scholarly journals Extensive Ca2+ leak through K4750Q cardiac ryanodine receptors caused by cytosolic and luminal Ca2+ hypersensitivity

2017 ◽  
Vol 149 (2) ◽  
pp. 199-218 ◽  
Author(s):  
Akira Uehara ◽  
Takashi Murayama ◽  
Midori Yasukochi ◽  
Michael Fill ◽  
Minoru Horie ◽  
...  
Keyword(s):  
Single Channel ◽  
Single Point ◽  
Ryanodine Receptors ◽  
Point Mutations ◽  
Hek293 Cells ◽  
Polymorphic Ventricular Tachycardia ◽  
Single Point Mutation ◽  
Control Input ◽  
Life Threatening ◽  
Functional Consequences

Various ryanodine receptor 2 (RyR2) point mutations cause catecholamine-induced polymorphic ventricular tachycardia (CPVT), a life-threatening arrhythmia evoked by diastolic intracellular Ca2+ release dysfunction. These mutations occur in essential regions of RyR2 that regulate Ca2+ release. The molecular dysfunction caused by CPVT-associated RyR2 mutations as well as the functional consequences remain unresolved. Here, we study the most severe CPVT-associated RyR2 mutation (K4750Q) known to date. We define the molecular and cellular dysfunction generated by this mutation and detail how it alters RyR2 function, using Ca2+ imaging, ryanodine binding, and single-channel recordings. HEK293 cells and cardiac HL-1 cells expressing RyR2-K4750Q show greatly enhanced spontaneous Ca2+ oscillations. An endoplasmic reticulum–targeted Ca2+ sensor, R-CEPIA1er, revealed that RyR2-K4750Q mediates excessive diastolic Ca2+ leak, which dramatically reduces luminal [Ca2+]. We further show that the K4750Q mutation causes three RyR2 defects: hypersensitization to activation by cytosolic Ca2+, loss of cytosolic Ca2+/Mg2+-mediated inactivation, and hypersensitization to luminal Ca2+ activation. These defects combine to kinetically stabilize RyR2-K4750Q openings, thus explaining the extensive diastolic Ca2+ leak from the sarcoplasmic reticulum, frequent Ca2+ waves, and severe CPVT phenotype. As the multiple concurrent defects are induced by a single point mutation, the K4750 residue likely resides at a critical structural point at which cytosolic and luminal RyR2 control input converge.

scholarly journals Molecular Basis of Ca2+ Activation of the Mouse Cardiac Ca2+ Release Channel (Ryanodine Receptor)

2001 ◽  
Vol 118 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Pin Li ◽  
S.R. Wayne Chen
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Molecular Basis ◽  
Single Channel ◽  
Single Point ◽  
Hek293 Cells ◽  
Single Point Mutation ◽  
Single Channels ◽  
Wild Type ◽  
Structural And Functional Properties

Activation of the cardiac ryanodine receptor (RyR2) by Ca2+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca2+. In this study, we investigated the role in Ca2+ sensing of the conserved glutamate 3987 located in the predicted transmembrane segment M2 of the mouse RyR2. Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca2+, as measured by using single-channel recordings in planar lipid bilayers and by [3H]ryanodine binding assay. However, this mutation did not alter the affinity of [3H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg2+, and was modified by ryanodine in a fashion similar to that of the wild-type channel. Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca2+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca2+ sensitivity to activation of the mouse RyR2 channel, and that Ca2+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results of this study also provide initial insights into the structural and functional properties of the mouse RyR2, which should be useful for studying RyR2 function and regulation in genetically modified mouse models.

scholarly journals Cryo-EM reveals local and global structural rearrangements in RYR mutants

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Kavita A. Iyer ◽  
Yifan Hu ◽  
Thomas Klose ◽  
Takashi Murayama ◽  
Montserrat Samsó
Keyword(s):  
Long Range ◽  
Conformational Changes ◽  
Large Scale ◽  
Single Point ◽  
Ryanodine Receptors ◽  
Critical Role ◽  
Point Mutations ◽  
Polymorphic Ventricular Tachycardia ◽  
Functional Studies ◽  
Single Point Mutations

Single-point mutations in ryanodine receptors (RYRs), large intracellular Ca2+ channels that play a critical role in EC coupling, are linked to debilitating and lethal disorders such as central core disease, malignant hyperthermia (for the skeletal isoform, RYR1), catecholaminergic polymorphic ventricular tachycardia, and ARVD2 (for the cardiac isoform, RYR2). Mutant RYRs result in elevated [Ca2+]cyto due to steady leak from the sarcoplasmic reticulum. To explore the nature of long-range allosteric mechanisms of malfunction, we determined the structure of two N-terminal domain mutants of RYR1, situated far away from the pore. Cryo-electron microscopy of the N-terminal subdomain A (NTDA) and subdomain C (NTDC) full-length mutants, RYR1 R163C (determined to 3.5 Å resolution), and RYR1 Y522S (determined to 4.0 Å resolution), respectively, reveal large-scale conformational changes in the cytoplasmic assembly under closed-state conditions (i.e., absence of activating Ca2+). The multidomain changes suggest that the mutations induce a preactivated state of the channel in R164C by altering the NTDA+/CD interface, and in Y522S by rearrangement of the α-helical bundle in NTDC. However, the extent of preactivation is considerably higher in Y522S as compared with R163C, which agrees with the increased severity of the Y522S mutation as established by various functional studies. The Y522S mutation represents loss of a spacer residue that is crucial for maintaining optimal orientation of α helices in NTDC, alteration of which has long-range effects felt as far away as ∼100 Å. Additionally, the structure of the Y522S mutant channel under open-state conditions also differs from RYR1 WT open channels. Our developing work with RYR mutants exhibits the diverse mechanisms by which these single-point mutations exert an effect on the channel’s function and highlight the complexity of the multidomain channel, as well as the need for targeted therapies.

scholarly journals Mutational Analysis of Quinolone-Resistant Determining Region gyrA and parC Genes in Quinolone-Resistant ESBL-Producing E. Coli

2021 ◽  
Vol 20 (3) ◽  
Author(s):  
Hairul Aini Hamzah ◽  
Rahmatullah Sirat ◽  
Mohammed Imad A. Mustafa Mahmud ◽  
Roesnita Baharudin
Keyword(s):  
Mutational Analysis ◽  
Single Point ◽  
Point Mutations ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Resistant Bacteria ◽  
Single Point Mutation ◽  
Phenotypic Resistance ◽  
Drug Effectiveness ◽  
E Coli

 Introduction: Co-resistance to quinolones among extended spectrum β[1]lactamase (ESBL)-producing E. coli commonly occurs in clinical settings. Quinolones act on DNA gyrase and DNA topoisomerase enzymes, which are coded by gyrA and parC genes, thus any mutation to the genes may affect the drug effectiveness. The objective of the study was to characterize gyrA and parC genes in quinolone-resistant E. coli isolates and correlated the mutations with their phenotypic resistance. Materials and Methods: Thirty-two quinolone-resistant (QR) and six quinolone-sensitive (QS) ESBL-E. coli isolates were identified by antibiotic susceptibility and minimum inhibitory concentration tests. Bioinformatics analysis were conducted to study any mutations occurred in the genes and generate their codon compositions. Results: All the QR ESBL-E. coli isolates were identified as multidrug-resistant bacteria. A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA, at codon 83, caused the substitution amino acid Ser83Leu. It is associated with a high level of resistance to nalidixic acid. However, double mutations Ser83Leu and Asp87Asn in the same region were significantly linked to higher levels of resistance to ciprofloxacin. Cumulative point mutations in gyrA and/or in parC were also correlated significantly (p<0.05) to increased resistance to ciprofloxacin. Conclusion: Together, the findings showed that the mutations in gyrA and parC genes handled the institution of intrinsic quinolone resistance in the ESBL-E. coli isolates. Thus, vigilant monitoring for emergence of new mutation in resistance genes may give an insight into dissemination of QR ESBL-E. coli in a particular region.

Different interactions of cardiac and skeletal muscle ryanodine receptors with FK-506 binding protein isoforms

1997 ◽  
Vol 272 (5) ◽  
pp. C1726-C1733 ◽  
Author(s):  
S. Barg ◽  
J. A. Copello ◽  
S. Fleischer
Keyword(s):  
Skeletal Muscle ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Ryanodine Receptors ◽  
Protein Isoforms ◽  
Channel Activity ◽  
Functional Interaction ◽  
Fk 506 ◽  
Receptor Isoforms ◽  
Functional Consequences

In the present study, we compare functional consequences of dissociation and reconstitution of binding proteins FKBP12 and FKBP12.6 with ryanodine receptors from cardiac (RyR2) and skeletal muscle (RyR1). The skeletal muscle RyR1 channel became activated on removal of endogenously bound FKBP12, consistent with previous reports. Both FKBP12 and FKBP12.6 rebind to FKBP-depleted RyR1 and restore its quiescent channel behavior by altering ligand sensitivity, as studied by single-channel recordings in planar lipid bilayers, and macroscopic behavior of the channels (ryanodine binding and net energized Ca2- uptake). By contrast, removal of FKBP12.6 from the cardiac RyR2 did not modulate the function of the channel using the same types of assays as for RyR1. FKBP12 or FKBP12.6 had no effect on channel activity of FKBP12.6-depleted cardiac RyR2, although FKBP12.6 rebinds. Our studies reveal important differences between the two ryanodine receptor isoforms with respect to their functional interaction with FKBP12 and FKBP12.6.

scholarly journals A Single P-loop Glutamate Point Mutation to either Lysine or Arginine Switches the Cation–Anion Selectivity of the CNGA2 Channel

2006 ◽  
Vol 127 (4) ◽  
pp. 375-389 ◽  
Author(s):  
Wei Qu ◽  
Andrew J. Moorhouse ◽  
Meenak Chandra ◽  
Kerrie D. Pierce ◽  
Trevor M. Lewis ◽  
...  
Keyword(s):  
Point Mutation ◽  
Single Channel ◽  
Noise Analysis ◽  
Critical Role ◽  
Reversal Potential ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Single Point Mutation ◽  
Cation Selectivity ◽  
Anion Selectivity

Cyclic nucleotide-gated (CNG) channels play a critical role in olfactory and visual transduction. Site-directed mutagenesis and inside-out patch-clamp recordings were used to investigate ion permeation and selectivity in two mutant homomeric rat olfactory CNGA2 channels expressed in HEK293 cells. A single point mutation of the negatively charged pore loop (P-loop) glutamate (E342) to either a positively charged lysine or arginine resulted in functional channels, which consistently responded to cGMP, although the currents were generally extremely small. The concentration–response curve of the lysine mutant channel was very similar to that of wild-type (WT) channels, suggesting no major structural alteration to the mutant channels. Reversal potential measurements, during cytoplasmic NaCl dilutions, showed that the lysine and the arginine mutations switched the selectivity of the channel from cations (PCl/PNa = 0.07 [WT]) to anions (PCl/PNa = 14 [Lys] or 10 [Arg]). Relative anion permeability sequences for the two mutant channels, measured with bi-ionic substitutions, were NO3− &gt; I− &gt; Br− &gt; Cl− &gt; F− &gt; acetate−, the same as those obtained for anion-selective GABA and glycine channels. The mutant channels also seem to have an extremely small single-channel conductance, measured using noise analysis of about 1–2 pS, compared to a WT value of about 29 pS. The results showed that it is predominantly the charge of the E342 residue in the P-loop, rather than the pore helix dipoles, which controls the cation–anion selectivity of this channel. However, the outward rectification displayed by both mutant channels in symmetrical NaCl solutions suggests that the negative ends of the pore helix dipoles may play a role in reducing the outward movement of Cl− ions through these anion-selective channels. These results have potential implications for the determinants of anion–cation selectivity in the large family of P-loop–containing channels.

scholarly journals Autism-associated mutation inhibits protein kinase C-mediated neuroligin-4X enhancement of excitatory synapses

2015 ◽  
Vol 112 (8) ◽  
pp. 2551-2556 ◽  
Author(s):  
Michael A. Bemben ◽  
Quynh-Anh Nguyen ◽  
Tongguang Wang ◽  
Yan Li ◽  
Roger A. Nicoll ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Single Point ◽  
Point Mutations ◽  
Genetic Mutation ◽  
Autism Spectrum ◽  
Repetitive Behaviors ◽  
Single Point Mutation ◽  
Kinase C ◽  
Human Neurons

Autism spectrum disorders (ASDs) comprise a highly heritable, multifarious group of neurodevelopmental disorders, which are characterized by repetitive behaviors and impairments in social interactions. Point mutations have been identified in X-linked Neuroligin (NLGN) 3 and 4X genes in patients with ASDs and all of these reside in their extracellular domains except for a single point mutation in the cytoplasmic domain of NLGN4X in which an arginine is mutated to a cysteine (R704C). Here we show that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707, and R704C completely eliminates T707 phosphorylation. Endogenous NLGN4X is intensely phosphorylated on T707 upon PKC stimulation in human neurons. Furthermore, a phospho-mimetic mutation at T707 has a profound effect on NLGN4X-mediated excitatory potentiation. Our results now establish an important interplay between a genetic mutation, a key posttranslational modification, and robust synaptic changes, which can provide insights into the synaptic dysfunction of ASDs.

scholarly journals Monitoring of Pyrethroid Resistance Allele Frequency in the Common Bed Bug (Cimex lectularius) in the Republic of Korea

2020 ◽  
Vol 58 (1) ◽  
pp. 99-102
Author(s):  
Susie Cho ◽  
Heung-Chul Kim ◽  
Sung-Tae Chong ◽  
Terry A. Klein ◽  
Deok Ho Kwon ◽  
...  
Keyword(s):  
Large Scale ◽  
Pyrethroid Resistance ◽  
Single Point ◽  
Point Mutations ◽  
Monitoring Program ◽  
Cimex Lectularius ◽  
Single Point Mutation ◽  
Resistance Allele ◽  
Bed Bugs ◽  
Bed Bug

Two-point mutations (V419L and L925I) on the voltage-sensitive sodium channel of bed bugs (<i>Cimex lectularius</i>) are known to confer pyrethroid resistance. To determine the status of pyrethroid resistance in bed bugs in Korea, resistance allele frequencies of bed bug strains collected from several US military installations in Korea and Mokpo, Jeollanamdo, from 2009-2019 were monitored using a quantitative sequencing. Most bed bugs were determined to have both of the point mutations except a few specimens, collected in 2009, 2012 and 2014, having only a single point mutation (L925I). No susceptible allele was observed in any of the bed bugs examined, suggesting that pyrethroid resistance in bed bug populations in Korea has reached a serious level. Large scale monitoring is required to increase our knowledge on the distribution and prevalence of pyrethroid resistance in bed bug populations in Korea. Based on present study, it is urgent to restrict the use of pyrethroids and to introduce effective alternative insecticides. A nation-wide monitoring program to determine the pyrethroid resistance level in bed bugs and to select alternative insecticides should be implemented.

Point mutations in the beta-subunit of cytochrome b558 leading to X- linked chronic granulomatous disease

Blood ◽  
1991 ◽  
Vol 77 (11) ◽  
pp. 2482-2487 ◽  
Author(s):  
BG Bolscher ◽  
M de Boer ◽  
A de Klein ◽  
RS Weening ◽  
D Roos
Keyword(s):  
Chronic Granulomatous Disease ◽  
Single Point ◽  
Genetic Defect ◽  
Point Mutations ◽  
Heme Iron ◽  
Beta Subunit ◽  
Stable Complex ◽  
Single Point Mutation ◽  
Granulomatous Disease ◽  
Cytochrome B558

The NADPH:O2 oxidoreductase of phagocytic leukocytes is an important enzyme for the bactericidal activity of these cells. Cytochrome b558 is a membrane component of this enzyme. In X-linked chronic granulomatous disease (Xb- CGD) the phagocytes are defective in the beta-subunit (gp91-phox) of this cytochrome. We have studied the genetic defect in a group of six X-linked CGD patients characterized by complete or partial loss of cytochrome b558 with the use of the polymerase chain reaction. All patients had a different single point mutation in the gp91-phox gene, indicating that the genetic defect in Xb- CGD is very heterogeneous. In one patient the mutation leads to a premature termination codon. In the other five cases these mutations predict incorporation of a different amino acid. The mutations were with one exception found in the N-terminal half of the protein, suggesting that this part of cytochrome b558 is important for the binding of the heme or for formation of a stable complex with p22-phox. Two histidyl residues were found that might be ligands of the heme iron.

scholarly journals mmCSM-AB: guiding rational antibody engineering through multiple point mutations

2020 ◽  
Vol 48 (W1) ◽  
pp. W125-W131 ◽  
Author(s):  
Yoochan Myung ◽  
Douglas E V Pires ◽  
David B Ascher
Keyword(s):  
Binding Affinity ◽  
Single Point ◽  
Point Mutations ◽  
Affinity Maturation ◽  
Antibody Engineering ◽  
Multiple Point ◽  
Single Point Mutation ◽  
Atomic Interaction ◽  
Therapeutic Class ◽  
Antibody Design

Abstract While antibodies are becoming an increasingly important therapeutic class, especially in personalized medicine, their development and optimization has been largely through experimental exploration. While there have been many efforts to develop computational tools to guide rational antibody engineering, most approaches are of limited accuracy when applied to antibody design, and have largely been limited to analysing a single point mutation at a time. To overcome this gap, we have curated a dataset of 242 experimentally determined changes in binding affinity upon multiple point mutations in antibody-target complexes (89 increasing and 153 decreasing binding affinity). Here, we have shown that by using our graph-based signatures and atomic interaction information, we can accurately analyse the consequence of multi-point mutations on antigen binding affinity. Our approach outperformed other available tools across cross-validation and two independent blind tests, achieving Pearson's correlations of up to 0.95. We have implemented our new approach, mmCSM-AB, as a web-server that can help guide the process of affinity maturation in antibody design. mmCSM-AB is freely available at http://biosig.unimelb.edu.au/mmcsm_ab/.

scholarly journals Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM

2020 ◽  
Vol 6 (31) ◽  
pp. eabb2964 ◽  
Author(s):  
Kavita A. Iyer ◽  
Yifan Hu ◽  
Ashok R. Nayak ◽  
Nagomi Kurebayashi ◽  
Takashi Murayama ◽  
...  
Keyword(s):  
Single Point ◽  
Ryanodine Receptors ◽  
Point Mutations ◽  
Salt Bridge ◽  
Structural Effect ◽  
Protein Loss ◽  
Structural Mechanism ◽  
Functional Studies ◽  
Equivalent Site ◽  
Intracellular Ca

Mutations in ryanodine receptors (RyRs), intracellular Ca2+ channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca2+ imaging, and cryo–electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca2+ binding site, which concur with the major “hyperactive” feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect.

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