Ryanodine receptor 1 (RyR1) is an isoform of ryanodine receptor predominantly expressed in skeletal muscle. It is a ~2MDa homotetrameric Ca²⁺ release channel with a large cytosolic domain and is in the membrane of the sarcoplasmic reticulum in skeletal muscle cells. RyR1 plays a key role in coordinating excitation-contraction (EC) coupling in skeletal muscle. The activity of the RyR1 channel is regulated by multiple factors, including phosphorylation, oxidation, and ligand binding, all of which tightly control the channel function. The cytosolic domain of RyR1 contains binding sites for these ligands, enabling allosteric regulation.
Malignant hyperthermia susceptibility (MHS) is a condition that predisposes individuals to an episode of malignant hyperthermia (MH), a pharmacogenetic shock syndrome triggered by the administration of volatile inhalational anesthetics such as halothane, isoflurane, and succinylcholine. Variants in the RYR1 gene is responsible for over 50% of MHS cases. To treat the rapid metabolic shock that occurs during an MH episode, dantrolene must be administered quickly. Dantrolene, the only approved drug for MH treatment, inhibits RyR1 and reduces Ca²⁺ influx into the cytoplasm of skeletal muscle cells. However, the detailed molecular mechanism by which dantrolene inhibits RyR1 has not been fully elucidated.
I purified rabbit RyR1 reconstituted in detergent micelles and subjected the vitrified protein-ligand samples to cryo-electron microscopy (cryoEM) in the presence of dantrolene and other RyR1 agonists, such as ATP, ADP, caffeine, and 4-chloro-m-cresol (4CmC; an MH-triggering molecule). I identified dantrolene binding in complex with ATP or ADP at the RY12 domain on RyR1. Additionally, multiple binding sites for 4CmC on RyR1 were identified. Following the initial characterization of the novel dantrolene and adenosine phosphate binding site in the RY12 domain, purified RyR1 was reconstituted in liposomes for single-channel planar lipid bilayer assays. These assays confirmed that either ATP or ADP is required at the dantrolene binding site for RyR1 inhibition. These findings led us to hypothesize that the novel drug and ATP/ADP binding site in the RY12 domain may also play a physiological role in sensing an increased ADP concentrations in skeletal muscle cells, particularly in the cytosolic compartment during muscle fatigue and pathological conditions. During EC COUPLING, ATP hydrolysis for muscle contraction increases cytosolic ADP concentrations above resting levels. I found that the RY12 site preferentially binds ADP rather than ATP when neither dantrolene nor 4CmC is present.
I also discovered that RyR1 forms an endogenous complex with calstabin1 (Cs1, also known as FK506-binding protein 12) and calmodulin (CaM), as observed through cryoEM analysis of rabbit RyR1 solubilized with digitonin and purified by sucrose gradient centrifugation. During these experiments, I noticed that RyR1s in digitonin non-specifically adhere to the air-water interface (AWI) between the sample buffer and atmospheric air on cryoEM grids, resulting in biased orientations of RyR1 particles in the micrographs. To mitigate this effect, glycyrrhizic acid was added to the purified RyR1 sample immediately before vitrification. I applied a similar strategy to prevent the protein from adhering to the AWI when solving the structure of iodinated bovine thyroglobulin (Tg), a ~660 kDa homodimeric soluble protein responsible for thyroid hormone biosynthesis in the thyroid gland. Thyroxines (T4) and iodotyrosines (mono- or di-iodotyrosine) were identified at hormonogenic sites on bovine Tg in the reconstructed EM map, and the analysis around the T4 sites allowed us to hypothesize the molecular mechanism of coupling reactions between two diiodotyrosine residues to synthesize T4 within the Tg molecule.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/180t-jb98 |
| Date | January 2024 |
| Creators | Kim, Kookjoo |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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