特征，可能为调节单个四聚体的开放概率提供了一种机制。
