Resource for the Visualization of Biological Complexity (RVBC)
Functional states of the ryanodine receptor (RyR) and 3D structure of the triad junction
RyRs function as intracellular calcium release channels that are particularly prevalent in muscle where they are associated with the sarcoplasmic reticulum (SR). RyRs are homotetramers constructed from a subunit of molecular mass 565 Kda, making these receptors the largest known ion channels (Samso and Wagenknecht, 1998; Fill and Copello, 2002; Wagenknecht and Samso, 2002). The Wagenknecht laboratory has been engaged in structural studies of isolated RyRs from skeletal and heart muscle by cryoelectron microscopy and three-dimensional reconstruction for the past 12 years (Liu et al., 2004; Radermacher et al., 1992). 3D reconstructions have shown that about 80% of the mass of the RyR is located in the cytoplasm as a complex assembly of ~10 distinct domains (Radermacher et al., 1994). Besides their function in releasing calcium, which is the immediate stimulus for muscle contraction, RyRs play a key role in excitation-contraction (E-C) coupling. E-C coupling refers to the process by which neuron-induced depolarization of the muscle plasma membrane (sarcolemma) and it invaginations known as transverse (t)-tubules) leads to release of Ca2+ from the SR via the RyRs. Perhaps the most important of the sarcolemma/t-tubule components is an L-type calcium channel known as the dihydropyridine receptor, which serves as the voltage sensor in e-c coupling (Rios and Pizarro, 1991;Jones, 2002). Interactions between the dihydropyridine receptor and the cytoplasmic region of the RyR are thought to mediate communication between the plasma membranes/transverse tubules and the transmembrane, ion-conducting region of the RyR in the SR. The figure below shows schematically the relationship of RyRs to DHPRs in skeletal muscle as well as some of the other protein components of the triad junction (CaM, calmodulin; FKBP, FK506-binding protein)
Triad junction schematic showing major components
The RyR is a ligand-gated channel. Physiologic ligand activators of the skeletal RyR include the dihydropyridine receptor (the voltage-sensor), Ca2+ at micromolar levels, and millimolar ATP. Inhibitors include Ca2+ and Mg2+ at millimolar concentrations. In a pioneering study that employed time-resolved cryo-microscopy, Unwin demonstrated that the acetylcholine receptor, also a ligand-gated ion channel, could be imaged in two of its functional states, a closed and an open state (Unwin, 1995). Time-resolved techniques were required because when the acetylcholine receptor is induced to open by physiologic agonists (in this case acetylcholine), the open state is short-lived, lasting only 50-100 ms before assuming a desensitized (inactivated) state. We are applying similar time-resolved cryo-EM methods to RyR.
Until recently it was not clear whether isolated RyRs inactivated, but now several studies have uncovered rather slow transitions (time constants ~100 ms to seconds, depending on experimental conditions) from the open form to states that have been described as either "inactivated"or "adapted" (Schiefer et al., 1995; Laver and Lamb, 1998;Valdivia et al., 1995) Gyorke and Fill, 1993) ; Lamb et al., 2000; Fill et al., 2000). Controversy currently exists regarding the precise nature and physiologic significance of these newly identified states of the RyR, and also regarding the disparate results that have been obtained in different laboratories. Evidence is mounting for a physiological role for inactivation of the heart isoform of the RyR to prevent uncontrolled calcium-induced calcium release (Wang et al., 2004).
RyRs also exhibit the somewhat unusual property of binding the regulator molecule, calmodulin, in both its apo and Ca2+-bound forms (Meissner, 2002; Tang et al., 2002). Interestingly, our structural analyses by single-particle cryo-EM have shown two discrete binding sites, separated by about 30 Ångstroms, for the two forms of calmodulin (Wagenknecht et al., 1997; Samso and Wagenknecht, 2002; Wagenknecht et al., 1997). We have hypothesized that calmodulin swithches between these two sites without dissociating from the ryanodine receptor.
Project 1: Three-dimensional reconstruction of closed, open and inactivated/adapted states of skeletal and cardiac RyRs.
Already we and others have deterined 3D reconstructions of of solubilized RyRs that had been incubated under conditions that should favor open and closed states of the receptor. Although the resolution of these reconstructions is presently limited to ~30 Å, nevertheless reproducible structural difference have been detected between the RyRs in the putatively open and closed states. An example for isoform 3 of RyR is shown below (Sharma et al., 2000). Notice, for example, that one of the peripheral domains (labeled "6"), which is far (> 100) from the ion-channel containing transmembrane regions, shows a marked difference between the two states of the receptor. Time-resolved methods are needed to confirm and extend these studies to other functional states of the receptor.
Functional states of the RyR
Project 2: 3D structure of complexes of RyR and protein ligands. RyRs are known to interact directly in with several proteins in skeletal muscle to form a signal-transducing complex known as the triad junction. One goal is to use the isolated RyR as an "assembly platform" to prepare complexes containing each of these ligands. Already, we have made progress in determining the binding locations of two ligands, FK506-binding protein and calmodulin, on the surface of the RyR (Wagenknecht et al., 1994; Wagenknecht et al., 1996; Wagenknecht et al., 1997; Samso and Wagenknecht, 2002; Sharma et al. 2004 (submitted)). Of particular interest is the interaction of the RyR with the dihydropyridine receptor (Samso et al., 1999), which is thought to represent the core of the signal-tranducing apparatus of e-c coupling. Sequence-specific antibodies represent another class of ligand that is being used to probe the structure of the RyR by means of in vitro assembly and 3D cryo-microscopy.
Project 3. Cryo-tomography of the skeletal muscle triad junction. Work in other laboratories has shown that it is feasible to isolate membrane fractions from skeletal muscles that contain functionally intact triads (Ikemoto et al., 1994). Low resolution microscopy on these preparations shows the presence of intact triads (i.e. sarcoplasmic reticulum-derived vesicles joined to transverse-tubule-derived vesicles with RyRs visible in the intermembrane space in the junctional regions) (Kim et al., 1990), and cryo-EM of frozen-hydrated triads yields images in which the bridging RyRs are directly visible. We determined the first tomograms from frozen-hydrated isolated triad junctions (Wagenknecht et al., 2002), see Figure below) The reconstruction resolved some of the individual ryanodine receptors and revealed their relationship to the dense mat of luminal density attributed to calsequestrin that underlies the RyR-enriched regions of the SR. Frozen-hydrated triads should be a readily amenable to the application of high-precision immuno-EM to identify protein components that are resolved in the tomograms. Time-resolved tomography is being applied to determined whether the structural configuration of calsequestrin changes as calcium reserves are depleted via release of Ca2+ through the RyRs.
Interpertative representation of the triad junction reconstructed by tomography
References
Fill M., Copello, J.A. (2002). Ryanodine Receptor Calcium Release Channels. Physiol Rev 82: 893-922.
Fill M., Zahradnikova,A., Villalba-Galea, C.A., Zahradnik, I., Escobar, A.L., Gyorke, S. (2000). Ryanodine receptor adaptation. J Gen Physiol 116: 873-882.
Gyorke, S., Fil, M. (1993). Ryanodine Receptor Adaptation - Control Mechanism of Ca2+- Induced Ca2+ Release in Heart. Science 260: 807-809.
Ikemoto, N., Yano, M., Elhayek, R., Antoniu, B., Morii, M. (1994). Chemical depolarization-induced SR calcium release in triads isolated from rabbit skeletal muscle. Biochemistry 33: 10961-10968.
Jones, S.W. (2002). Calcium channels: when is a subunit not a subunit? J Physiol (Lond)545: 334.
Kim, K.C., Caswel,l A.H., Brunschwig, J.-P., Brandt, N.R. (1990). Identification of a new subpopulation of triad junctions isolated from skeletal muscle; morphological correlations with intact muscle. J Membrane Biol 113: 221-235.
Lamb, G.D., Laver, D.R., Stephenson, D.G. (2000). Questions about adaptation in ryanodine receptors. J. Gen. Physiol. 116: 883-890.
Laver, D.R., Lamb, G.D. (1998). Inactivation of Ca2+ release channels (ryanodine receptors RyR1 and RyR2) with rapid steps in [Ca2+] and voltage. Biochem. J. 74: 2352-2364.
Liu, Z., Zhang, J., Wang, R.W., Chen, S.R.W., Wagenknecht, T. (2004). Location of divergent region 2 on the three-dimensional structure of cardiac muscle ryanodine receptor/calcium release channel. J. Mol. Biol. 338: 533-545.
Meissner, G. (2002). Regulation of mammalian ryanodine receptors. Frontiers in Bioscience 7: d2080.
Radermacher, M., Rao, V., Grassucci, R., Frank, J., Timerman, A.P., Fleischer, S., Wagenknecht, T. (1994), Cryo-electron microscopy and three-dimensional reconstruction of the calcium release channel ryanodine receptor from skeletal muscle. J. Cell Biol. 127: 411-423.
Radermacher, M., Wagenknecht, T., Grassucci, R., Frank, J., Inui, M., Chadwick, C., Fleischer, S. (1992). Cryo-EM of the native structure of the calcium release channel/ryanodine receptor from sarcoplasmic reticulum. Biophys. J. 61: 936-940.
Rios, E., Pizarro, G. (1991). Voltage sensor of excitation-contraction coupling in skeletal muscle. Physiol. Rev. 71: 849-908.
Samso, M., Trujillo, R., Gurrola, G.B., Validivia, H.H., Wagenknecht, T. (1999). Three-dimensional location of the imperatoxin A binding site on the ryanodine receptor. J. Cell Biol. 146: 493-499.
Samso, M., Wagenknecht, T. (1998). Contributions of electron microscopy and single-particle techniques to the determination of the ryanodine receptor three-dimensional structure. J. Struct. Biol. 121: 172-180.
Samso, M., Wagenknecht, T. (2002). Apocalmodulin and Ca2+-calmodulin bind to neighboring locations on the ryanodine receptor. J. Biol. Chem.277: 1349-1353.
Schiefer, A., Meissner, G., Isenberg, G. (1995). Ca2+ activation and Ca2+ inactivation of canine reconstituted cardiac sarcoplasmic reticulum Ca2+-release channels. J. Physiol -London 489: 337-348.
Sharma, M.R., Jeyakumar, L.H,, Fleischer, S., Wagenknecht, T. (2000). Three-dimensional structure of yanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy. J. Biol. Chem.275: 9485-9491.
Tang, W., Sencer, S., Hamilton, S.L. (2002). Calmodulin modulation of proteins involved in excitation-contraction coupling [Review]. Frontiers in Bioscience 7: D1583-D1589.
Unwin, N. (1995). Acetylcholine receptor channel imaged in the open state. Nature 373: 37-43.
Valdivia, H.H., Kaplan, J.H., Ellis-Davies, G.C., Lederer, W.J. (1995) Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2+ and phosphorylation. Science 267: 1997-2000.
Wagenknecht, T., Berkowitz, J., Grassucci, R., Timerman, A.P., Fleischer, S. (1994). Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy. Biophys. J. 67: 2286-2295.
Wagenknecht, T., Grassucci, R., Berkowitz, J., Wiederrecht, G.J., Xin, H.-B., Fleischer, S. (1996). Cryoelectron microscopy resolves FK506-binding protein sites on the skeletal muscle ryanodine receptor. Biophys. J. 70: 1709-1715.
Wagenknecht, T., Hsieh, C.-E., Rath, B.K., Fleischer, S., Marko, M. (2002). Electron Tomography of Frozen-Hydrated Isolated Triad Junctions. Biophys. J. 83: 2491-2501.
Wagenknecht, T., Radermacher, M., Grassucci, R., Berkowitz, J., Xin, H.-B., Fleischer, S. (1997). Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor. J. Biol. Chem. 272: 32463-32471.
Wagenknecht, T., Samso, M. (2002). Three-dimensional reconstruction of ryanodine receptors [Review]. Frontiers in Bioscience 7: D1464-D1474.
Wang, S.Q., Stern, M.D., Rios, E., Cheng, H.P. (2004). The quantal nature of Ca2+ sparks and in situ operation of the ryanodine receptor array in cardiac cells. Proc. Natl. Acad. Sci. USA 101: 3979-3984.