RESUMEN
Protein structure prediction and modeling provide a tool for understanding protein functions by computationally constructing protein structures from amino acid sequences and analyzing them. With help from protein prediction tools and web servers, users can obtain the three-dimensional protein structure models and gain knowledge of functions from the proteins. In this chapter, we will provide several examples of such studies. As an example, structure modeling methods were used to investigate the relation between mutation-caused misfolding of protein and human diseases including epilepsy and leukemia. Protein structure prediction and modeling were also applied in nucleotide-gated channels and their interaction interfaces to investigate their roles in brain and heart cells. In molecular mechanism studies of plants, rice salinity tolerance mechanism was studied via structure modeling on crucial proteins identified by systems biology analysis; trait-associated protein-protein interactions were modeled, which sheds some light on the roles of mutations in soybean oil/protein content. In the age of precision medicine, we believe protein structure prediction and modeling will play more and more important roles in investigating biomedical mechanism of diseases and drug design.
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Encéfalo/metabolismo , Biología Computacional/métodos , Epilepsia/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Medicina de Precisión/métodos , Secuencia de Aminoácidos , Encéfalo/patología , Caspasa 9/química , Caspasa 9/genética , Caspasa 9/metabolismo , Caveolina 3/química , Caveolina 3/genética , Caveolina 3/metabolismo , Epilepsia/genética , Epilepsia/patología , Estudio de Asociación del Genoma Completo , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Ligandos , Oryza/genética , Fitomejoramiento , Canales de Potasio/química , Canales de Potasio/genética , Canales de Potasio/metabolismo , Unión Proteica , Conformación Proteica , Receptores de GABA-A/química , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Alineación de Secuencia , Programas InformáticosRESUMEN
The newborn heart adapts to postnatal life by shifting from a fetal glycolytic metabolism to a mitochondrial oxidative metabolism. Abcc9, an ATP-binding cassette family member, increases expression concomitant with this metabolic shift. Abcc9 encodes a membrane-associated receptor that partners with a potassium channel to become the major potassium-sensitive ATP channel in the heart. Abcc9 also encodes a smaller protein enriched in the mitochondria. We now deleted exon 5 of Abcc9 to ablate expression of both plasma membrane and mitochondria-associated Abcc9-encoded proteins, and found that the myocardium failed to acquire normal mature metabolism, resulting in neonatal cardiomyopathy. Unlike wild-type neonatal cardiomyocytes, mitochondria from Ex5 cardiomyocytes were unresponsive to the KATP agonist diazoxide, consistent with loss of KATP activity. When exposed to hydrogen peroxide to induce cell stress, Ex5 neonatal cardiomyocytes displayed a rapid collapse of mitochondria membrane potential, distinct from wild-type cardiomyocytes. Ex5 cardiomyocytes had reduced fatty acid oxidation, reduced oxygen consumption and reserve. Morphologically, Ex5 cardiac mitochondria exhibited an immature pattern with reduced cross-sectional area and intermitochondrial contacts. In the absence of Abcc9, the newborn heart fails to transition normally from fetal to mature myocardial metabolism.-Fahrenbach, J. P., Stoller, D., Kim, G., Aggarwal, N., Yerokun, B., Earley, J. U., Hadhazy, M., Shi, N.-Q., Makielski, J. C., McNally, E. M. Abcc9 is required for the transition to oxidative metabolism in the newborn heart.
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Corazón/fisiología , Miocitos Cardíacos/metabolismo , Consumo de Oxígeno/fisiología , Receptores de Sulfonilureas/metabolismo , Animales , Animales Recién Nacidos , Cardiomiopatías/congénito , Membrana Celular/metabolismo , Ácidos Grasos/metabolismo , Femenino , Canales KATP/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismoRESUMEN
BACKGROUND: Estrogen has been shown to mediate protection in female hearts against ischemia-reperfusion (I-R) stress. Composed by a Kir6.2 pore and an SUR2 regulatory subunit, cardiac ATP-sensitive potassium channels (KATP) remain quiescent under normal physiological conditions but they are activated by stress stimuli to confer protection to the heart. It remains unclear whether KATP is a regulatory target of estrogen in the female-specific I-R signaling pathway. In this study, we aimed at delineating the molecular mechanism underlying estrogen modulation on KATP channel activity during I-R. MATERIALS AND METHODS: We employed KATP knockout mice in which SUR2 is disrupted (SUR2KO) to characterize their I-R response using an in vivo occlusion model. To test the protective effects of estrogen, female mice were ovariectomized and implanted with 17ß-estradiol (E2) or placebo pellets (0.1 µg/g/day, 21-day release) before receiving an I-R treatment. Comparative proteomic analyses were performed to assess pathway-level alterations between KO-IR and WT-IR hearts. RESULTS AND DISCUSSION: Echocardiographic results indicated that KO females were pre-disposed to cardiac dysfunction at baseline. The mutant mice were more susceptible to I-R stress by having bigger infarcts (46%) than WT controls (31%). The observation was confirmed using ovariectomized mice implanted with E2 or placebo. However, the estrogen-mediated protection was diminished in KO hearts. Expression studies showed that the SUR2 protein level, but not RNA level, was up-regulated in WT-IR mice relative to untreated controls possibly via PTMs. Our antibodies detected different glycosylated SUR2 receptor species after the PNGase F treatment, suggesting that SUR2 could be modified by N-glycosylation. We subsequently showed that E2 could further induce the formation of complex-glycosylated SUR2. Additional time-point experiments revealed that I-R hearts had increased levels of N-glycosylated SUR2; and DPM1, the first committed step enzyme in the N-glycosylation pathway. Comparative proteomic profiling identified 41 differentially altered protein hits between KO-IR and WT-IR mice encompassing those related to estrogen biosynthesis. CONCLUSIONS: Our findings suggest that KATP is likely a downstream regulatory target of estrogen and it is indispensable in female I-R signaling. Increasing SUR2 expression by N-glycosylation mediated by estrogen may be effective to enhance KATP channel subunit expression in I-R.
RESUMEN
Consistent left-right asymmetry requires specific ion currents. We characterize a novel laterality determinant in Xenopus laevis: the ATP-sensitive K(+)-channel (K(ATP)). Expression of specific dominant-negative mutants of the Xenopus Kir6.1 pore subunit of the K(ATP) channel induced randomization of asymmetric organ positioning. Spatio-temporally controlled loss-of-function experiments revealed that the K(ATP) channel functions asymmetrically in LR patterning during very early cleavage stages, and also symmetrically during the early blastula stages, a period when heretofore largely unknown events transmit LR patterning cues. Blocking K(ATP) channel activity randomizes the expression of the left-sided transcription of Nodal. Immunofluorescence analysis revealed that XKir6.1 is localized to basal membranes on the blastocoel roof and cell-cell junctions. A tight junction integrity assay showed that K(ATP) channels are required for proper tight junction function in early Xenopus embryos. We also present evidence that this function may be conserved to the chick, as inhibition of K(ATP) in the primitive streak of chick embryos randomizes the expression of the left-sided gene Sonic hedgehog. We propose a model by which K(ATP) channels control LR patterning via regulation of tight junctions.
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Tipificación del Cuerpo , Embrión de Pollo/crecimiento & desarrollo , Canales KATP/fisiología , Xenopus laevis/embriología , Animales , Blastómeros/metabolismo , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/fisiología , Uniones Estrechas/fisiologíaRESUMEN
RATIONALE: Cardioprotective pathways may involve a mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel but its composition is not fully understood. OBJECTIVE: We hypothesized that the mitoK(ATP) channel contains a sulfonylurea receptor (SUR)2 regulatory subunit and aimed to identify the molecular structure. METHODS AND RESULTS: Western blot analysis in cardiac mitochondria detected a 55-kDa mitochondrial SUR2 (mitoSUR2) short form, 2 additional short forms (28 and 68 kDa), and a 130-kDa long form. RACE (Rapid Amplification of cDNA Ends) identified a 1.5-Kb transcript, which was generated by a nonconventional intraexonic splicing (IES) event within the 4th and 29th exons of the SUR2 mRNA. The translated product matched the predicted size of the 55-kDa short form. In a knockout mouse (SUR2KO), in which the SUR2 gene was disrupted, the 130-kDa mitoSUR2 was absent, but the short forms remained expressed. Diazoxide failed to induce increased fluorescence of flavoprotein oxidation in SUR2KO cells, indicating that the diazoxide-sensitive mitoK(ATP) channel activity was associated with 130-kDa-based channels. However, SUR2KO mice displayed similar infarct sizes to preconditioned wild type, suggesting a protective role for the remaining short form-based channels. Heterologous coexpression of the SUR2 IES variant and Kir6.2 in a K(+) transport mutant Escherichia coli strain permitted improved cell growth under acidic pH conditions. The SUR2 IES variant was localized to mitochondria, and removal of a predicted mitochondrial targeting sequence allowed surface expression and detection of an ATP-sensitive current when coexpressed with Kir6.2. CONCLUSIONS: We identify a novel SUR2 IES variant in cardiac mitochondria and provide evidence that the variant-based channel can form an ATP-sensitive conductance and may contribute to cardioprotection.
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Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Empalme Alternativo/fisiología , Isquemia Miocárdica/genética , Miocitos Cardíacos/fisiología , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Droga/genética , Receptores de Droga/metabolismo , Animales , Células Cultivadas , Exones/genética , Flavoproteínas/metabolismo , Biblioteca de Genes , Humanos , Ratones , Ratones Noqueados , Mitocondrias/fisiología , Isquemia Miocárdica/metabolismo , Miocitos Cardíacos/citología , Oxidación-Reducción , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores de SulfonilureasRESUMEN
Based on autopsy data collected in Southern China from 2001-2006, 975 cases of sudden unexplained nocturnal death syndrome (SUNDS) were surveyed. Genetic screening of SCN5A gene encoding the voltage dependent cardiac sodium channel was performed in 74 SUNDS cases. The annual occurrence rate of SUNDS in the area was estimated to be about 1 per 100,000 people. About 80.6% of deaths occurred between the ages of 21 to 40 years and the case number peaked at age 30 years. In 75.4% of cases with witnesses, victims died asleep between 11 PM and 4 AM and they showed predominantly abrupt respiratory distress shortly preceding death. The monthly distribution of emergency fever cases in the area during the same period was positively correlated to that of SUNDS cases (r(s) = 0.611, P = 0.035). Four polymorphisms in SCN5A were identified in both SUNDS and control groups. Compared with controls, the allele frequency of C5457 and C3666 + 69 were significant higher in SUNDS (P < 0.005) while the genotypes of both 5457CC (P = 0.012, OR = 2.0, 95% CI = 1.3-3.2) and 3666+69CC (P = 0.004, OR = 2.1, 95% CI = 1.3-3.3) in SUNDS cases were significantly higher. This is the first report of an epidemiological survey and SCN5A gene screening in SUNDS in the Han population of China. The genotypes of 5457CC and 3666+69CC in SCN5A gene may be Chinese SUNDS susceptible polymorphisms.
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Muerte Súbita/epidemiología , Mutación , Polimorfismo de Nucleótido Simple , Sueño , Canales de Sodio/genética , Adolescente , Adulto , Distribución por Edad , Estudios de Casos y Controles , China/epidemiología , Etnicidad/genética , Femenino , Fiebre/epidemiología , Frecuencia de los Genes , Pruebas Genéticas , Genotipo , Humanos , Masculino , Canal de Sodio Activado por Voltaje NAV1.5 , Reacción en Cadena de la Polimerasa , Distribución por Sexo , TemperaturaRESUMEN
The sulfonylurea receptor-2 (SUR2) is a subunit of ATP-sensitive potassium channels (K(ATP)) in heart. Mice with the SUR2 gene disrupted (SUR2m) are constitutively protected from ischemia-reperfusion (I/R) cardiac injury. This was surprising because K(ATP), either sarcolemmal or mitochondrial or both, are thought to be important for cardioprotection. We hypothesized that SUR2m mice have an altered mitochondrial phenotype that protects against I/R. Mitochondrial membrane potential (ΔΨ(m)), tolerance to Ca(2+) load, and reactive oxygen species (ROS) generation were studied by fluorescence-based assays, and volumetric changes in response to K(+) were measured by light scattering in isolated mitochondria. For resting SUR2m mitochondria compared with wild type, the ΔΨ(m) was less polarized (46.1 ± 0.4 vs. 51.9 ± 0.6%), tolerance to Ca(2+) loading was increased (163 ± 2 vs. 116 ± 2 µM), and ROS generation was enhanced with complex I [8.5 ± 1.2 vs. 4.9 ± 0.2 arbitrary fluorescence units (afu)/s] or complex II (351 ± 51.3 vs. 166 ± 36.2 afu/s) substrates. SUR2m mitochondria had greater swelling in K(+) medium (30.2 ± 3.1%) compared with wild type (14.5 ± 0.6%), indicating greater K(+) influx. Additionally, ΔΨ(m) decreased and swelling increased in the absence of ATP in SUR2m, but the sensitivity to ATP was less compared with wild type. When the mitochondria were subjected to hypoxia-reoxygenation, the decrease in respiration rates and respiratory control index was less in SUR2m. ΔΨ(m) maintenance in the SUR2m intact myocytes was also more tolerant to metabolic inhibition. In conclusion, the cardioprotection observed in the SUR2m mice is associated with a protected mitochondrial phenotype resulting from enhanced K(+) conductance that partially dissipated ΔΨ(m). These results have implications for possible SUR2 participation in mitochondrial K(ATP).
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Metabolismo Energético , Mitocondrias Cardíacas/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio/metabolismo , Receptores de Droga/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Hipoxia de la Célula , Respiración de la Célula , Genotipo , Luz , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Ratones Mutantes , Dilatación Mitocondrial , Daño por Reperfusión Miocárdica/metabolismo , Fenotipo , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/genética , Especies Reactivas de Oxígeno/metabolismo , Receptores de Droga/genética , Dispersión de Radiación , Espectrometría de Fluorescencia , Receptores de Sulfonilureas , Factores de TiempoRESUMEN
Sulfonylurea receptor-containing ATP-sensitive potassium (K(ATP)) channels have been implicated in cardioprotection, but the cell type and constitution of channels responsible for this protection have not been clear. Mice deleted for the first nucleotide binding region of sulfonylurea receptor 2 (SUR2) are referred to as SUR2 null since they lack full-length SUR2 and glibenclamide-responsive K(ATP) channels in cardiac, skeletal, and smooth muscle. As previously reported, SUR2 null mice develop electrocardiographic changes of ST segment elevation that were shown to correlate with coronary artery vasospasm. Here we restored expression of the cardiomyocyte SUR2-K(ATP) channel in SUR2 null mice by generating transgenic mice with ventricular cardiomyocyte-restricted expression of SUR2A. Introduction of the cardiomyocyte SUR2A transgene into the SUR2 null background restored functional cardiac K(ATP) channels. Hearts isolated from rescued mice, referred to as MLC2A, had significantly reduced infarct size (27 ± 3% of area at risk) compared with SUR2 null mice (36 ± 3% of area at risk). Compared with SUR2 null hearts, MLC2A hearts exhibited significantly improved cardiac function during the postischemia reperfusion period primarily because of preservation of low diastolic pressures. Additionally, restoration of cardiac SUR2-K(ATP) channels significantly reduced the degree and frequency of ST segment elevation episodes in MLC2A mice. Therefore, cardioprotective mechanisms both dependent and independent of SUR2-K(ATP) channels contribute to cardiac function.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Electrocardiografía , Canales KATP/metabolismo , Miocitos Cardíacos/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Droga/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Membrana Celular/metabolismo , Vasoespasmo Coronario/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Infarto del Miocardio/metabolismo , Canales de Potasio de Rectificación Interna/genética , Receptores de Droga/genética , Transducción de Señal/fisiología , Receptores de SulfonilureasRESUMEN
The cardiac sarcolemmal ATP-sensitive potassium channel (K(ATP)) consists of a Kir6.2 pore and an SUR2 regulatory subunit, which is an ATP-binding cassette (ABC) transporter. K(ATP) channels have been proposed to play protective roles during ischemic preconditioning. An SUR2 mutant mouse was previously generated by disrupting the first nucleotide-binding domain (NBD1), where a glibenclamide action site was located. In the mutant ventricular myocytes, a non-conventional glibenclamide-insensitive (10 microM), ATP-sensitive current (I(KATPn)) was detected in 33% of single-channel recordings with an average amplitude of 12.3+/-5.4 pA per patch, an IC(50) to ATP inhibition at 10 microM and a mean burst duration at 20.6+/-1.8 ms. Newly designed SUR2 isoform- or variant-specific antibodies identified novel SUR2 short forms in the sizes of 28 and 68 kDa in addition to a 150-kDa long form in the sarcolemmal membrane of wild-type (WT) heart. We hypothesized that channels constituted by these short forms that lack NBD1 confer I(KATPn). The absence of the long form in the mutant corresponded to loss of the conventional glibenclamide-sensitive K(ATP) currents (I(KATP)) in isolated cardiomyocytes and vascular smooth muscle cells but the SUR2 short forms remained intact. Nested exonic RT-PCR in the mutant indicated that the short forms lacked NBD1 but contained NBD2. The SUR2 short forms co-immunoprecipitated with Kir6.1 or Kir6.2 suggesting that the short forms may function as hemi-transporters reported in other eukaryotic ABC transporter subgroups. Our results indicate that different K(ATP) compositions may co-exist in cardiac sarcolemmal membrane.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Gliburida/farmacología , Activación del Canal Iónico/efectos de los fármacos , Canales KATP/metabolismo , Miocardio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio/metabolismo , Receptores de Droga/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Animales , Anticuerpos , Especificidad de Anticuerpos/efectos de los fármacos , Western Blotting , Línea Celular , Inmunoprecipitación , Cinética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Proteínas Mutantes/metabolismo , Canales de Potasio/química , Canales de Potasio de Rectificación Interna/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Ratas , Receptores de Droga/química , Receptores de SulfonilureasRESUMEN
Targeting of ion channels to caveolae, a subset of lipid rafts, allow cells to respond efficiently to extracellular signals. Hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 4 is a major subunit for the cardiac pacemaker. Caveolin-3 (Cav3), abundantly expressed in muscle cells, is responsible for forming caveolae. P104L, a Cav3 mutant, has a dominant negative effect on wild type (WT) Cav3 and associates with limb-girdle muscular dystrophy and cardiomyopathy. HCN4 was previously shown to localize to lipid rafts, but how caveolae regulate the function of HCN4 is unknown. We hypothesize that Cav3 associates with HCN4 and regulates the function of HCN4 channel. In this study, we applied whole-cell patch clamp analysis, immunostaining, biotinylation, and immunoprecipitation methods to investigate this hypothesis. The immunoprecipitation results indicated an association of HCN4 and Cav3 in the heart and in HEK293 cells. Our immunostaining results showed that HCN4 colocalized with Cav3 but only partially colocalized with P104L in HEK293 cells. Transient expression of Cav3, but not P104L, in HEK 293 cells stably expressing HCN4 caused a 45% increase in HCN4 current (IHCN4) density. Transient expression of P104L caused a two-fold increase in the activation time constant for IHCN4 and shifted the voltage of the steady-state inactivation to a more negative potential. We conclude that HCN4 associates with Cav3 to form a HCN4 macromolecular complex. Our results indicated that disruption of caveolae using P104L alters HCN4 function and could cause a reduction of cardiac pacemaker activity.
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Caveolina 3/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Proteínas Musculares/metabolismo , Animales , Caveolina 3/genética , Línea Celular , Regulación de la Expresión Génica , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Ratones , Mutación , Miocardio/metabolismo , Marcapaso Artificial , Canales de Potasio , Transporte de ProteínasRESUMEN
In the vasculature, ATP-sensitive potassium channels (KATP) channels regulate vascular tone. Mice with targeted gene disruptions of KATP subunits expressed in vascular smooth muscle develop spontaneous coronary vascular spasm and sudden death. From these models, it was hypothesized that the loss of KATP channel activity in arterial vascular smooth muscle was responsible for coronary artery spasm. We now tested this hypothesis using a transgenic strategy where the full-length sulfonylurea receptor containing exon 40 was expressed under the control of a smooth muscle-specific SM22alpha promoter. Two transgenic founder lines were generated and independently bred to sulfonylurea receptor 2 (SUR2) null mice to generate mice that restored expression of KATP channels in vascular smooth muscle. Transgenic expression of the sulfonylurea receptor in vascular smooth muscle cells was confirmed by detecting mRNA and protein from the transgene. Functional restoration was determined by recording pinacidil-based KATP current by whole cell voltage clamping of isolated aortic vascular smooth muscle cells isolated from the transgenic restored mice. Despite successful restoration of KATP channels in vascular smooth muscle, transgene-restored SUR2 null mice continued to display frequent episodes of spontaneous ST segment elevation, identical to the phenotype seen in SUR2 null mice. As in SUR2 null mice, ST segment elevation was frequently followed by atrioventricular heart block. ST segment elevation and coronary perfusion pressure in the restored mice did not differ significantly between transgene-negative and transgene-positive SUR2 null mice. We conclude that spontaneous coronary vasospasm and sudden death in SUR2 null mice arises from a coronary artery vascular smooth muscle-extrinsic process.
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Transportadoras de Casetes de Unión a ATP/fisiología , Adenosina Trifosfato/farmacología , Vasoespasmo Coronario/etiología , Músculo Liso Vascular/metabolismo , Canales de Potasio de Rectificación Interna/fisiología , Transportadoras de Casetes de Unión a ATP/análisis , Animales , Vasoespasmo Coronario/metabolismo , Electrocardiografía , Ratones , Ratones Transgénicos , Mutación , Canales de Potasio de Rectificación Interna/genética , Receptores de Droga , Receptores de SulfonilureasRESUMEN
Endogenous bioelectrical signaling coordinates cell behaviors toward correct anatomical outcomes. Lack of a model explaining spatialized dynamics of bioelectric states has hindered the understanding of the etiology of some birth defects and the development of predictive interventions. Nicotine, a known neuroteratogen, induces serious defects in brain patterning and learning. Our bio-realistic computational model explains nicotine's effects via the disruption of endogenous bioelectrical gradients and predicts that exogenous HCN2 ion channels would restore the endogenous bioelectric prepatterns necessary for brain patterning. Voltage mapping in vivo confirms these predictions, and exogenous expression of the HCN2 ion channel rescues nicotine-exposed embryos, resulting in normal brain morphology and molecular marker expression, with near-normal learning capacity. By combining molecular embryology, electrophysiology, and computational modeling, we delineate a biophysical mechanism of developmental brain damage and its functional rescue.
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Encéfalo/embriología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Modelos Neurológicos , Nicotina/toxicidad , Animales , Tipificación del Cuerpo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Larva , Aprendizaje , Ratones , Microinyecciones , Xenopus laevisRESUMEN
Laterality is a basic characteristic of all life forms, from single cell organisms to complex plants and animals. For many metazoans, consistent left-right asymmetric patterning is essential for the correct anatomy of internal organs, such as the heart, gut, and brain; disruption of left-right asymmetry patterning leads to an important class of birth defects in human patients. Laterality functions across multiple scales, where early embryonic, subcellular and chiral cytoskeletal events are coupled with asymmetric amplification mechanisms and gene regulatory networks leading to asymmetric physical forces that ultimately result in distinct left and right anatomical organ patterning. Recent studies have suggested the existence of multiple parallel pathways regulating organ asymmetry. Here, we show that an isoform of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of ion channels (hyperpolarization-activated cyclic nucleotide-gated channel 4, HCN4) is important for correct left-right patterning. HCN4 channels are present very early in Xenopus embryos. Blocking HCN channels (Ih currents) with pharmacological inhibitors leads to errors in organ situs. This effect is only seen when HCN4 channels are blocked early (pre-stage 10) and not by a later block (post-stage 10). Injections of HCN4-DN (dominant-negative) mRNA induce left-right defects only when injected in both blastomeres no later than the 2-cell stage. Analysis of key asymmetric genes' expression showed that the sidedness of Nodal, Lefty, and Pitx2 expression is largely unchanged by HCN4 blockade, despite the randomization of subsequent organ situs, although the area of Pitx2 expression was significantly reduced. Together these data identify a novel, developmental role for HCN4 channels and reveal a new Nodal-Lefty-Pitx2 asymmetric gene expression-independent mechanism upstream of organ positioning during embryonic left-right patterning.
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Hyperpolarization-activated cyclic-nucleotide gated channel (HCN) proteins are important regulators of both neuronal and cardiac excitability. Among the 4 HCN isoforms, HCN4 is known as a pacemaker channel, because it helps control the periodicity of contractions in vertebrate hearts. Although the physiological role of HCN4 channel has been studied in adult mammalian hearts, an earlier role during embryogenesis has not been clearly established. Here, we probe the embryonic roles of HCN4 channels, providing the first characterization of the expression profile of any of the HCN isoforms during Xenopus laevis development and investigate the consequences of altering HCN4 function on embryonic pattern formation. We demonstrate that both overexpression of HCN4 and injection of dominant-negative HCN4 mRNA during early embryogenesis results in improper expression of key patterning genes and severely malformed hearts. Our results suggest that HCN4 serves to coordinate morphogenetic control factors that provide positional information during heart morphogenesis in Xenopus.
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Cardiac ATP-sensitive potassium channels (KATP) are found in both the sarcoplasmic reticulum (sarcKATP) and the inner membrane of mitochondria (mitoKATP). SarcKATP are composed of a pore containing subunit Kir6.2 and a regulatory sulfonylurea receptor subunit (SUR2), but the composition of mitoKATP remains unclear. An unusual intra-exonic splice variant of SUR2 (SUR2A-55) was previously identified in mitochondria of mammalian heart and brain, and by analogy with sarcKATP we proposed SUR2A-55 as a candidate regulatory subunit of mitoKATP. Although SUR2A-55 lacks the first nucleotide binding domain (NBD) and 2 transmembrane domains (TMD), it has a hybrid TMD and retains the second NBD. It resembles a hemi-ABC transporter suggesting it could multimerize to function as a regulatory subunit. A putative mitochondrial targeting signal in the N-terminal domain of SUR2A-55 was removed by truncation and when co-expressed with Kir6.1 and Kir6.2 it targeted to the plasma membrane and yielded KATP currents. Single channel conductance, mean open time, and burst open time of SUR2A-55 based KATP was similar to the full-length SUR2A based KATP. However, the SUR2A-55 KATP were 70-fold less sensitive to block by ATP, and twice as resistant to intracellular Ca (2+) inhibition compared with the SUR2A KATP, and were markedly insensitive to KATP drugs, pinacidil, diazoxide, and glybenclamide. These results suggest that the SUR2A-55 based channels would tend to be open under physiological conditions and in ischemia, and could account for cardiac and mitochondrial phenotypes protective for ischemia.
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Adenosina Trifosfato/metabolismo , Fenómenos Electrofisiológicos , Canales KATP/metabolismo , Mitocondrias/metabolismo , Miocardio/citología , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Sulfonilureas/metabolismo , Animales , Calcio/metabolismo , Masculino , Ratones , Isoformas de Proteínas/metabolismo , RatasRESUMEN
Adenosine triphosphate-sensitive potassium (K(ATP)) channels are thought to mediate the stress response by sensing intracellular ATP concentration. Cardiomyocyte K(ATP) channels are composed of the pore-forming Kir6.2 subunit and the regulatory sulfonylurea receptor 2 (SUR2). We studied the response to acute isoproterenol in SUR2 null mice as a model of acute adrenergic stress and found that the episodic coronary vasospasm observed at baseline in SUR2 null mice was alleviated. Similar results were observed following administration of a nitric oxide donor consistent with a vasodilatory role. Langendorff-perfused hearts were subjected to global ischemia, and hearts from SUR2 null mice exhibited significantly reduced infarct size (54+/-4 versus 30+/-3%) and improved cardiac function compared to control mice. SUR2 null mice have hypertension and develop cardiac hypertrophy. However, despite longstanding hypertension, fibrosis was absent in SUR2 null mice. SUR2 null mice were administered nifedipine to block baseline coronary vasospasm, and hearts from nifedipine-treated SUR2 null mice exhibited increased infarct size compared to untreated SUR2 null mice (42+/-3% versus 54+/-3%). We conclude that conventional sarcolemmal cardiomyocyte K(ATP) channels containing full-length SUR2 are not required for mediating the response to acute cardiovascular stress.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Enfermedades Cardiovasculares/genética , Enfermedad Aguda , Agonistas Adrenérgicos beta/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Cardiomegalia/genética , Enfermedades Cardiovasculares/etiología , Vasoespasmo Coronario/genética , Vasoespasmo Coronario/patología , Citoprotección/efectos de los fármacos , Citoprotección/genética , Predisposición Genética a la Enfermedad , Isoproterenol/farmacología , Canales KATP/genética , Masculino , Ratones , Ratones Noqueados , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Óxido Nítrico/farmacología , Canales de Potasio de Rectificación Interna , Receptores de Droga , Estrés Fisiológico/complicaciones , Estrés Fisiológico/genética , Receptores de SulfonilureasRESUMEN
Alternative splicing allows multiple mRNAs to be generated from a single gene, which in turn can be translated into a group of diverse proteins with different roles and structures. The outcome of alternative splicing leads to the co-existence of multiple splice variants of a gene at different concentrations in different tissues. The pore-forming subunit of the K(ATP) channel (K(IR)6.x) and the regulatory sulfonylurea receptor (SUR(x)) subunits exist in a 4:4 stoichiometry to form hetero-octameric ATP-sensitive potassium channel (K(ATP)) channels, which are widely distributed in various types of tissues at either the plasma membrane (cellK(ATP)) or mitochondrial inner membrane (the mitochondrial form of K(ATP) channel, mitoK(ATP)). They perform important physiological functions in regulating insulin secretion in pancreatic beta-cells, providing ischemic protection in heart and brain, and regulating vascular tone in smooth muscles. Two separate genes, the regulatory subunit protein I (SUR1) and the regulatory subunit protein II (SUR2) encode the high- and low-affinity SUR, respectively. This review summarizes the current studies on the function and distribution of the SUR isoforms and alternative splice variants, and to a lesser extent the K(IR)6.x subunits. The different isoforms and splice variants allow for many K(ATP) channel combinations, and therefore, increases the channel diversity and the possibility of complexity in function.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Empalme Alternativo , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Receptores de Droga/genética , Receptores de Droga/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Animales , Humanos , Precondicionamiento Isquémico , Complejo Mediador , Ratones , Ratones Noqueados , Canales de Potasio/efectos de los fármacos , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Subunidades de Proteína , Transporte de Proteínas , Receptores de Droga/efectos de los fármacos , Receptores de Sulfonilureas , Transactivadores/efectos de los fármacosRESUMEN
SHAM-sensitive (STO) alternative respiration is present in the xylose-metabolizing, Crabtree-negative yeast, Pichia stipitis, but its pathway components and physiological roles during xylose metabolism are poorly understood. We cloned PsSTO1, which encodes the SHAM-sensitive terminal oxidase (PsSto1p), by genome walking from wild-type CBS 6054 and subsequently deleted PsSTO1 by targeted gene disruption. The resulting sto1-delta deletion mutant, FPL-Shi31, did not contain other isoforms of Sto protein that were detectable by Western blot analysis using an alternative oxidase monoclonal antibody raised against the Sto protein from Sauromatum guttatum. Levels of cytochromes b, c, c(1) and a.a(3) did not change in the sto1-delta mutant, which indicated that deleting PsSto1p did not alter the cytochrome pool. Interestingly, the sto1-delta deletion mutant stopped growing earlier than the parent and produced 20% more ethanol from xylose. Heterologous expression of PsSTO1 in Saccharomyces cerevisiae increased its total oxygen consumption rate and imparted cyanide-resistant oxygen uptake but did not enable growth on ethanol, indicating that PsSto1p is not coupled to ATP synthesis. We present evidence that the mitochondrial NADH dehydrogenase complex (Complex I) was present in wild-type CBS 6054 but was bypassed in the cells during xylose metabolism. Unexpectedly, deleting PsSto1p led to the use of Complex I in the mutant cells when xylose was the carbon source. We propose that the non-proton-translocating NAD(P)H dehydrogenases are linked to PsSto1p in xylose-metabolizing cells and that this non-ATP-generating route serves a regulatory function in the complex redox network of P. stipitis.
Asunto(s)
NADH NADPH Oxidorreductasas/metabolismo , Pichia/metabolismo , Xilosa/metabolismo , Secuencia de Aminoácidos , Western Blotting , Clonación Molecular , Complejo I de Transporte de Electrón , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Eliminación de Gen , Genes Fúngicos , Datos de Secuencia Molecular , Mutación , NADH NADPH Oxidorreductasas/análisis , Consumo de Oxígeno/efectos de los fármacos , Pichia/efectos de los fármacos , Pichia/genética , Saccharomyces cerevisiae/metabolismo , Salicilamidas/farmacología , Alineación de SecuenciaRESUMEN
XYL3, which encodes a D-xylulokinase (EC 2.7.1.17), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of XYL3 eliminated D-xylulokinase activity, but D-ribulokinase activity was still present. Southern analysis of P. stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes. Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that XYL3 is critical for ethanol production from xylose but that P. stipitis has another pathway for xylose assimilation. Expression of XYL3 using its P. stipitis promoter increased Saccharomyces cerevisiae D-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro, D-xylulokinase activity in recombinant S. cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of XYL3 in S. cerevisiae.