Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are responsible for the release of Ca 2þ from intracellular stores during excitation-contraction coupling in both cardiac and skeletal muscle. RyRs are the largest known ion channels (.2MDa) and exist as three mammalian isoforms (RyR 1 -3), all of which are homotetrameric proteins that interact with and are regulated by phosphorylation, redox modifications, and a variety of small proteins and ions. Most RyR channel modulators interact with the large cytoplasmic domain whereas the carboxy-terminal portion of the protein forms the ion-conducting pore. Mutations in RyR2 are associated with human disorders such as catecholaminergic polymorphic ventricular tachycardia whereas mutations in RyR1 underlie diseases such as central core disease and malignant hyperthermia. This chapter examines the current concepts of the structure, function and regulation of RyRs and assesses the current state of understanding of their roles in associated disorders.

Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We previously identified a subset of cancers harboring homozygous deletion of the glycolytic enzyme Enolase (ENO1) with exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small molecule Enolase inhibitor, POMHEX, can selectively kill ENO1 -deleted glioma cells at low nanomolar concentrations and eradicate intracranial orthotopic ENO1 -deleted tumors in mice at doses well-tolerated in non-human primates. Our data provide in vivo proof-of-principal for the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential across a range of therapeutic settings.

Mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (RyR1) die when exposed to short periods of temperature elevation (≥ 37 °C). We demonstrate that treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevents heat-induced sudden death in Y524S mice. The AICAR protection is independent of AMPK activation and results from a newly identified action on the mutant RyR1 to reduce Ca2+ leak, preventing Ca2+ dependent increases in both reactive oxygen and reactive nitrogen species that act to further increase resting Ca2+ concentrations. If unchecked, the temperature driven increases in resting Ca2+ and ROS/RNS create an amplifying cycle that ultimately triggers sustained muscle contractions, rhabdomyolysis and death. Although antioxidants are effective in reducing this cycle in vitro, only AICAR prevents the heat induced death in vivo. Our findings suggest that AICAR is likely to be effective in prophylactic treatment of humans with enhanced susceptibility to exercise/heat-induced sudden death associated with RyR1 mutations.

During layerwise growth of crystals, capillarity governs the generation of new crystal layers. Theory predicts that the line tension of the layer edge determines, via the characteristic two-dimensional capillary length L(c), the rates of generation and initial growth of the new layers. To test the correlation between L(c) and the rate of layer generation, we used in situ Tapping Mode Atomic Force Microscopy (TM-AFM) to study the generation and spreading of layers during crystallization of rhombohedral, R3, porcine insulin. We show that crystallization of this insulin form is uniquely suitable for such an investigation due to the linear kinetics of step growth it exhibits. This linear kinetics reflects the abundance of the incorporation sites along the rough steps, the lack of long-range step-step interactions, and the transport control of the growth kinetics. The kinetic coefficients are 7 x 10(-)(3) and 4 x 10(-)(2) cm s(-)(1), respectively, in the absence and presence of the cosolvent acetone-somewhat high for proteins and comparable to values for inorganic systems. We show that (i). the relevant capillary length, the size of a critical quadrangular 2D nucleus L(c), is the main scaling factor for the density of growth steps, while (ii). all steps longer than L(c) grow with a rate determined only by the supersaturation and independent of their length. We explain the divergence of (ii). from theoretical predictions with the high supersaturations typical of the growth of this protein system.

Mutations in the RYR1 gene cause severe myopathies. Mice with an I4895T mutation in the type 1 ryanodine receptor/Ca2+ release channel (RyR1) display muscle weakness and atrophy, but the underlying mechanisms are unclear. Here we show that the I4895T mutation in RyR1 decreases the amplitude of the sarcoplasmic reticulum (SR) Ca2+ transient, resting cytosolic Ca2+ levels, muscle triadin content and calsequestrin (CSQ) localization to the junctional SR, and increases endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and mitochondrial ROS production. Treatment of mice carrying the I4895T mutation with a chemical chaperone, sodium 4-phenylbutyrate (4PBA), reduces ER stress/UPR and improves muscle function, but does not restore SR Ca2+ transients in I4895T fibres to wild type levels, suggesting that decreased SR Ca2+ release is not the major driver of the myopathy. These findings suggest that 4PBA, an FDA-approved drug, has potential as a therapeutic intervention for RyR1 myopathies that are associated with ER stress.