Structural and functional interactions between the EF hand domain and S2-S3 loop in the type-1 ryanodine receptor ion channel.

Previous cryo-electron micrographs suggested that the skeletal muscle Ca release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface.

Point mutations in RyR2 Ca2+-binding residues of human cardiomyocytes cause cellular remodelling of cardiac excitation contraction-coupling.

Aims: CRISPR/Cas9 gene edits of cardiac ryanodine receptor (RyR2) in human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) provide a novel platform for introducing mutations in RyR2 Ca2+-binding residues and examining the resulting excitation contraction (EC)-coupling remodelling consequences.

Methods And Results: Ca2+-signalling phenotypes of mutations in RyR2 Ca2+-binding site residues associated with cardiac arrhythmia (RyR2-Q3925E) or not proven to cause cardiac pathology (RyR2-E3848A) were determined using ICa- and caffeine-triggered Ca2+ releases in voltage-clamped and total internal reflection fluorescence-imaged wild type and mutant cardiomyocytes infected with sarcoplasmic reticulum (SR)-targeted ER-GCaMP6 probe. (i) ICa- and caffeine-triggered Fura-2 or ER-GCaMP6 signals were suppressed, even when ICa was significantly enhanced in Q3925E and E3848A mutant cardiomyocytes; (ii) spontaneous beating (Fura-2 Ca2+ transients) persisted in mutant cells without the SR-release signals; (iii) while 5-20 mM caffeine failed to trigger Ca2+-release in voltage-clamped mutant cells, only ∼20% to ∼70% of intact myocytes responded respectively to caffeine; (iv) and 20 mM caffeine transients, however, activated slowly, were delayed, and variably suppressed by 2-APB, FCCP, or ruthenium red.

CRISPR/Cas9 Gene Editing of RYR2 in Human iPSC-Derived Cardiomyocytes to Probe Ca Signaling Aberrancies of CPVT Arrhythmogenesis.

Human-induced pluripotent stem cells (hiPSCs) provide a powerful platform to study biophysical and molecular mechanisms underlying the pathophysiology of genetic mutations associated with cardiac arrhythmia. Human iPSCs can be generated by reprograming of dermal fibroblasts of normal or diseased individuals and be differentiated into cardiac myocytes. Obtaining biopsies from patients afflicted with point mutations causing arrhythmia is often a cumbersome process even when patients are available.

Do CPVT-linked mutations alter RYR2 regulation by cytosolic Ca2+ in cardiomyocytes?

It is controversial whether the cardiac type-2 ryanodine receptor harboring a catecholaminergic polymorphic ventricular tachycardia-associated point mutation is regulated by luminal or cytosolic Ca. This commentary discusses new findings supporting the cytosolic Ca-dependent regulation.

Association between neurosarcoidosis with autonomic dysfunction and anti-ganglionic acetylcholine receptor antibodies.

Objective: To determine whether autonomic dysfunction in neurosarcoidosis is associated with anti-ganglionic acetylcholine receptor (gAChR) antibodies, which are detected in autoimmune autonomic ganglionopathy.

Methods: We retrospectively extracted cases of sarcoidosis from 1787 serum samples of 1,381 patients between 2012 and 2018. Anti-gAChR antibodies against the α3 and β4 subunit were measured by luciferase immunoprecipitation to confirm the clinical features of each case.

Dextran sulphate inhibits an association of prions with plasma membrane at the early phase of infection.

The defining characteristic of prion diseases is conversion of a cellular prion protein (PrP) to an abnormal prion protein (PrP). The exogenous attachment of PrP to the surface of a target cell is critical for infection. However, the initial interaction of PrP with the cell surface is poorly characterized.

Prion protein interacts with the metabotropic glutamate receptor 1 and regulates the organization of Ca signaling.

Cellular prion protein (PrP) is a membrane protein that is highly conserved among mammals and mainly expressed on the cell surface of neurons. Despite its reported interactions with various membrane proteins, no functional studies have so far been carried out on it, and its physiological functions remain unclear. Neuronal cell death has been observed in a PrP-knockout mouse model expressing Doppel protein, suggesting that PrP might be involved in Ca signaling.

Molecular Insights into Calcium Dependent Regulation of Ryanodine Receptor Calcium Release Channels.

Ryanodine receptor calcium release channels (RyRs) play central roles in controlling intracellular calcium concentrations in excitable and non-excitable cells. RyRs are located in the sarcoplasmic or endoplasmic reticulum, intracellular Ca storage compartment, and release Ca during cellular action potentials or in response to other cellular stimuli. Mammalian cells express three structurally related isoforms of RyR.

A central core disease mutation in the Ca-binding site of skeletal muscle ryanodine receptor impairs single-channel regulation.

Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca-mediated activation of skeletal muscle Ca release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca release measurements, single-channel recordings, and computational methods.

Nonsense mutation in causes normal-pressure hydrocephalus with ciliary abnormalities.

Objective: To identify genes related to normal-pressure hydrocephalus (NPH) in one Japanese family with several members with NPH.

Methods: We performed whole-exome sequencing (WES) on a Japanese family with multiple individuals with NPH and identified a candidate gene. Then we generated knockout mouse using CRISPR/Cas9 to confirm the effect of the candidate gene on the pathogenesis of hydrocephalus.

Ca-mediated activation of the skeletal-muscle ryanodine receptor ion channel.

Cryo-electron micrograph studies recently have identified a Ca-binding site in the 2,200-kDa ryanodine receptor ion channel (RyR1) in skeletal muscle. To clarify the role of this site in regulating RyR1 activity, here we applied mutational, electrophysiological, and computational methods. Three amino acid residues that interact directly with Ca were replaced, and these RyR1 variants were expressed in HEK293 cells.

Whole-exome sequencing and gene-based rare variant association tests suggest that PLA2G4E might be a risk gene for panic disorder.

Panic disorder (PD) is characterized by recurrent and unexpected panic attacks, subsequent anticipatory anxiety, and phobic avoidance. Recent epidemiological and genetic studies have revealed that genetic factors contribute to the pathogenesis of PD. We performed whole-exome sequencing on one Japanese family, including multiple patients with panic disorder, which identified seven rare protein-altering variants.

Two EF-hand motifs in ryanodine receptor calcium release channels contribute to isoform-specific regulation by calmodulin.

The mammalian ryanodine receptor Ca release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca. This suggests isoform-specific domains are involved in RyR regulation by CaM.

Malignant hyperthermia-associated mutations in the S2-S3 cytoplasmic loop of type 1 ryanodine receptor calcium channel impair calcium-dependent inactivation.

Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca and inactivated by higher (∼1 mM) Ca To gain insight into a mechanism underlying Ca-dependent inactivation of RyR1 and its relationship with skeletal muscle diseases, we constructed nine recombinant RyR1 mutants carrying malignant hyperthermia or centronuclear myopathy-associated mutations and determined RyR1 channel activities by [H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca-dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W, and R4736Q) in the cytoplasmic loop between the S2 and S3 transmembrane segments (S2-S3 loop) greatly reduced Ca-dependent channel inactivation.

Inhibition of CaMKII does not attenuate cardiac hypertrophy in mice with dysfunctional ryanodine receptor.

In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s) leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM) and by phosphorylation of Ca2+/CaM-dependent protein kinase II (CaMKII). Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2ADA) impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2ADA mutation.

Two regions of the ryanodine receptor calcium channel are involved in Ca(2+)-dependent inactivation.

Skeletal (RyR1) and cardiac muscle (RyR2) isoforms of ryanodine receptor calcium channels are inhibited by millimollar Ca(2+), but the affinity of RyR2 for inhibitory Ca(2+) is ~10 times lower than that of RyR1. Previous studies demonstrated that the C-terminal quarter of RyR has critical domain(s) for Ca(2+) inactivation. To obtain further insights into the molecular basis of regulation of RyRs by Ca(2+), we constructed and expressed 18 RyR1-RyR2 chimeras in HEK293 cells and determined the Ca(2+) activation and inactivation affinities of these channels using the [(3)H]ryanodine binding assay.

Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor.

Cardiac ryanodine receptor (RyR2) is a homotetramer of 560 kDa polypeptides regulated by calmodulin (CaM), which decreases its open probability at diastolic and systolic Ca(2+) concentrations. Point mutations in the CaM-binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2(ADA)) in mice result in severe cardiac hypertrophy, poor left ventricle contraction and death by postnatal day 16, suggesting that CaM inhibition of RyR2 is required for normal cardiac function. Here, we report on Ca(2+) signalling properties of enzymatically isolated, Fluo-4 dialysed whole cell clamped cardiac myocytes from 10-15-day-old wild-type (WT) and homozygous Ryr2(ADA/ADA) mice.

Cardiac hypertrophy associated with impaired regulation of cardiac ryanodine receptor by calmodulin and S100A1.

The cardiac ryanodine receptor (RyR2) is inhibited by calmodulin (CaM) and S100A1. Simultaneous substitution of three amino acid residues (W3587A, L3591D, F3603A; RyR2ADA) in the CaM binding domain of RyR2 results in loss of CaM inhibition at submicromolar (diastolic) and micromolar (systolic) Ca²⁺, cardiac hypertrophy, and heart failure in Ryr2ADA/ADA mice. To address whether cardiac hypertrophy results from the elimination of CaM and S100A1 inhibition at diastolic or systolic Ca²⁺, a mutant mouse was generated with a single RyR2 amino acid substitution (L3591D; RyR2D).

Mechanical regulation of native and the recombinant calcium channel.

L-type calcium channels are modulated by a host of mechanisms that include voltage, calcium ions (Ca(2+) dependent inactivation and facilitation), cytosolic proteins (CAM, CAMKII, PKA, PKC, etc.), and oxygen radicals. Here we describe yet another Ca(2+) channel regulatory mechanism that is induced by pressure-flow (PF) forces of ∼25dyn/cm(2) producing 35-60% inhibition of channel current.

Role of prostaglandin E2 in bacterial growth in women with endometriosis.

Study Question: Can prostaglandin E(2) (PGE(2)) in menstrual and peritoneal fluid (PF) promote bacterial growth in women with endometriosis?

Summary Answer: PGE(2) promotes bacterial growth in women with endometriosis.

What Is Known Already: Menstrual blood of women with endometriosis is highly contaminated with Escherichia coli (E. coli) compared with that of non-endometriotic women: E.

Protective role of interferon regulatory factor 3-mediated signaling against prion infection.

Abnormal prion protein (PrP(Sc)) generated from the cellular isoform of PrP (PrP(C)) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, acquired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood.