This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.SUPPORT: NIH R01 R40615 Structure of channels in excitation-contraction coupling , 8/15/00-8/14/05. Terence Wagenknecht, Wadsworth CenterNIH R01 HL55438 Modulatory mechanisms of Ryanodine receptor adaptation , 3/8/96-8/31/04Hector Valdivia, Department of Physiology, University of WisconsinNIH HL76826 Modulation of cardiac E-C coupling , 4/1/04-3/31/09.Hector Valdivia, Department of Physiology, University of WisconsinABSTRACT 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 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. 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). The mechanism of excitation-contraction (e-c) coupling is a major research problem among muscle physiologists. Ryanodine receptors play a key role in the signal-transducing events of the process. Our research will provide structural details of the e-c coupling apparatus using what are perhaps the only methods available for obtaining this information. Several muscle diseases are known to involve mutations of the ryanodine receptor, and there is evidence that RyRs play roles in heart disease and brain function as well. Our entire research program on RyRs depends on single-particle image processing methodology (TRD#3) and cryo-EM technology (TRD#2), as described in detail above. Electron tomography of frozen-hydrated subcellular structures (TRD#1) is also essential to one component of our program (Project 3 above).Project 1: Three-dimensional reconstruction of closed, open and inactivated/adapted states of skeletal and cardiac RyR Closed states of the RyR are easy to achieve by simply withholding activators. Open states will be induced by rapid addition of activators followed by freezing for cryo-EM at defined time intervals of sufficiently short duration that inactivated/adapted states will not form. The technology developed in TRD#2 is necessary to carry out the proposed experiments. There is good reason to anticipate that interesting structural changes will be observable between the closed and open states. Our own group and a group at Baylor have published cryo-EM studies of RyRs in putatively open (but possibly inactivated) states that were obtained treating the receptor with appropriate levels of Ca2+ and nucleotide Sharma et al., 2000; Serysheva et al., 1999). Signficant structural differences were detected between the two states, not only in the transmembrane regions as might be expected, but, surprisingly, also in the cytoplasmic regions at distances quite distant (>100 ) from the ion channel. To confirm and improve upon these findings, the RyR should be studied in the open form at several time points following addition of activators. We plan to activate RyRs by exposing them to the activator, Ca2+, by flash photolysis of caged Ca2+ (Ca2+-Dinitrophen) as has been done for electrophysiologyl experiments on RyRs (Gyorke et al., 1994). Alternatively, the microdroplet spray approach could also be used to expose RyRs on the grid to activators contained in the spray. The pre-mix approach may be preferable if we find that reaction times greater than 1 second are sufficiently short to trap the open state of RyR. The time-resolved approach may prove to be indispensable for reasons other than preventing formation of undesirable, slow-forming conformational states of the RyR. All of the cryo-EM studies on the RyR to date have been conducted on purified, detergent-solubilized material. The presence of detergent and the lack of a membrane bilayer in these preparations could lead to conclusions that are not valid for the receptor in its natural environment. We have found that at reduced levels of detergents, the closed form of the receptor remains soluble for short periods (minutes) of time (Wagenknecht et al., 1997; Wagenknecht et al., 1994). However, our initial experiments to characterize the open form of the RyR at low concentrations of detergent often showed unacceptably high amounts of apparently aggregated receptors on the EM grid. By performing these same experiments using time-resolved methods we will be able time to reduce the time of exposure to low detergent levels to less than 1 second; this should greatly reduce solubility problems, and allow even lower levels of detergent to be used. The advantages of rapid-dilution to remove detergents could be substantial and of general applicability to membrane proteins or, more generally to any macromolecule that becomes prone to aggregation upon a change in solution conditions or addition of a ligand (e.g. as was indeed found for a low pH-induced conformational state of Semliki Forest virus (Fuller et al., 1995). Eventually, we expect to be able to study the RyRs in a lipid bilayer environment where functional activities can be monitored in parallel with structural work. Some preliminary success has already been achieved in imaging RyRs reconstituted into bilayers (Liu and Wagenknecht, 1999). More generally, for macromolecular machines that are metastable, such as RyR, precautions to reduce exposure to the carbon surface of the EM grid might be beneficial to retention of native structure even in non-time-resolved experiments (Trujillo et al., 2004) As discussed above, evidence is accumulating that RyRs can exist in at least three major conformations: closed, open, and inactivated (and/or adapted). Probably there are multiple states within each of these major categories (Laver and Lamb, 1998). As our understanding of the transitions between the various states improves, it should become feasible to characterize the structure of the inactivated (or adapted) state by cryo-EM and image reconstruction. It may even prove feasible to follow the kinetics of structural transitions undergone by the receptor in response to conditions that mimic those experienced by the receptor in vivo.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. 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). Thus, calmodulin could be considered to be a resident regulatory component of the RyR; a similar role for calmodulin has been postulated for other ion channels (Budde et al., 2002). Interestingly, our structural analyses by single-particle cryo-EM have shown two discrete binding sites, separated by about 30 ngstroms, for the two forms of calmodulin (Samso and Wagenknecht, 2002; Wagenknecht et al., 1997). We have hypothesized that calmodulin swithches between these two sites without dissociating from the ryanodine receptor. We propose to test this hypothesis using time-resolved cryo-EM to induce the putative switch of binding sites in the absence of exogenous calmodulin.Since the equilibrium constants for ligands vary widely, it is frequently desirable to perform the assembly reactions using RyR and ligand at concentrations that are higher than optimal for EM (~10-7 M for RyR) so as to maximize the fraction of RyR that has bound ligand. The development of rapid dilution techniques of time-resolved cryo-microscopy (TRD2) will allow RyR ligands with lower affintities (Kd>10-7) to be incubated at concentrations appropriate for obtaining high yields (>90%), and then to be rapidly diluted for cryo-EM within a time frame that is much slower than the rate of ligand dissociation. The use of the rapid dilution technique for obtaining high yields of in vitro assembly products for cryo-microscopy may well prove to be its most powerful application. Project 3. Cryo-tomography of the skeletal muscle triad junctionWork 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. During the current grant period we determined the first tomograms from frozen-hydrated isolated triad junctions (Wagenknecht et al., 2002) 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. A potentially interesting application of time-resolved tomography would be to look for changes in density attributed to calsequestrin as calcium reserves are depleted via release of Ca2+ through the RyRs.References1. Budde T, Meuth S, Pape HC (2002) Calcium-dependent inactivation of neuronal calcium channels [Review]. Nature Reviews Neuroscience 3: 873-883.2. Fill M, Copello JA (2002) Ryanodine Receptor Calcium Release Channels. Physiol Rev 82: 893-922.3. Fill M, Zahradnikova A, Villalba-Galea CA, Zahradnik I, Escobar AL, Gyorke S (2000) Ryanodine receptor adaptation. J Gen Physiol 116: 873-882.4. Fuller SD, Berriman JA, Butcher SJ, Gowen BE (1995) Low pH induces swiveling of the glycoprotein heterodimers in the Semliki Forest virus spike complex. Cell 81: 715-725.5. Gyorke S, Fill M (1993) Ryanodine Receptor Adaptation - Control Mechanism of Ca2+- Induced Ca2+ Release in Heart. Science 260: 807-809.6. Gyorke S, Velez P, Suarez-Isla BA, Fill M (1994) Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2+. Biophys J 66: 1879-1886.7. 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: 10961-10968.8. Jones SW (2002) Calcium channels: when is a subunit not a subunit? J Physiol (Lond) 545: 334.9. Kim KC, Caswell AH, Brunschwig J-P, Brandt NR (1990) Identification of a new subpopulation of triad junctions isolated from skeletal muscle; morphological correlations with intact muscle. J Membrane Biol 113: 221-235.10. Lamb GD, Laver DR, Stephenson DG (2000) Questions about adaptation in ryanodine receptors. J Gen Physiol 116: 883-890.11. 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Nature 373: 37-43.27. Valdivia HH, Kaplan JH, Ellis-Davies GC, Lederer WJ (1995) Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2+ and phosphorylation. Science 267: 1997-2000.28. Wagenknecht T, Berkowitz J, Grassucci R, Timerman AP, Fleischer S (1994) Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy. Biophys J 67: 2286-2295.29. Wagenknecht T, Grassucci R, Berkowitz J, Wiederrecht GJ, Xin H-B, Fleischer S (1996) Cryoelectron microscopy resolves FK506-binding protein sites on the skeletal muscle ryanodine receptor. Biophys J 70: 1709-1715.30. Wagenknecht T, Hsieh CE, Rath BK, Fleischer S, Marko M (2002) Electron Tomography of Frozen-Hydrated Isolated Triad Junctions. Biophys J 83: 2491-2501.31. 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. 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