From the Wadsworth Center for Laboratories and
Research, New York State Department of Health, Albany, New York
12201-0509, the ** Department of Biomedical Sciences, School of Public
Health, State University of New York, Albany, New York 12222, and
the
Department of Molecular Biology, Vanderbilt University,
Nashville, Tennessee 37235
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ABSTRACT |
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The three-dimensional structure of the cardiac
muscle ryanodine receptor (RyR2) is described and compared with its
skeletal muscle isoform (RyR1). Previously, structural studies of RyR2 have not been as informative as those for RyR1 because optimal conditions for electron microscopy, which require low levels of phospholipid, are destabilizing for RyR2. A simple procedure was devised for diluting RyR2 (in phospholipid-containing buffer) into a
lipid-free buffer directly on the electron microscope grid, followed by
freezing within a few seconds. Cryoelectron microscopy of RyR2 so
prepared yielded images of sufficient quality for analysis by single
particle image processing. Averaged projection images for RyR2, as well
as for RyR1, prepared under the same conditions, were found to be
nearly identical in overall dimensions and appearance at the resolution
attained, 30 Å. An initial three-dimensional reconstruction of RyR2
was determined (resolution
41 Å) and compared with previously
reported reconstructions of RyR1. Although they looked similar, which
is consistent with the similarity found for the projection images, and
with expectations based on the 66% amino acid sequence identity of the
two isoforms, structural differences near the corners of the
cytoplasmic assembly were observed in both two- and three-dimensional
studies.
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INTRODUCTION |
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Ryanodine receptors (RyRs)1 are the major calcium release channels associated with the sarcoplasmic reticulum in striated muscle (for recent, reviews, see Refs. 1-5), and they are the largest ion channels known. The major receptor isoforms from skeletal (RyR1) and heart (RyR2) muscle are composed of four copies of a very large protein subunit (565 kDa) that has 66% amino acid sequence homology between the two isoforms. In addition to the large subunit, RyR1 and RyR2 are each associated with much smaller (12 kDa), isoform-specific modulatory polypeptides, also present in four copies per receptor, which have been identified as FK506-binding proteins (6-8). A third RyR isoform, RyR3, is expressed in a wide range of tissues, including mammalian brain and skeletal muscle. In fact, all three isoforms are expressed in multiple cell types.
In both skeletal and cardiac muscle, RyRs are thought to play a central
role in excitation-contraction coupling by releasing Ca2+
into the myoplasm, and by mediating signal transduction between the
plasma membrane/transverse- (T) tubule system and the sarcoplasmic reticulum. Dihydropyridine receptors (DHPRs), which are
membrane-spanning voltage sensors in the plasma membrane/t-tubule
system, communicate with the RyRs in the apposing junctional face
membrane of the sarcoplasmic reticulum. Together, the RyRs and DHPRs
are thought to form the major components of the signal transducing
apparatus of excitation-contraction coupling. RyRs are present at
specialized regions of the sarcoplasmic reticulum, the junctional face
membranes of the terminal cisternae, which are closely apposed to the
t-tubule. RyRs possess large cytoplasmic assemblies that span the 12
nm gap between the two membrane systems and had been visualized in electron micrographs of thin-sectioned muscle (9) long before their
identification as RyRs (10-12).
As for the RyR, different isoforms of the DHPR are also present in heart and skeletal muscle, and current evidence suggests that the DHPR and RyR together are responsible for the different mechanisms of E-C coupling in the two types of muscle (13, 14). In skeletal muscle, DHPRs are thought to be physically coupled to RyRs and control their activity by a conformational coupling mechanism. In contrast, cardiac DHPRs function as voltage-regulated Ca2+ channels that control RyR activity by a Ca2+-induced Ca2+ release mechanism.
Several lines of evidence have shown that the single channel properties of RyR1 and RyR2 that have been incorporated into lipid bilayers are similar but not identical. For example, differences are present between the two isoforms regarding their sensitivities to Ca2+ activation/inactivation and Mg2+ inhibition (15).
To help understand the different mechanisms of E-C coupling it is essential to characterize the structural differences of the two RyR isoforms, as well as those of the DHPR, and to relate these differences to functional consequences. With regard to the RyR, the overall sequence identity of RyR1 and RyR2 is 66% (16-20), but three subregions show much less homology and are, therefore, likely to be responsible for the functional differences of the two isoforms. Mutagenesis of these regions together with functional expression in cultured myocytes will clarify which regions are responsible for the different properties of RyR1 and RyR2 (14).
The three-dimensional architecture of solubilized RyR1 has been investigated by cryoelectron microscopy and single-particle image processing methods (21-28, 31). Although the resolution of such studies has been limited to 30-40 Å, numerous structural details have been resolved for RyR1, and for RyR1 complexed with some of the proteins with which it interacts in vivo (25).
To further understand the structural basis of the differing mechanisms of E-C coupling in heart and skeletal muscle, we have attempted to characterize RyR2 by electron microscopy. Until recently these studies have been unsuccessful owing to structural instability of RyR2 as compared with RyR1. Here we describe a procedure for imaging of RyR2 by cryoelectron microscopy, and provide the first three-dimensional reconstruction of RyR2.
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MATERIALS AND METHODS |
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Preparation of RyRs--
Cardiac RyR2s were purified from dog
heart ventricles (12) by sucrose density gradient centrifugation (10,
29). The pooled peak fractions containing RyR2 were in a buffer
containing 0.02 M Tris-HCl (pH 7.4), 1 M NaCl,
2 mM dithiothreitol, 4 µg/ml leupeptin, 0.5% CHAPS,
0.25% soybean phospholipid (SBL), and 20% sucrose. For optimal
electron microscopy it was necessary to concentrate the receptor, which
was accomplished by adsorption to heparin-agarose (11) at reduced NaCl
(0.2 M, which was attained by dilution of the pooled peak
fractions into NaCl-free buffer), followed by elution into the gradient
buffer supplemented with 1.0 µg/ml pepstatin, 100 µM
phenylmethylsulfonyl fluoride, and lacking sucrose (buffer A). RyR1 was
purified from rabbit skeletal muscle as described previously (10, 25,
29), and the final buffer was modified to contain 1% CHAPS and 0.5%
SBL.
Preparation of Grids for Cryoelectron Microscopy-- The following procedure was followed for collecting the microscopy data used to determine the two-dimensional projection averages. RyR2 in buffer A was diluted so that the final concentrations of CHAPS and SBL were 0.5% (w/v) and 0.25% (w/v), respectively, and EGTA was present at 1 mM to maintain the receptor in a closed state. All other buffer components were the same as for buffer A. To avoid prolonged exposure to lipid-free conditions, 0.5 µl of RyR2 (0.25-0.50 mg/ml) was diluted 10-fold into 4.5 µl of buffer B (same as buffer A except that SBL was absent), on the grid. The grid was then immediately rinsed by holding the specimen side on a drop of buffer C (same as buffer A, except that no SBL was present and [CHAPS] = 0.5%). For purposes of comparison, grids of RyR1 were prepared using the identical procedure. The procedure employed for obtaining the data used for computing the three-dimensional image reconstruction differed by the absence of EGTA in the buffers.
Cryoelectron Microscopy--
Grids were transferred while under
liquid nitrogen to a Gatan (model 626) cryoholder. Micrographs were
recorded with the objective lens underfocussed by 1.8-2.0 µm at a
nominal magnification of 38,000 × and with a net electron dose
estimated at 10 e/Å2, using a
Philips EM420 transmission electron microscope equipped with a
goniometer stage and low dose kit, and operated at 100 kV. Specimen
temperatures were maintained at
176 ± 2 °C.
Image Processing-- The suitability of the micrographs for image processing was checked by optical diffraction for image defects (astigmatism, specimen charging, and drift) and appropriate defocus (31). Selected micrographs were digitized using a Perkin-Elmer PDS 1010A flatbed scanning microdensitometer (Orbital Sciences, Pomona, CA) with a step size of 20 µm corresponding to 5.3 Å on the object scale. The SPIDER software package (30) was used for all image processing. The procedures used for averaging images of receptors in the 4-fold symmetric orientation and for determining difference images have been previously described (23, 31).
It was found that micrographs of RyR2 contain images of apparently well preserved receptors in many different orientations, thereby obviating the requirement of tilting the specimen grid (37, 38). RyR1 in the present experimental conditions shows a strong propensity for the orientations displaying 4-fold symmetry and would require tilting the specimen grid to determine a three-dimensional reconstruction, in agreement with our earlier structural analysis of RyR1. Therefore, for RyR2 we employed a method of three-dimensional reconstruction applicable to macromolecules that display multiple orientations on the specimen grid that was developed by Penczek et al. (32). In this method, experimental images of the macromolecule are matched to images that are computed by projecting a pre-existing three-dimensional model over a sampling of all possible orientations, and a three-dimensional reconstruction is determined from the experimental images based upon the assigned viewing directions. By iterating the procedure using the output of the previous iteration as the input model for the current one, any bias caused by the initial model is reduced. We have applied this algorithm to micrographs of frozen hydrated RyR2 using a previous reconstruction of RyR1 as the starting model (31). This starting model was thus used as template structure to compute 48 reference projections so as to cover the entire angular space evenly. Initially, a set of 4,143 images of individual receptors ("experimental projections," e.g. see Fig. 1A) were cross-correlated with this reference set of 48 quasi-evenly spaced projections. This was done to classify the total number of orientations present in the complete experimental data set. The projection angles of the experimental projections were assigned as those corresponding to the reference projection yielding the highest cross-correlation coefficient. Those images showing the highest correlation with the reference projections were retained for further consideration and refinement of orientation parameters. From these images an initial reconstruction was computed which used 1264 reference projections derived from the starting model. Subsequent iterations were correlated with 1264 projections derived from current reconstruction of RyR2. As a check on the success of the orientational assignments, experimental projection images that were assigned to a given reference projection were rotationally and translationally aligned to one another so that an averaged image could be computed (e.g. Fig. 1B) and compared with the matching projection computed from the final three-dimensional reconstruction (e.g. Fig. 1C). The final reconstruction was computed from 600 particle images, and additional averaging was attained by enforcing 4-fold symmetry. To display the three-dimensional reconstruction of RyR2, a threshold was chosen as described previously (31). The final resolution was estimated by Fourier shell correlation with the cut-off value of 0.5 (33). A nominal resolution of 41 Å was obtained. ![]() |
RESULTS AND DISCUSSION |
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Electron Microscopy of RyR2-- Initial attempts to image RyR2 by cryoelectron microscopy using the same procedures as were employed previously for RyR1 (21-23) were unsuccessful; nearly all of the RyR2 complexes appeared to be distorted or aggregated (Fig. 1A), and few images displaying the expected 4-fold symmetry were apparent. For these studies the receptors were isolated in a buffer containing detergent (0.5-1.0% CHAPS), but lacking phospholipid. Similar results by the negative staining technique have been reported (34). Since earlier studies demonstrated an irreversible loss in ryanodine binding activity upon removal of phospholipid from purified RyR2 (12, 35), we surmised that the omission of lipids might be responsible for the apparent loss in structural integrity. However, phospholipid at the levels commonly employed in the purification of RyR2, 2.5-5 mg/ml, interferes with cryoelectron microscopy by reducing the contrast to unacceptably low levels.
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Two-dimensional Image Analysis of RyR2-- Images of frozen hydrated RyR2s in the 4-fold symmetric orientation were selected from micrographs such as that shown in Fig. 2B, and the particles were aligned by correlation methods. The aligned images were subjected to correspondence analysis and classification techniques, and two principal classes of roughly equal numbers were identified which correspond to receptors lying either face up or face down on the carbon support surface of the microscope grid. The same behavior has been observed previously for RyR1 (31). Averages of the aligned and classified RyR2 for each of the two classes were computed, and one of them is shown in Fig. 3A. The face-up and face-down averages, which show the three-dimensional density distribution of the receptor projected along opposing directions of the 4-fold symmetry axis, are simply mirror images of each other, and are otherwise essentially identical. The reproducibility of the averaged images extended to 32 Å as assessed by the Fourier ring-correlation method (33, 36).
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Three-dimensional Reconstruction-- An initial reconstruction of RyR2 has been determined by a projection matching algorithm (32). The three-dimensional structure of RyR2 is shown in Fig. 4A, represented as a solid surface in three orientations. Corresponding views of RyR1 from previous work (31) are shown in Fig. 4B. Both reconstructions were low-pass filtered to match the limiting resolution of 41 Å obtained for RyR2, which is lower than was obtained for RyR1 (31). Probably the lower resolution of RyR2 is due to the lower signal-to-noise ratio of RyR2 resulting from the presence of residual lipids on the electron microscope grid, and by the paucity of non-4-fold symmetric views of RyR2 available for inclusion in the reconstruction. Improvements in the resolution of ryanodine receptor reconstructions by the projection matching technique (32) will likely require significantly larger data sets to compensate for these effects. Additionally, micrographs will have to be recorded at several values of objective lens defocus. Following this strategy, a resolution of about 15 Å has been attained for another non-crystalline macromolecular complex, the prokaryotic ribosome.2
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ACKNOWLEDGEMENTS |
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We thank Dr. Phillip Williams (Department of Surgery, Vanderbilt University Medical School) for supplying the canine hearts used in this study. We gratefully acknowledge the use and technical assistance of the Wadsworth Center's electron microscopy and image processing core facilities.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AR40615 (to T. W.) and HL32711 and the Muscular Dystrophy Association (to S. F).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of American Heart Association Fellowship 960124. To whom all correspondence should be addressed.
¶ Supported by National Institutes of Health Grant GM29169 (Joachim Frank).
1 The abbreviations used are: RyR, ryanodine receptor; RyR1, skeletal muscle ryanodine receptors; RyR2, cardiac muscle ryanodine receptors; E-C coupling, excitation-contraction coupling; DHPR, dihydropyridine receptor; SBL, soybean phospholipid; CHAPS, 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2 J. Frank, personal communication.
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REFERENCES |
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