Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.

Ryanodine receptor type 1 (RyR1) produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 Å resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic “inner branches” and the transmembrane “inner helices”). Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 Å diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right-handed bundle structures along a common 4-fold axis.

A change in cytosolic Ca 2ϩ concentration serves as a signal for modulating a wide range of cellular activities (1-3). A major mechanism for increasing cytosolic Ca 2ϩ includes release of Ca 2ϩ from internal stores (endoplasmic or sarcoplasmic reticulum, ER or SR) 1 via a genetic superfamily of Ca 2ϩ release channels including inositol 1,4,5-trisphosphate receptors (IP 3 R) and ryanodine receptors (RyR) (4 -6). A prominent functional property of all of these channels is exquisite sensitivity to reduction and oxidation by sulfhydryl reagents (7-12). The functional consequences of sulfhydryl modification of RyRs include phases of activation and inhibition, revealing that multiple classes of sulfhydryl groups residing on Cys residues of all three isoforms of RyR channel complexes are important for native functioning and subject to chemical modification (11,12). However, defining a role for sulfhydryl redox chemistry in RyR function has been controversial since the initial suggestion that sulfhydryl oxidation is a key step in channel activation (13). A plausible physiological role for redox control of ER/SR Ca 2ϩ release channels and its attendant mechanism has remained elusive.It is known that glutathione (GSH) and glutathione disulfide (GSSG) constitute the major redox buffer system of skeletal muscle and many non-muscle cells (14, 15). In the typical mammalian cell, the ratio of [GSH]/[GSSG] in the cytosol is Ն30:1, thereby maintaining very reduced redox potential (RP) of approximately Ϫ220 mV (16). By contrast, the RP of the ER lumen is significantly more oxidized (approximately Ϫ180 mV) and is maintained with a 3:1 to 1:1 ratio of [GSH]/[GSSG] (16, 17). Thus, the typical microsomal membrane within which the RyR and IP 3 R reside is normally subject to a large transmembrane RP difference of 40 -50 mV with the lumen much more oxidized than the cytosol (16,17).To study redox regulation of RyR channel activity, the bilayer lipid membrane (BLM) preparation affords precise control of the redox state on both the cytoplasmic (cis) and luminal (trans) faces of the reconstituted channel by adjustment of the [GSH]/[GSSG] ratio to form varied redox potentials. In the present work, we provide direct evidence that RyR1 channel activity follows transmembrane redox potential. Chemical labeling studies with CPM indicate previously identified hyperreactive sulfhydryl moieties within the RyR1 complex (18,19) constitute an essential component of a unique transmembrane redox sensor. EXPERIMENTAL PROCEDURESPreparation of SR Membranes--Sarcoplasmic reticulum membrane vesicles were prepared from back and hind limb skeletal muscles of New Zealand White rabbits according to the method of Saito et al. (20) with some modifications. During the SR preparation, GSH and GSSG were included in the homogenization buffer, and the glutathione RP was made to Ϫ220 mV, which mimics the typical cytoplasmic RP in vivo. The preparations were stored in 10% sucrose, 10 mM Hepes, pH 7.4, at Ϫ80°C until needed.GSH and GSSG Stock Solutions-GSH was dissolve...

Triclosan (TCS), a high-production-volume chemical used as a bactericide in personal care products, is a priority pollutant of growing concern to human and environmental health. TCS is capable of altering the activity of type 1 ryanodine receptor (RyR1), but its potential to influence physiological excitation–contraction coupling (ECC) and muscle function has not been investigated. Here, we report that TCS impairs ECC of both cardiac and skeletal muscle in vitro and in vivo. TCS acutely depresses hemodynamics and grip strength in mice at doses ≥12.5 mg/kg i.p., and a concentration ≥0.52 μM in water compromises swimming performance in larval fathead minnow. In isolated ventricular cardiomyocytes, skeletal myotubes, and adult flexor digitorum brevis fibers TCS depresses electrically evoked ECC within ∼10–20 min. In myotubes, nanomolar to low micromolar TCS initially potentiates electrically evoked Ca 2+ transients followed by complete failure of ECC, independent of Ca 2+ store depletion or block of RyR1 channels. TCS also completely blocks excitation-coupled Ca 2+ entry. Voltage clamp experiments showed that TCS partially inhibits L-type Ca 2+ currents of cardiac and skeletal muscle, and [ 3 H]PN200 binding to skeletal membranes is noncompetitively inhibited by TCS in the same concentration range that enhances [ 3 H]ryanodine binding. TCS potently impairs orthograde and retrograde signaling between L-type Ca 2+ and RyR channels in skeletal muscle, and L-type Ca 2+ entry in cardiac muscle, revealing a mechanism by which TCS weakens cardiac and skeletal muscle contractility in a manner that may negatively impact muscle health, especially in susceptible populations.

Homer proteins are adapters that physically bind and functionally couple target proteins (1, 2). Homer1, Homer2, and Homer3 are encoded by three mammalian genes whose expression is dynamically regulated by cellular activity (2) and can attain high levels of protein in the nervous system where their functional regulation of excitatory signaling has been studied (3-5). A common element of structure of all Homer proteins is an N-terminal Enabled/Vasp homology domain (EVH1) essential for binding Homer ligands (6 -7). Crystallographic analysis of the EVH1 domain binding surface has revealed its specific association with polyproline ligands (7) that have previously been defined within plasmalemmal glutamate receptors including mGluR1a 1 and mGluR5a/b (1), inositol 1,4,5-trisphosphate receptors (IP 3 R) localized within endoplasmic and sarcoplasmic reticulum (SR) membranes (1), and cytoplasmic Shank proteins that are part of the N-methyl-D-aspartate receptor-associated PSD-95 complex (8, 9). A C-terminal coiled-coil domain is responsible for Homer self-multimerization (10 -13). Although full-length Homer proteins are constitutively expressed in a number of tissues, immediate-early gene products of the Homer1 gene including Homer1a and Ania 3 lack the CC domain (3, 10). "Short form" Homer1a is rapidly and transiently induced by physiological synaptic stimuli that evoke long term potentiation in the hippocampus (3, 10) or in striatum by the addition of dopaminergic drugs (3). Thus, a use-dependent exchange of multimeric and protomeric short forms of Homer appears to be responsible for dynamic regulation of context-dependent signaling in neurons. The functional influences of Homer adaptors on targeted protein have not been identified. Multi-PDZ domain proteins have been reported to cluster membrane ion channels with the result that the channel become active, but this effect is mimicked by agents that otherwise cross-link the channel (14). Homer binding to mGluR1 was recently reported to define agonist-independent activity of the receptor (15); however, this report did not define a molecular mechanism. The direct consequence of forming Homer-IP 3 R complexes on the dynamics of endoplasmic reticulum/SR Ca 2ϩ release and its possible contribution to temporal and spatial aspects of Ca 2ϩ signals remain unclear.Recently all three Homer mRNAs have also been detected in striated muscle, and Homer protein has been identified within skeletal and cardiac muscle (4, 5). Moreover, putative Homer ligand sequences are found within both the type 1 ryanodine receptor (RyR1) and the ␣ 1S -subunit of the dihydropyridine receptor (DHPR; CACNA1S) of skeletal muscle (7). RyR1 assembles as tetrameric structures within junctional regions of SR where it forms large organized arrays (16). RyR1 forms physical associations with ␣ 1S -DHPR that are essential for engaging reciprocal signaling units that are essential for excitation-contraction (E-C) coupling, a process whereby depolarization in the T-tubules triggers Ca 2ϩ release from SR resulting i...

Polychlorinated biphenyls (PCBs) with unsymmetrical chlorine substitutions and multiple ortho-substitutions that restrict rotation around the biphenyl bond may exist in two stable enantiomeric forms. Stereospecific binding and functional modification of specific biological signaling targets have not been previously described for PCB atropisomers. We report that (-)-2,2′,3,3′,6,6′-hexachlorobiphenyl ((-)-PCB 136) enhances the binding of [3H]ryanodine to high affinity sites on ryanodine receptors type 1 (RyR1) and type 2 (RyR2) (EC50s∼ 0.95 μM), whereas (+)-PCB 136 is inactive at ≤10μM. (-)-PCB 136 induces a rapid release of Ca2+ from microsomal vesicles by selective sensitization of RyRs, an effect not antagonized by (+)-PCB 136. (-)-PCB 136 (500nM) enhances the activity of reconstituted RyR1 channels 3-fold by stabilizing the open and destabilizing the closed conformational states. The enantiomeric specificity is also demonstrated in intact HEK 293 cells expressing RyR1 where exposure to (-)-PCB 136 (100nM; 12hr) sensitizes responses to caffeine, whereas (+)-PCB 136 does not. These data show enantiomeric specificity of (-)-PCB 136 toward a broadly expressed family of microsomal Ca2+ channels that may extend to other chiral non-coplanar PCBs and related structures. Evidence for enantioselective enrichment of PCBs in biological tissues that express RyR1 and RyR2 channels may provide new mechanistic leads about their toxicological impacts on human health.

Mutation T4825I in the type 1 ryanodine receptor (RYR1(T4825I/+)) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1(T4826I/+) (Het) or homozygous RYR1(T4826I/T4826I) (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca(2+)](rest), and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.

Polychlorinated biphenyls (PCBs) pose significant risk to the developing human brain; however, mechanisms of PCB developmental neurotoxicity (DNT) remain controversial. Two widely posited mechanisms are tested here using PCBs identified in pregnant women in the MARBLES cohort who are at increased risk for having a child with a neurodevelopmental disorder (NDD). As determined by gas chromatography-triple quadruple mass spectrometry, the mean PCB level in maternal serum was 2.22 ng/mL. The 12 most abundant PCBs were tested singly and as a mixture mimicking the congener profile in maternal serum for activity at the thyroid hormone receptor (THR) and ryanodine receptor (RyR). Neither the mixture nor the individual congeners (2 fM to 2 μM) exhibited agonistic or antagonistic activity in a THR reporter cell line. However, as determined by equilibrium binding of [ 3 H]ryanodine to RyR1enriched microsomes, the mixture and the individual congeners (50 nM to 50 μM) increased RyR activity by 2.4−19.2-fold. 4-Hydroxy (OH) and 4-sulfate metabolites of PCBs 11 and 52 had no TH activity; but 4-OH PCB 52 had higher potency than the parent congener toward RyR. These data support evidence implicating RyRs as targets in environmentally triggered NDDs and suggest that PCB effects on the THR are not a predominant mechanism driving PCB DNT. These findings provide scientific rationale regarding a point of departure for quantitative risk assessment of PCB DNT, and identify in vitro assays for screening other environmental pollutants for DNT potential.