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PubMed2004

Steroid sulfotransferase 2A1 gene transcription is regulated by steroidogenic factor 1 and GATA-6 in the human adrenal.

Sulfonation is a phase II conjugation reaction responsible for the biotransformation of many compounds including steroids, bile acids, and drugs. Humans are presently known to express at least five cytosolic sulfotransferase (SULT) enzymes, of which only two are hydroxysteroid SULT, SULT2A1, commonly known as steroid sulfotransferase, and the cholesterol sulfotransferase SULT2B1. SULT2A1 is highly expressed in the adrenal where it is responsible for the sulfation of hydroxysteroids including conversion of dehydroepiandrosterone to dehydroepiandrosterone sulfate and in the liver where it is responsible for sulfation of bile acids and circulating hydroxysteroids. Little is known concerning the transcriptional regulation of human SULT2A1 in adrenal. Herein we demonstrate the role of two transcription factors, steroidogenic factor 1 (SF1) and GATA-6, in the regulation of SULT2A1 transcription. These transcription factors were quantified by real-time RT-PCR in normal human adrenal tissue. Transient transfection assays with deleted and mutated SULT2A1 promoter constructs allowed for the determination of specific SF1 and GATA binding cis-regulatory elements necessary for transactivation of SULT2A1 promoter, and binding was confirmed by EMSA analysis. Both SF1 and GATA-6 were positive regulators of SULT2A1 promoter constructs. These data support the hypothesis that adrenal SULT2A1 expression is regulated by SF1 and GATA-6.

Adrenal Glands↗
PubMed1990

Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene.

To examine the functional relationship between distinct cis-active elements within the distal enhancer region of the rat PRL gene, we have used deletional and mutational analysis of that region in transient transfection studies in GH3 pituitary tumor cells. Results from these studies demonstrate that the region of the PRL distal enhancer containing the Pit-1-binding sites is critical not only for enhancer activity and the response to cAMP, but also for the response to estradiol. An interaction of the estrogen receptor with factors conferring basal enhancer activity is suggested by studies with a mutant distal enhancer region in which the PRL estrogen response element was converted to a palindromic estrogen response element. To directly examine potential interactions, cotransfection studies using PRL distal enhancer reporter gene constructs and expression vectors for Pit-1 and rat estrogen receptor were performed in two heterologous cell lines. The activity of the reporter gene under the control of the PRL distal enhancer linked to either the thymidine kinase promoter or the PRL proximal promoter was not significantly altered by cotransfection with the Pit-1 expression vector in COS-1 or RAT-1 cells. Coexpression of these reporter constructs and an expression vector for estrogen receptor resulted in only a slight response to estradiol. However, when both Pit-1 and estrogen receptor were cotransfected with the distal enhancer reporter gene, a marked induction was observed in response to estradiol, and this activity was dependent upon the concentration of the Pit-1 expression vector.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Multiple prolactin (PRL) receptor cytoplasmic residues and Stat1 mediate PRL signaling to the interferon regulatory factor-1 promoter.

The Nb2 PRL receptor (PRL-R) is known to mediate PRL signaling to the interferon (IFN) regulatory factor-1 (IRF-1) gene via the family of signal transducers and activators of transcription or Stats. To analyze the components of the PRL-R/Stat/IRF-1 signaling pathway, various PRL-R, Stat, and IRF-1-CAT reporter constructs were transiently cotransfected into COS cells. First, mutations in the IFNgamma-activated sequence (GAS), either multimerized or in the context of the 1.7-kb IRF-1 promoter, failed to mediate a PRL response, showing that the IRF-1 GAS is a target of PRL signaling. Next, pairwise alanine substitutions into conserved residues in the proline-rich motif or Box 1 region and two tyrosine mutations, Y308F and Y382F, in the PRL-R intracellular domain all impaired PRL signaling to multimerized GAS or to the 1.7-kb IRF-1 promoter. Furthermore, these PRL-R mutants mediated reduced Stat1 binding to the IRF-1 GAS. Transfection of Stat1 further enhanced PRL signaling to the IRF-1 promoter, suggesting that Stat1 is a positive mediator of PRL action. These studies show that both membrane proximal and distal residues of the PRL-R are involved in signaling to the IRF-1 gene. Further, Stat1 and the GAS element are important for PRL activation of the IRF-1 gene.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of Ser530 facilitates hormone-dependent transcriptional activation of the chicken progesterone receptor.

Phosphorylation regulates the activities of many proteins, including transcription factors. However, the evidence for the significance of phosphorylation in steroid hormone receptor action is mainly indirect. In this study, one of the hormone-induced phosphorylation sites of chicken progesterone receptor, Ser530, was mutated to alanine, a nonphosphorylatable amino acid, and the transcriptional activity of the mutant receptor was compared with that of wild type in a transient cotransfection assay. The results showed that this mutation resulted in reduced transcriptional activity of chicken progesterone receptor at low hormone concentrations but did not affect the maximal activity of the receptor at saturating levels of hormone, suggesting that the phosphorylation at Ser530 influences the response of the receptor to its ligand. The decreased sensitivity of the mutant receptor is not due to a decrease in hormone-binding affinity, leading to our hypothesis that Ser530 phosphorylation stabilizes the receptor in its active state, perhaps by preventing its reassociation with heat shock proteins or by maintaining a conformation suitable for interaction with other transcription factors.

Animals↗
PubMed2002

Roles of the N- and C-termini of GLUT4 in endocytosis.

In insulin target cells, the predominantly expressed glucose transporter isoform GLUT4 recycles between distinct intracellular compartments and the plasma membrane. To characterize putative targeting signals within GLUT4 in a physiologically relevant cell type, we have analyzed the trafficking of hemagglutinin (HA)-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells. Mutation of the C-terminal dileucine motif (LL489/90) did not affect the cell-surface expression of HA-GLUT4. However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) resulted in substantial increases, whereas deletion of 37 or 28 of the 44 C-terminal residues led to substantial decreases in cell-surface HA-GLUT4 in both the basal and insulin-stimulated states. Studies with wortmannin and coexpression of a dominant-negative dynamin GTPase mutant indicate that these effects appear to be primarily due to decreases and increases, respectively, in the rate of endocytosis. Yeast two-hybrid analyses revealed that the N-terminal phenylalanine-based targeting signal in GLUT4 constitutes a binding site for medium chain adaptins mu1, mu2, and mu3A, implicating a role of this motif in the targeting of GLUT4 to clathrin-coated vesicles.

Adipocytes↗
PubMed2003

Stimulation of type-X collagen gene transcription by retinoids occurs in part through the BMP signaling pathway.

BACKGROUND: Chondrocyte maturation and hypertrophy during endochondral bone formation are stimulated by both retinoids and bone morphogenetic proteins (BMPs). The type-X collagen gene, which is expressed only in hypertrophic chondrocytes, provides an excellent marker for chondrocyte maturation. We previously identified a 651-base-pair region of the type-X collagen promoter that is essential for its activation by BMP. We examined the relationship between the retinoid and BMP signaling pathways in transcriptional stimulation of the type-X collagen gene to determine whether they act independently or interact to regulate endochondral bone formation. METHODS: Prehypertrophic chondrocytes from embryonic chick sterna cultured in the presence or absence of retinoic acid or BMP-2 were transiently transfected with plasmids containing various mutations of the type-X collagen promoter directing expression of a luciferase reporter gene. In addition, real-time polymerase chain reaction was used to examine the effects of retinoic acid on expression of genes encoding BMP-2, 4, and 6. RESULTS: The previously identified BMP-responsive region of the type-X collagen promoter also mediated stimulation by physiological concentrations of retinoic acid in prehypertrophic chondrocytes. Systematic deletion mutagenesis of the BMP/retinoid-responsive region of the type-X collagen promoter identified distinct regions that are responsible for promoter stimulation by retinoids and BMP. Retinoic acid rapidly and dramatically stimulated accumulation of BMP-2 and BMP-6 messenger RNAs. CONCLUSIONS: These results suggest that, while retinoic acid appears to stimulate type-X collagen gene transcription in part by stimulating the BMP signaling pathway, it also acts in part through mechanisms that are independent of BMP.

Animals↗
PubMed2002

Structural bioinformatics: methods, concepts and applications to blood coagulation proteins.

Structural and theoretical analyses of proteins are central to the understanding of complex molecular mechanisms and are fundamental to the drug discovery process. Computational techniques yield useful insights into an ever-wider range of biomolecular systems. Protein three-dimensional structures and molecular functions can be predicted in some circumstances, while experimental structures can be analyzed in depth via such computational approaches. Non-covalent binding of biomolecules can be understood by considering structural, thermodynamic and kinetic issues, and theoretical simulations of such events can be attempted. The central role of electrostatic interactions with regard to protein function, structure and stability has been investigated and some electrostatic properties can be modeled theoretically. Computer methods thus help to prioritize, design, analyze and rationalize biochemical experiments. Cardiovascular diseases and associated blood coagulation disorders are leading causes of death worldwide. Blood coagulation involves more than 30 proteins that interact specifically with various degrees of affinity. Many of these molecules can also bind transiently to phospholipid surfaces. Numerous point mutations in the genes of coagulation proteins and regulators have been identified. Understanding the coagulation cascade, its regulation and the impact of mutations is required for the development of new therapies and diagnostic tools. In this review, we describe concepts and methods pertaining to the field of structural bioinformatics. We provide examples of applications of these approaches to blood coagulation proteins and show that such studies can give insights about molecular mechanisms contributing to cardiovascular disease susceptibility.

Blood Coagulation↗
PubMed2001

alpha2-chimaerin, a Cdc42/Rac1 regulator, is selectively expressed in the rat embryonic nervous system and is involved in neuritogenesis in N1E-115 neuroblastoma cells.

Neuronal differentiation involves Rac and Cdc42 GTPases. alpha-Chimaerin, a Rac/Cdc42 regulator, occurs as alpha1- and alternatively spliced Src homology 2 (SH2) domain-containing alpha2-isoforms. alpha2-chimaerin mRNA was highly expressed in the rat embryonic nervous system, especially in early postmitotic neurons. alpha1-chimaerin mRNA was undetectable before embryonic day 16.5. Adult alpha2-chimaerin mRNA was restricted to neurons within specific brain regions, with highest expression in the entorhinal cortex. alpha2-chimaerin protein localized to neuronal perikarya, dendrites, and axons. The overall pattern of alpha2-chimaerin mRNA expression resembles that of cyclin-dependent kinase regulator p35 (CDK5/p35) which participates in neuronal differentiation and with which chimaerin interacts. To determine whether alpha2-chimaerin may have a role in neuronal differentiation and the relevance of the SH2 domain, the morphological effects of both chimaerin isoforms were investigated in N1E-115 neuroblastoma cells. When plated on poly-lysine, transient alpha2-chimaerin but not alpha1-chimaerin transfectants formed neurites. Permanent alpha2-chimaerin transfectants generated neurites whether or not they were stimulated by serum starvation, and many cells were enlarged. Permanent alpha1-chimaerin transfectants displayed numerous microspikes and contained F-actin clusters, a Cdc42-phenotype, but generated few neurites. In neuroblastoma cells, alpha2-chimaerin was predominantly soluble with some being membrane-associated, whereas alpha1-chimaerin was absent from the cytosol, being membrane- and cytoskeleton-associated, paralleling their subcellular distribution in brain. Transient transfection with alpha2-chimaerin mutated in the SH2 domain (N94H) generated an alpha1-chimaerin-like phenotype, protein partitioned in the particulate fraction, and in NGF-stimulated pheochromocytoma cell line 12 (PC12) cells, neurite formation was inhibited. These results indicate a role for alpha2-chimaerin in morphological differentiation for which its SH2 domain is vital.

Aging↗
PubMedDate not supplied

E2F and GATA-1 are required for the Sertoli cell-specific promoter activity of the follicle-stimulating hormone receptor gene.

The follicle-stimulating hormone receptor (FSHR) gene is expressed in Sertoli cells in males and in granulosa cells in females. Cis-acting sequences and associated binding factors responsible for the transcription of the TATA-less FSHR gene in Sertoli cells were analyzed with dimethylsulfate (DMS) footprinting assays and electrophoretic mobility shift assay (EMSA). In vivo footprints in the core promoter using nuclear proteins from Sertoli cells identified several protected sequences, including an inverted GATA (TATC, -88/-85), and an E2F (TTTCGCG, -45/-39) motif. EMSA showed the presence of one or more sequence-specific proteins interacting with these potential regulatory elements. Antibody-supershift assays as well as competition assays further revealed that testis-specific GATA-1 recognized the inverted GATA element. The functional role of the potential cis-acting elements was analyzed by transient transfection assays with and without mutations of the putative elements. The mutational analysis indicated that the GATA and E2F elements were each required for optimal promoter activity. The effects of each of the promoter elements was examined in transfections in which mutations were made in each of the known regulatory sites, including the E box, GATA, and E2F sites in various combinations. All of these sites contribute to the maximum promoter activity such that mutations of the E box, GATA, and E2F sites eliminated nearly all promoter activity.

Animals↗
PubMed2000

Active cis-elements of PCNA Promoter in HeLa, HepG2, L-02 and MCF-7 Cell Lines.

Proliferating cell nuclear antigen (PCNA) is an auxiliary factor of DNA polymerase delta and epsilon and functions in DNA replication and repair. Using PCR methods, 17 sites within the human PCNA promoter from 60 to 538 were subjected to a 8 bp substitution mutagenesis. Wild type promoter and each mutated promoters were inserted into luciferase expression vector pGL2-Basic. These nonmutated and mutated PCNA promoters were assayed by transient transfection of the plasmids into HeLa, HepG2, L-02 and MCF-7 cell line, respectively. It was found that several sites were common cis acting elements and several sites were cell-specific cis acting elements. Data were further presented using in vitro transcription assay with HeLa and HepG2 nuclear extract.

Journal Article↗
PubMed1991

Essential AP-1 and PEA3 binding elements in the human urokinase enhancer display cell type-specific activity.

We have characterized a transcriptional enhancer of the human urokinase-type plasminogen activator (uPA) gene in three transformed human cell lines: HeLa, HepG2 and HT1080. The enhancer is located approximately 2 kbp upstream of the mRNA cap site and is active in all three cell lines. By footprinting and gel retardation analysis we found that it contained two binding sites for transcription factor AP-1, encoded by the fos and jun proto-oncogene families. The most upstream of these sites was juxtaposed to a binding site for PEA3, a product of the ets/Spi proto-oncogene family. By transient transfection analysis of deletions, point mutations and subcloned fragments, we found these sites to be crucial for enhancer activity. However, the sites displayed differences in activity in the three different cell lines. The downstream AP-1 site was almost exclusively responsible for enhancer activity in HeLa cells, whereas the AP-1/PEA3 site played a major role in HT1080 and HepG2 cells. The implications of our findings for the known regulation of uPA expression by transforming oncogenes, adenovirus E1A protein and glucocorticoids are discussed.

Base Sequence↗
PubMed1989

Activation of the interleukin-2 receptor alpha gene: regulatory role for DNA-protein interactions flanking the kappa B enhancer.

The human interleukin-2 receptor alpha (IL2R alpha) gene is transcriptionally activated by both phorbol esters and the HTLV-I trans-activator (Tax) protein through a mechanism that involves the interaction of inducible DNA binding proteins with a kappa B-like enhancer element (-267 to -256). Using mutated IL2R alpha promoter constructs in transient transfection and DNA binding assays, we now demonstrate that sequences located immediately upstream and downstream of the kappa B enhancer also contribute to the regulation of IL2R alpha gene expression. One upstream sequence termed UE-1 is preferentially required for phorbol ester relative to Tax-induced activation and specifically interacts with a constitutively expressed 56-kD cellular factor. In contrast, two overlapping downstream elements between nucleotides -252 and -239 appear to be required for both phorbol ester and Tax-induced activation. One of these elements, an Sp1-like sequence, binds a constitutively expressed 100-kD T-cell protein consistent in size with Sp1 isolated from HeLa cells. The second element, located between the kappa B and Sp1 sites, resembles the decanucleotide core of the serum response element (SRE) from the c-fos gene and interacts with a constitutively expressed factor. Together, these findings implicate a functional role for multiple constitutively expressed DNA binding proteins, in addition to the inducible kappa B-specific factors, in the overall regulation of IL2R alpha gene activation.

Base Sequence↗
PubMed1993

Neuron-specific expression of the human dopamine beta-hydroxylase gene requires both the cAMP-response element and a silencer region.

Dopamine beta-hydroxylase (DBH), the enzyme catalyzing the conversion of dopamine to norepinephrine, is specifically expressed in adrenergic and noradrenergic neurons in the central nervous system. DNase I hypersensitive sites were found in the 5'-flanking region of the DBH gene in noradrenergic human neuroblastoma SK-N-BE(2)C cells, but not in DBH-nonexpressing HeLa cells. We report here that the 4.3-kilobase upstream sequence of the human DBH gene confers cell type-specific expression as assessed by transient expression assay. Furthermore, deletional and mutational analyses revealed two genetic regulatory elements required for the regulation of cell type specificity. First, deletion of the cAMP-response element (CRE) abolished > 95% of the transcriptional activity by the DBH upstream promoter, thus implicating the CRE as an essential positive genetic element. Second, deletion of a region between -490 and -263 base pairs resulted in 10-fold increase of reporter gene activity only in HeLa cells, indicating that this region contains a cell-specific silencer. A 13-base pair fragment residing within that region shows 77% sequence identity with the neuron-specific silencer motif recently identified in two neuronal genes, i.e. SCG10 and type II sodium channel genes. We propose that the interplay between the CRE and this neuron-specific silencer region plays an important role in the tissue-specific expression of the DBH gene in noradrenergic cells.

Animals↗
PubMed1995

Distinct roles for arginines in transmembrane helices 6 and 7 of the thyrotropin-releasing hormone receptor.

The thyrotropin-releasing hormone (TRH) receptor (TRH-R) is a member of the seven-transmembrane region, G protein-coupled receptor family. Arg-283 and Arg-306, in transmembrane helices 6 and 7, respectively, are putatively in positions homologous to those of residues that are important for agonist and antagonist binding in receptors for neurotransmitters. These arginines were mutated and the mutant receptors were transiently expressed in COS-1 cells. The affinity of the R306K TRH-R was similar to that of the wild-type (WT) TRH-R, whereas no specific binding was detected in cells expressing R306A, R306E, or R306L TRH-Rs. Because TRH stimulated inositol phosphate (IP) formation to similar maximal extents in cells expressing WT and Arg-306 mutant TRH-Rs, relative potencies were used to estimate the relative affinities of the receptors. The EC50 values for stimulation of R306A, R306E, and R306L TRH-Rs were 1500-, 1200-, and 3000-fold higher than that for the WT TRH-R. No specific binding was measurable in COS-1 cells expressing R283K, R283H, or R283A TRH-Rs, whereas maximal TRH stimulation of IP formation was to levels 64%, 42%, or < 1%, respectively, of that in cells expressing WT TRH-Rs; for R283K and R283H TRH-Rs, EC50 values were 6300- and 50,000-fold higher, respectively, than that for the WT TRH-R. In AtT-20 cells stably expressing R283A TRH-Rs, the binding affinity was 39,000-fold lower than that of the WT TRH-R and the number of receptors was estimated to be 0.88 x 10(6)/cell, but TRH did not stimulate IP formation. Thus, in the TRH-R, Arg-306 appears to be important for binding but not for activation, whereas Arg-283 appears to be important for binding and activation.

Animals↗
PubMed1994

Protein-DNA interactions within DNase I-hypersensitive sites located downstream of the HIV-1 promoter.

We have examined by in vitro footprinting a region located downstream of the human immunodeficiency virus, type 1 (HIV-1) promoter found to be hypersensitive to DNase I digestion in vivo. Recognition sites for several constitutive or inducible DNA binding factors were identified. Three AP-1 binding sites and an AP-3-like motif were situated within the R-U5 region of the long terminal repeat. A novel purine-rich motif (5'-GAAAGC-GAAAGDD-3' (D represents G, A, or T residues)), which interacts with a nuclear factor designated downstream binding factor 1 (DBF1), and two juxtaposed Sp-1 binding sites were located in the untranslated sequence immediately downstream of the 5'-long terminal repeat. Genomic footprinting of these sequence elements in the HIV-1 chronically infected cell lines revealed that the DBF1 and Sp-1 sites are occupied in vivo. Furthermore, transient transfection assays showed that point mutations in the DBF1 binding site decreased significantly the HIV-1 basal promoter activity. Taken together, these results suggest that the DBF1 play a role in the HIV-1 transcription regulation.

Base Sequence↗
PubMed1993

The transcription of a mammalian voltage-gated potassium channel is regulated by cAMP in a cell-specific manner.

The transcript of Kv1.5, a Shaker-like delayed rectifier K+ channel cloned in our laboratory, is regulated in both tissue and developmentally specific manners. In this study we characterized the 5'-flanking region of the Kv1.5 gene. The gene lacks a canonical TATA box, has several transcription start sites, and the 5'-noncoding sequence is intronless. A cAMP response element (CRE) consensus signal was identified in the 5'-noncoding region. cAMP regulates the expression of Kv1.5 gene in a cell-specific manner. In primary cardiac cells, cAMP induces a 6-fold increase in the steady state levels of Kv1.5 transcript. However, in GH3 cells cAMP induces a 5-6-fold decrease in steady state levels of Kv1.5 transcript. The half-life of Kv1.5 transcript is 37 min and is not affected by cAMP. Nuclear run-on experiments show that in GH3 cells, cAMP reduces the transcription rate of Kv1.5 gene. Transient transfection assays using 5'-deletion mutations of Kv1.5 5'-flanking sequences revealed that the CRE located at +636 can confer the cAMP inducibility to Kv1.5 reporter gene constructs and binds to CRE-binding protein (CREB) and CRE modulator protein (CREM) in electromobility gel shift assays. Furthermore, KCl-induced depolarization can increase the steady state levels of Kv1.5 transcript in primary atrial cells and decrease it in GH3 cells. We conclude that cAMP and depolarization play an important role in regulating K+ channel expression and thus may induce long term effects on the pattern of electrical activity of excitable cells.

Animals↗
PubMed1993

The D domain of the thyroid hormone receptor alpha 1 specifies positive and negative transcriptional regulation functions.

Four structural domains are characteristic of the members of the thyroid/steroid receptor superfamily. Of these, the A/B and D domains are the least conserved. We have investigated the role of two clusters of positively charged amino acids within the D domain of the thyroid hormone receptor alpha 1 (TR alpha 1). The sequences Lys134-Arg-Lys and Arg188-Arg-Lys, individually or together, were substituted to the neutral residues TIT in three mutants named alpha 1-1, alpha 1-2, and alpha 1-3, respectively. Subcellular localization of transiently transfected wild-type and mutated TRs was monitored by immunostaining, using a TR alpha 1-specific antibody. The wild-type and the alpha 1-2 TRs were detected exclusively in the nucleus, in the presence or absence of thyroid hormone. In contrast, the alpha 1-1 and alpha 1-3 mutants accumulated in both cytoplasm and nucleus, underscoring the importance of the Lys134-Arg-Lys residues for correct nuclear targeting. More importantly, although the mutants had unimpaired DNA- and hormone-binding activities, all three had lost positive and negative transcriptional regulatory functions. Thus, transactivation and repression functions can be entirely dissociated from the other properties of the receptor. In addition, substitution of either one of the positively charged amino acid clusters was sufficient to convert the native TR alpha 1 into a dominant, thyroid hormone-dependent receptor antagonist. These observations, which underline the functional relevance of the D domain for TR alpha 1 function, may also have implications for the autosomal dominant syndrome of generalized resistance to thyroid hormone.

Amino Acid Sequence↗
PubMed2002

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.

Animals↗