Nonuniform and Variable Arrangements of Ryanodine Receptors Within Mammalian Ventricular Couplons

Abstract

T he type 2 ryanodine receptor (RYR2) is an integral membrane protein of the cardiomyocyte sarcoplasmic reticulum (SR) that functions as a Ca 2+ -activated Ca 2+ ion channel. Each receptor is a homotetramer, measuring roughly 29×29×12 nm, which can be readily identified in electron micrographs based on its location within the dyadic cleft and on its size and shape. 1,2 Rotary shadowing studies of type 1 ryanodine receptors (RYR1) in skeletal muscle triads 3 and numerous transmission electron micrographs of cardiac muscle 4 left the impression that the tetramers filled the dyadic cleft, forming a defect-free crystalline array, often referred to as a checkerboard. The array's formation is thought to be an intrinsic property of the protein reflecting the homotetramer's 4-fold symmetry whereby adjacent tetramers were noncovalently connected through their adjacent clamp domains.5 This is also thought to provide the structural basis for interprotein allosteric interactions. 6,7 Electron tomography and super-resolution fluorescence microscopy later revealed that the dyad contained subarrays that did not completely fill the cleft, and although neither technique had the resolution to determine the position and orientation of individual tetramers, the super resolution study assumed a regular checkerboard array when fitting their data. 8,9 A single-tilt tomogram with higher resolution indicated that the subarrays were unlikely to be fitted with a simple checkerboard. 10 RYR1 tetramers, purified from skeletal muscle and inserted in artificial bilayers, spontaneously formed 2 different types of array that depended on the free Mg 2+ concentration. Using a nominally Mg 2+ -free buffer, the tetramers formed a checkerboard, but with the addition of 4 mmol/L Mg 2+ , the tetramers were more densely packed in a side-by-side orientation although there was no physical contact between them. 11,12 The organization of the tetramers at the expected intracellular free Mg 2+ concentration of ≈1 mmol/L was not investigated. Whether RYR2 behaves similarly, and if such changes can occur in vivo, is unknown.In this study, we examined dual-tilt tomograms to visualize the position directly of individual RYR2 tetramers in adult rat ventricular myocytes. When fixed in situ, where the Mg 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 2+ 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 dis...

Size Matters: Ryanodine Receptor Cluster Size Affects Arrhythmogenic Sarcoplasmic Reticulum Calcium Release

“…Not surprisingly, the number of RyRs/cluster correlates with the physical cluster size (Figure 2B), although there is significant variation, which may reflect differences in relative RyR packing 2, 3, 5. The clusters were typically asymmetric with the major axis roughly 50% longer than the minor axis (Figure 2C), and the major axis tended to be more transverse in orientation (Figure 2D; Figure S5).…”

“…First we assume that our mean cluster ΔF/F 0 of 1.39 (Figure 1D) corresponds to the average (or most frequent) RyR cluster size estimated from several prior studies that range mostly between 50 and 150 RyR/cluster, which in some high‐resolution cases were clusters of clusters or superclusters 2, 3, 4, 5, 6, 27. The mean ΔF/F 0 of 1.39 is indicated by a vertical broken line in Figure 1E.…”

“…Confocal resolution used here cannot directly resolve individual RyRs within a junctional cluster as is possible with superresolution or electron microscopy 2, 3, 4, 5, 6. However, the relative F‐FKBP fluorescence intensity (ΔF/F 0 ) within 250 nm around the local cluster peak intensity (Figure 1D) provides a direct quantitative readout that is proportional to the number of RyRs within that local cluster (or supercluster; Figure 1E).…”

“…High‐resolution imaging has shown that RyR clusters can range from 1 to 400 per cluster,2, 3, 4, 5, 6 but terminology merits further discussion. Seminal quantitative electron microscopy by Franzini‐Armstrong et al4 inferred a typical RyR cluster in rat ventricular myocyte junctions to be 267 in circular arrays, requiring a fully packed checkerboard array of 553 nm diameter (for 30‐nm center‐center RyR distance) 41, 42.…”

“…RyR sensitivity to cytosolic Ca 2+ ([Ca 2+ ] i ) is increased in genetic diseases (catecholaminergic polymorphic ventricular tachycardia), on increased SR Ca 2+ content, during heart failure, and on certain posttranslational modifications (eg, phosphorylation or oxidation), all of which promote diastolic SR Ca 2+ leak and triggered arrhythmias 1. In cardiac myocytes, RyRs form clusters in which some RyRs are tightly packed in checkerboard arrays (corner to corner) with others clustered in a less organized manner within junctional clefts between the SR and sarcolemma 2, 3, 4, 5, 6. These junctional clefts are cylinders 100 to 200 nm in diameter along transverse tubules and surface sarcolemma, with a depth comparable to the height of the RyR protruding from the SR (≈13 nm).…”