Mouse heart Na+ channels: primary structure and function of two isoforms and alternatively spliced variants

2002 ◽  
Vol 282 (3) ◽  
pp. H1007-H1017 ◽  
Author(s):  
Thomas Zimmer ◽  
Christian Bollensdorff ◽  
Volker Haufe ◽  
Eckhard Birch-Hirschfeld ◽  
Klaus Benndorf
Keyword(s):  
Cardiac Cells ◽  
Mouse Heart ◽  
Cdna Clones ◽  
Intracellular Loop ◽  
Sequence Comparisons ◽  
Electrophysiological Properties ◽  
Ventricular Cells ◽  
Alternatively Spliced ◽  
Main Portion ◽  
And Function

We isolated two full-length cDNA clones from the adult murine heart that encode two different voltage-gated Na+ channels: mH1 and mH2. Sequence comparisons indicated that mH1 is highly homologous to rat SCN5A, whereas mH2 is highly homologous to SCN4A, expressed in rat skeletal muscle. Electrophysiological properties of mH1 channels strongly resembled the tetrodotoxin (TTX)-resistant Na+ current of mouse ventricular cells, whereas mH2 channels activated at more positive potentials and were highly sensitive to TTX [50% inhibitory constant (IC50) = 11 nM]. We found that mH2 is not expressed in cardiac cells of neonatal mice, but appears to be upregulated during the development. Besides these Na+channel isoforms, we also detected two alternatively spliced mH1 variants that were characterized by deletions within the sequence coding for the intracellular loop between domains II and III. One of the shortened channels, mH1–2, developed Na+ currents indistinguishable from those of mH1. The other splice variant (mH1–3) did not form functional channels. Quantitative reverse transcriptase-polymerase chain reaction indicated that RNA preparations of the adult mouse heart contain 54% mH1, 25% mH1–2, 16% mH2, and 5% mH1–3. Conclusively, mH1 generates the main portion of the mouse cardiac TTX-resistant Na+ current and mH2 is a candidate for TTX-sensitive currents previously described in adult cardiomyocytes. Furthermore, the presence of mH1–2 and mH1–3 transcripts indicates that alternative splicing plays a role in the regulation of functional Na+ channels in cardiomyocytes.

scholarly journals New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sara Bitam ◽  
Ahmad Elbahnsi ◽  
Geordie Creste ◽  
Iwona Pranke ◽  
Benoit Chevalier ◽  
...  
Keyword(s):  
Phosphinic Acid ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Transmembrane Conductance Regulator ◽  
Intracellular Loop ◽  
Respiratory Cells ◽  
Cftr Protein ◽  
Dynamics Simulations ◽  
And Function ◽  
Molecular Mode

AbstractC407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

scholarly journals Structure and expression of human IgG FcRII(CD32). Functional heterogeneity is encoded by the alternatively spliced products of multiple genes.

1989 ◽  
Vol 170 (4) ◽  
pp. 1369-1385 ◽  
Author(s):  
D G Brooks ◽  
W Q Qiu ◽  
A D Luster ◽  
J V Ravetch
Keyword(s):  
Structural Heterogeneity ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Membrane Glycoproteins ◽  
Igg Binding ◽  
Genes Expression ◽  
Alternatively Spliced ◽  
Membrane Spanning ◽  
High Degree ◽  
Membrane Spanning Domains

The structural heterogeneity of the human low affinity receptor for IgG, FcRII(CD32), has been elucidated through the isolation, characterization, and expression of cDNA clones derived from myeloid and lymphoid RNA. These clones predict amino acid sequences consistent with integral membrane glycoproteins with single membrane spanning domains. The extracellular domains display sequence homology to other Fc gamma Rs and members of the Ig supergene family. A minimum of three genes (Fc gamma RIIa, IIa', and Fc gamma RIIb) encode these transcripts, which demonstrate highly related extracellular and membrane spanning domains. IIa/IIa' differ substantially in the intracytoplasmic domain from IIb. Alternative splicing of the IIb gene generates further heterogeneity in both NH2- and COOH-terminal domains of the predicted proteins. Comparison to the murine homologues of these molecules reveals a high degree of conservation between the products of one of these genes, Fc gamma RIIb, and the murine beta gene in primary sequence, splicing pattern, and tissue distribution. In contrast, the sequence of IIa' indicates its relationship to the beta-like genes, with mutation giving rise to a novel cytoplasmic domain, while IIa is a chimera of both alpha- and beta-like genes. Expression of these cDNA molecules by transfection results in the appearance of IgG binding molecules that bear the epitopes defined by the FcRII(CD32) mAbs previously described.

Abstract 250: Haploinsufficiency of Muscle-Enriched A-Type Lamin Interacting Protein (MLIP) Manifests as Enlarged Dilated Hearts

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Patrick Burgon ◽  
Julia Lockwood ◽  
Glenn Wells ◽  
Alexandre Blais
Keyword(s):  
Heart Development ◽  
Left Ventricular ◽  
Lamin A ◽  
Interacting Protein ◽  
Cellular Hypertrophy ◽  
Cellular Source ◽  
Alternatively Spliced ◽  
Normal Body Weight ◽  
Heart Size ◽  
And Function

Approximately 116 unique mutations in the lamin A/C gene have been described to date that are associated with dilated cardiomyopathy. We recently reported the discovery of MLIP through its interaction with lamin A/C. MLIP is expressed ubiquitously and most abundantly in heart, skeletal and smooth muscle of amniotes (mammals, reptiles and birds) and has no paralogous homologue suggesting no functional redundancy. The MLIP gene encodes at least seven, alternatively spliced, LMNA-interacting factors that possess several structural motifs not found in any other protein. The MLIP isoforms pattern of expression differs between each of the tissues with heart being the most heterogeneous. Down-regulation of lamin A/C expression by shRNA results in the up-regulation and mis-localization of MLIP. In addition to interacting and co-localizing with lamin A/C we also demonstrated that MLIP localizes to micro-domains in the nucleus with promyelocytic leukemia protein (PML) in close proximity to chromatin. MLIP's biological function still remains elusive. Eight week old hemizygous MLIP null mice develop enlarged hearts with a significant increase in heart to body weights (MLIP+/+ 5.62mg/g vs MLIP+/- 10.73mg/g, p<0.0001 n=7) with an overall 30% increase in the anterior-posterior ventricle length of MLIP hearts while maintaining a normal body weight (Figure). Echocardiographic analysis of MLIP+/- mice revealed that their hearts as having a significant (p3.93mm with a significant (p=0.011, n=12) reduction of left ventricular fractional shorting (LVFS) 31% when compare to littermate controls. Histological analysis of the hearts showed no overt phenotype other than an overall increase in the size of the MLIP+/- hearts. The cellular source for the increase in heart size and mass remains to be determined if it is the product of an increase in the number of cardiomyocytes due to aberrant hyperplasia or an increase in cardiomyocyte size through cellular hypertrophy. In conclusion, MLIP is a newly discovered lamin interacting protein that may serve as a transcriptional regulator that impact genes involved in heart development, growth and function and provides a new signaling paradigm.

Bioengineered Cardiac Grafts

Circulation ◽  
2000 ◽  
Vol 102 (suppl_3) ◽  
Author(s):  
Jonathan Leor ◽  
Sharon Aboulafia-Etzion ◽  
Ayelet Dar ◽  
Lilia Shapiro ◽  
Israel M. Barbash ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Left Ventricular ◽  
Cardiac Cells ◽  
Sham Operation ◽  
Complete Disappearance ◽  
3 Dimensional ◽  
Infarcted Myocardium ◽  
Lv Remodeling ◽  
And Function ◽  
Conducive Environment

Background —The myocardium is unable to regenerate because cardiomyocytes cannot replicate after injury. The heart is therefore an attractive target for tissue engineering to replace infarcted myocardium and enhance cardiac function. We tested the feasibility of bioengineering cardiac tissue within novel 3-dimensional (3D) scaffolds. Methods and Results —We isolated and grew fetal cardiac cells within 3D porous alginate scaffolds. The cell constructs were cultured for 4 days to evaluate viability and morphology before implantation. Light microscopy revealed that within 2 to 3 days in culture, the dissociated cardiac cells form distinctive, multicellular contracting aggregates within the scaffold pores. Seven days after myocardial infarction, rats were randomized to biograft implantation (n=6) or sham-operation (n=6) into the myocardial scar. Echocardiography study was performed before and 65±5 days after implantation to assess left ventricular (LV) remodeling and function. Hearts were harvested 9 weeks after implantation. Visual examination of the biograft revealed intensive neovascularization from the neighboring coronary network. Histological examination revealed the presence of myofibers embedded in collagen fibers and a large number of blood vessels. The specimens showed almost complete disappearance of the scaffold and good integration into the host. Although control animals developed significant LV dilatation accompanied by progressive deterioration in LV contractility, in the biograft-treated rats, attenuation of LV dilatation and no change in LV contractility were observed. Conclusions —Alginate scaffolds provide a conducive environment to facilitate the 3D culturing of cardiac cells. After implantation into the infarcted myocardium, the biografts stimulated intense neovascularization and attenuated LV dilatation and failure in experimental rats compared with controls. This strategy can be used for regeneration and healing of the infarcted myocardium.

scholarly journals Two Alternatively Spliced Transcripts from the Drosophila period Gene Rescue Rhythms Having Different Molecular and Behavioral Characteristics

1998 ◽  
Vol 18 (11) ◽  
pp. 6505-6514 ◽  
Author(s):  
Yuzhong Cheng ◽  
Barbara Gvakharia ◽  
Paul E. Hardin
Keyword(s):  
Type A ◽  
Behavioral Characteristics ◽  
Cdna Clones ◽  
Type B ◽  
Differential Splicing ◽  
Rnase Protection ◽  
Period Gene ◽  
Alternatively Spliced ◽  
Transgenic Flies

ABSTRACT The period (per) and timeless(tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The pergene is thought to encode three transcripts via differential splicing (types A, B, and C) that give rise to three proteins. Since the threeper mRNA types were based on the analysis of cDNA clones, we tested whether these mRNA types were present in vivo by RNase protection assays and reverse transcriptase-mediated PCR. The results show that per generates two transcript types that differ only by the presence (type A) or absence (type B′) of an alternative intron in the 3′ untranslated region. Transgenic flies containing transgenes that produce only type B′ transcripts (perB′ ), type A transcripts (perA ), or both transcripts (perG ) rescue locomotor activity rhythms with average periods of 24.7, 25.4, and 24.4 h, respectively. Although no appreciable differences in type A and type B′ mRNA cycling were observed, a slower accumulation of PER in flies making only type A transcripts suggests that the intron affects the translation ofper mRNA.

scholarly journals The Emerging Roles of the RNA Binding Protein QKI in Cardiovascular Development and Function

2021 ◽  
Vol 9 ◽  
Author(s):  
Xinyun Chen ◽  
Jianwen Yin ◽  
Dayan Cao ◽  
Deyong Xiao ◽  
Zhongjun Zhou ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Property ◽  
Cardiovascular Development ◽  
Potential Link ◽  
Alternatively Spliced ◽  
Unique Cell ◽  
Carboxy Terminal ◽  
And Function

RNA binding proteins (RBPs) have a broad biological and physiological function and are critical in regulating pre-mRNA posttranscriptional processing, intracellular migration, and mRNA stability. QKI, also known as Quaking, is a member of the signal transduction and activation of RNA (STAR) family, which also belongs to the heterogeneous nuclear ribonucleoprotein K- (hnRNP K-) homology domain protein family. There are three major alternatively spliced isoforms, QKI-5, QKI-6, and QKI-7, differing in carboxy-terminal domains. They share a common RNA binding property, but each isoform can regulate pre-mRNA splicing, transportation or stability differently in a unique cell type-specific manner. Previously, QKI has been known for its important role in contributing to neurological disorders. A series of recent work has further demonstrated that QKI has important roles in much broader biological systems, such as cardiovascular development, monocyte to macrophage differentiation, bone metabolism, and cancer progression. In this mini-review, we will focus on discussing the emerging roles of QKI in regulating cardiac and vascular development and function and its potential link to cardiovascular pathophysiology.

The development expression of the rat alpha-vascular and gamma-enteric smooth muscle isoactins: isolation and characterization of a rat gamma-enteric actin cDNA

1988 ◽  
Vol 8 (12) ◽  
pp. 5224-5231
Author(s):  
K M McHugh ◽  
J L Lessard
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Actin ◽  
The Other ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Muscle Actin ◽  
Adult Rats ◽  
Sequence Comparisons ◽  
Isolation And Characterization ◽  
Sequence Similarities

We have isolated and characterized two cDNA clones from whole rat stomach, pRV alpha A-19 and pRE gamma A-11, which are specific for the alpha-vascular and gamma-enteric smooth muscle isoactins, respectively. The rat gamma-enteric smooth muscle actin contains a single amino acid substitution of a proline for a glutamine at position 359 of the mature peptide when compared with the chicken gizzard gamma-actin sequence (J. Vandekerckhove and K. Weber, FEBS Lett. 102:219, 1979). Sequence comparisons of the 5' and 3' untranslated (UT) regions of the two smooth muscle actin cDNAs demonstrate that these regions contain no apparent sequence similarities. Additional comparisons of the 5' UT regions of the two smooth muscle actin cDNAs to all other known actin sequences reveal no apparent sequence similarities for the rat gamma-enteric isoactin within the 15 base pairs of sequence currently available, while the rat alpha-vascular isoactin contains two separate sequences which are similar to sequences within the 5' UT regions of the human and chicken alpha-vascular actin genes. A similar comparison of the 3' UT regions of the two smooth muscle actins demonstrates that the alpha-vascular isoactins do not contain the high degree of cross-species sequence conservation observed for the other isoactins and that the gamma-enteric isoactin contains an inverted sequence of 52 nucleotides which is similar to a sequence found within the 3' UT regions of the human, chicken, and rat beta-cytoplasmic isoactins. These observations complicate the apparent cross-species conservation of isotype specificity of these domains previously observed for the other actin isoforms. Northern blot analysis of day 15 rat embryos and newborn, day 19 postbirth, and adult rats demonstrates that the day 15 rat embryo displays low to undetectable levels of smooth muscle isoactin mRNA expression. By birth, the stomach and small intestine show dramatic increases in alpha-vascular and gamma-enteric actin expression. These initially high levels of expression decrease through day 19 to adulthood. In the adult rat, the uterus and aorta differ in their content of smooth muscle isoactin mRNA. These results demonstrate that the gamma-enteric and alpha-vascular isoactin mRNAs are coexpressed to various degrees in tissues which contain smooth muscle.

scholarly journals Cardiac Transmembrane Ion Channels and Action Potentials: Cellular Physiology and Arrhythmogenic Behavior

2020 ◽  
Author(s):  
András Varró ◽  
Jakub Tomek ◽  
Norbert Nagy ◽  
Laszlo Virag ◽  
Elisa Passini ◽  
...  
Keyword(s):  
Ion Channel ◽  
Action Potentials ◽  
Cardiac Electrophysiology ◽  
Current Knowledge ◽  
Animal Experiments ◽  
Cardiac Cells ◽  
Cellular Mechanisms ◽  
Electrophysiological Properties ◽  
Channel Expression ◽  
Ionic Mechanisms

Cardiac arrhythmias are among the leading causes of mortality. They often arise from alterations in the electrophysiological properties of cardiac cells, and their underlying ionic mechanisms. It is therefore critical to further unravel the patho-physiology of the ionic basis of human cardiac electrophysiology in health and disease. In the first part of this review, current knowledge on the differences in ion channel expression and properties of the ionic processes that determine the morphology and properties of cardiac action potentials and calcium dynamics from cardiomyocytes in different regions of the heart are described. Then the cellular mechanisms promoting arrhythmias in congenital or acquired conditions of ion channel function (electrical remodelling) are discussed. The focus is human relevant findings obtained with clinical, experimental and computational studies, given that interspecies differences make the extrapolation from animal experiments to the human clinical settings difficult. Deepening the understanding of the diverse patholophysiology of human cellular electrophysiology will help developing novel and effective antiarrhythmic strategies for specific subpopulations and disease conditions.

scholarly journals Phenotypic Screen with the Human Secretome Identifies FGF16 as Inducing Proliferation of iPSC-Derived Cardiac Progenitor Cells

2019 ◽  
Vol 20 (23) ◽  
pp. 6037 ◽  
Author(s):  
Karin Jennbacken ◽  
Fredrik Wågberg ◽  
Ulla Karlsson ◽  
Jerry Eriksson ◽  
Lisa Magnusson ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cardiac Fibroblasts ◽  
Cardiac Regeneration ◽  
Cardiac Cells ◽  
Mouse Heart ◽  
Cardiac Progenitor Cells ◽  
High Sequence Identity ◽  
Paracrine Factors ◽  
Human Cardiomyocytes ◽  
Phenotypic Screen

Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated naïve cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.

scholarly journals Characterization and Reconstitution of Drosophila γ-Tubulin Ring Complex Subunits

2000 ◽  
Vol 151 (7) ◽  
pp. 1513-1524 ◽  
Author(s):  
Ruwanthi N. Gunawardane ◽  
Ona C. Martin ◽  
Kan Cao ◽  
Lijun Zhang ◽  
Kimberly Dej ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Sequence Motifs ◽  
Microtubule Nucleation ◽  
Sequence Comparisons ◽  
Baculovirus Expression ◽  
Ring Complex ◽  
And Function ◽  
First Time ◽  
Centrosomal Proteins

The γ-tubulin ring complex (γTuRC) is important for microtubule nucleation from the centrosome. In addition to γ-tubulin, the Drosophila γTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small γ-tubulin complex (γTuSC) and the γTuRC, while the remaining subunits are found only in the γTuRC. To study γTuRC assembly and function, we first reconstituted γTuSC using the baculovirus expression system. Using the reconstituted γTuSC, we showed for the first time that this subcomplex of the γTuRC has microtubule binding and capping activities. Next, we characterized two new γTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known γTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with γTuSC. However, this interaction is insufficient for γTuRC assembly.

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