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ABSTRACT: This retrospective study examined the relationships among 10 academic predictors and first-time success on the NCLEX-RN in a sample of 92 bachelor of science in nursing minority and culturally diverse generic/traditional students at a large minority-serving, urban, public university. Predictors included the Test of Essential Academic Skills (overall, science, and reading), science grade point average (GPA), cumulative GPA, and scores on various standardized exams: Kaplan, HESI, and ATI. Discriminant analysis found science GPA of >3.50 and ATI B of 60 or above to be the best predictors of passing NCLEX-RN. Based on statistically significant differences between NCLEX-RN pass and fail scores, good indicators of NCLEX-RN success were scores of 50 or above on Kaplan and 950 or above on HESI. Overall, the Test of Essential Academic Skills did not predict students' NCLEX-RN outcomes.
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Bacharelado em Enfermagem , Estudantes de Enfermagem , Avaliação Educacional , Humanos , Licenciamento em Enfermagem , Estudos RetrospectivosRESUMO
Coronavirus disease 2019 (COVID-19) emerged in 2019 and rapidly became a global pandemic, infecting millions and killing hundreds of thousands. The disease altered the practices of hospitals, clinics, and patients. These changes have implications for advanced practice registered nurses (APRNs). APRNs must remain current on best practices for treatment and diagnosis of COVID-19 while being cognizant of changes to their scope of practice. As the pandemic continues, APRNs will remain on the front lines treating patients with COVID-19 while also caring for vulnerable populations within the community. To provide high-quality care, APRNs must use a multifaceted approach that heeds ongoing updates to evidence-based practice.
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The large conductance calcium-sensitive potassium (BK) channel is widely expressed in many organs and tissues, but its in vivo physiological functions have not been fully defined. Here we report a genetic locus associated with a human syndrome of coexistent generalized epilepsy and paroxysmal dyskinesia on chromosome 10q22 and show that a mutation of the alpha subunit of the BK channel causes this syndrome. The mutant BK channel had a markedly greater macroscopic current. Single-channel recordings showed an increase in open-channel probability due to a three- to fivefold increase in Ca(2+) sensitivity. We propose that enhancement of BK channels in vivo leads to increased excitability by inducing rapid repolarization of action potentials, resulting in generalized epilepsy and paroxysmal dyskinesia by allowing neurons to fire at a faster rate. These results identify a gene that is mutated in generalized epilepsy and paroxysmal dyskinesia and have implications for the pathogenesis of human epilepsy, the neurophysiology of paroxysmal movement disorders and the role of BK channels in neurological disease.
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Coreia/genética , Epilepsia Generalizada/genética , Canais de Potássio Cálcio-Ativados/genética , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Pré-Escolar , Coreia/complicações , Cromossomos Humanos Par 10 , Sequência Conservada , Cricetinae , Cricetulus , Epilepsia Generalizada/complicações , Feminino , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Canais de Potássio Ativados por Cálcio de Condutância Alta , Dados de Sequência Molecular , Mutação , Oócitos/fisiologia , Linhagem , Canais de Potássio Cálcio-Ativados/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Xenopus laevisRESUMO
Na(+)-glucose cotransporter (SGLT) mRNAs have been detected in many organs of the body, but, apart from kidney and intestine, transporter expression, localization, and functional activity, as well as physiological significance, remain elusive. Using a SGLT-specific molecular imaging probe, α-methyl-4-deoxy-4-[(18)F]fluoro-D-glucopyranoside (Me-4-FDG) with ex vivo autoradiography and immunohistochemistry, we mapped in vivo the regional distribution of functional SGLTs in rat brain. Since Me-4-FDG is not a substrate for GLUT1 at the blood-brain barrier (BBB), in vivo delivery of the probe into the brain was achieved after opening of the BBB by an established procedure, osmotic shock. Ex vivo autoradiography showed that Me-4-FDG accumulated in regions of the cerebellum, hippocampus, frontal cortex, caudate nucleus, putamen, amygdala, parietal cortex, and paraventricular nucleus of the hypothalamus. Little or no Me-4-FDG accumulated in the brain stem. The regional accumulation of Me-4-FDG overlapped the distribution of SGLT1 protein detected by immunohistochemistry. In summary, after the BBB is opened, the specific substrate for SGLTs, Me-4-FDG, enters the brain and accumulates in selected regions shown to express SGLT1 protein. This localization and the sensitivity of these neurons to anoxia prompt the speculation that SGLTs may play an essential role in glucose utilization under stress such as ischemia. The expression of SGLTs in the brain raises questions about the potential effects of SGLT inhibitors under development for the treatment of diabetes.
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Encéfalo/metabolismo , Transportador 1 de Glucose-Sódio/metabolismo , Animais , Autorradiografia/métodos , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagem , Feminino , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Imuno-Histoquímica/métodos , RNA Mensageiro/genética , Cintilografia , Ratos , Ratos Sprague-Dawley , Transportador 1 de Glucose-Sódio/genética , Distribuição TecidualRESUMO
Excitatory amino acid transporters (EAATs) remove glutamate from synapses. They maintain an efficient synaptic transmission and prevent glutamate from reaching neurotoxic levels. Glutamate transporters couple the uptake of one glutamate to the cotransport of three sodium ions and one proton and the countertransport of one potassium ion. The molecular mechanism for this coupled uptake of glutamate and its co- and counter-transported ions is not known. In a crystal structure of the bacterial glutamate transporter homolog, GltPh, only two cations are bound to the transporter, and there is no indication of the location of the third sodium site. In experiments using voltage clamp fluorometry and simulations based on molecular dynamics combined with grand canonical Monte Carlo and free energy simulations performed on different isoforms of GltPh as well on a homology model of EAAT3, we sought to locate the third sodium-binding site in EAAT3. Both experiments and computer simulations suggest that T370 and N451 (T314 and N401 in GltPh) form part of the third sodium-binding site. Interestingly, the sodium bound at T370 forms part of the binding site for the amino acid substrate, perhaps explaining both the strict coupling of sodium transport to uptake of glutamate and the ion selectivity of the affinity for the transported amino acid in EAATs.
Assuntos
Transportador 3 de Aminoácido Excitatório/química , Sódio/química , Animais , Sítios de Ligação , Cátions/química , Simulação por Computador , Transportador 3 de Aminoácido Excitatório/genética , Transportador 3 de Aminoácido Excitatório/metabolismo , Humanos , Modelos Moleculares , Método de Monte Carlo , Mutação , Oócitos , Estrutura Terciária de Proteína , Sódio/metabolismo , Especificidade por Substrato , Xenopus laevisRESUMO
Sodium-glucose cotransporters (SGLTs) are secondary active transporters belonging to the SLC5 gene family. SGLT1, a well-characterized member of this family, electrogenically transports glucose and galactose. Human SGLT3 (hSGLT3), despite sharing a high amino acid identity with human SGLT1 (hSGLT1), does not transport sugar, although functions as a sugar sensor. In contrast to humans, two different genes in mice and rats code for two different SGLT3 proteins, SGLT3a and SGLT3b. We previously cloned and characterized mouse SGLT3b (mSGLT3b) and showed that, while it does transport sugar like SGLT1, it likely functions as a physiological sugar sensor like hSGLT3. In this study, we cloned mouse SGLT3a (mSGLT3a) and characterized it by expressing it in Xenopus laevis oocytes and performing electrophysiology and sugar transport assays. mSGLT3a did not transport sugar, and sugars did not induce currents at pH 7.4, though acidic pH induced inward currents that increased in the presence of sugar. Moreover, mutation of residue 457 from glutamate to glutamine resulted in a Na(+)-dependent transport of sugar that was inhibited by phlorizin. To corroborate our results in oocytes, we expressed and characterized mSGLT3a in mammalian cells and confirmed our findings. In addition, we cloned, expressed, and characterized rat SGLT3a in oocytes and found characteristics similar to mSGLT3a. In summary, acidic pH induces currents in mSGLT3a, and sugar-induced currents are increased at acidic pH, but wild-type SGLT3a does not transport sugar.
Assuntos
Carboidratos/fisiologia , Proteínas de Transporte de Sódio-Glucose/genética , Proteínas de Transporte de Sódio-Glucose/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Transporte Biológico/fisiologia , Células CHO , Cricetinae , Concentração de Íons de Hidrogênio , Intestino Delgado/metabolismo , Intestino Delgado/fisiologia , Rim/metabolismo , Rim/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Mutação/genética , Florizina/farmacologia , Prótons , RNA Mensageiro/genética , RNA Ribossômico 18S/genética , Ratos , Sódio/metabolismo , Xenopus laevisRESUMO
SGLT1 is a sodium/glucose cotransporter that moves two Na(+) ions with each glucose molecule per cycle. SGLT3 proteins belong to the same family and are described as glucose sensors rather than glucose transporters. Thus, human SGLT3 (hSGLT3) does not transport sugar, but extracellular glucose depolarizes the cell in which it is expressed. Mouse SGLT3b (mSGLT3b), although it transports sugar, has low apparent sugar affinity and partially uncoupled stoichiometry compared with SGLT1, suggesting that mSGLT3b is also a sugar sensor. The crystal structure of the Vibrio parahaemolyticus SGLT showed that residue Gln(428) interacts directly with the sugar. The corresponding amino acid in mammalian proteins, 457, is conserved in all SGLT1 proteins as glutamine. In SGLT3 proteins, glutamate is the most common residue at this position, although it is a glycine in mSGLT3b and a serine in rat SGLT3b. To test the contribution of this residue to the function of SGLT3 proteins, we constructed SGLT3b mutants that recapitulate residue 457 in SGLT1 and hSGLT3, glutamine and glutamate, respectively. The presence of glutamine at residue 457 increased the apparent Na(+) and sugar affinities, whereas glutamate decreased the apparent Na(+) affinity. Moreover, glutamate transported more cations per sugar molecule than the wild type protein. We propose a model where cations are released intracellularly without the release of sugar from an intermediate state. This model explains the uncoupled charge:sugar transport phenotype observed in wild type and G457E-mSGLT3b compared with SGLT1 and the sugar-activated cation transport without sugar transport that occurs in hSGLT3.
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Modelos Químicos , Proteínas de Transporte de Sódio-Glucose/química , Proteínas de Transporte de Sódio-Glucose/metabolismo , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/fisiologia , Glucose , Humanos , Camundongos , Mutação de Sentido Incorreto , Ligação Proteica , Ratos , Proteínas de Transporte de Sódio-Glucose/genética , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismoRESUMO
GABA transporters play an important but poorly understood role in neuronal inhibition. They can reverse, but this is widely thought to occur only under pathological conditions. Here we use a heterologous expression system to show that the reversal potential of GAT-1 under physiologically relevant conditions is near the normal resting potential of neurons and that reversal can occur rapidly enough to release GABA during simulated action potentials. We then use paired recordings from cultured hippocampal neurons and show that GABAergic transmission is not prevented by four methods widely used to block vesicular release. This nonvesicular neurotransmission was potently blocked by GAT-1 antagonists and was enhanced by agents that increase cytosolic [GABA] or [Na(+)] (which would increase GAT-1 reversal). We conclude that GAT-1 regulates tonic inhibition by clamping ambient [GABA] at a level high enough to activate high-affinity GABA(A) receptors and that transporter-mediated GABA release can contribute to phasic inhibition.
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Inibidores da Captação de GABA , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Ácido gama-Aminobutírico/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Células CHO , Células Cultivadas , Cloretos/metabolismo , Cricetinae , Cricetulus , Citosol/efeitos dos fármacos , Citosol/metabolismo , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Proteínas da Membrana Plasmática de Transporte de GABA/fisiologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/fisiologia , Sódio/metabolismo , Sódio/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Termodinâmica , Transfecção , Ácido gama-Aminobutírico/fisiologiaRESUMO
Diabetic nephropathy (DN) represents a major public health cost. Tight glycemic and blood pressure control can dramatically slow, but not stop, the progression of the disease, and a large number of patients progress toward end-stage renal disease despite currently available interventions. An early and key event in the development of DN is loss of podocyte function (or glomerular visceral epithelial cells) from the kidney glomerulus, where they contribute to the integrity of the glomerular filtration barrier. Recent evidence suggests that podocytes can be the direct target of circulating hormones, lipids, and adipokines that are affected in diabetes. We review the clinical and experimental evidence implicating novel endocrine and metabolic pathways in the pathogenesis of podocyte dysfunction and the development of DN.
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Nefropatias Diabéticas/patologia , Podócitos/patologia , Adipocinas/fisiologia , Adulto , Albuminúria/etiologia , Albuminúria/fisiopatologia , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/fisiopatologia , Nefropatias Diabéticas/prevenção & controle , Hormônios/fisiologia , Humanos , Hipoglicemiantes/uso terapêutico , Glomérulos Renais/fisiopatologia , Glomérulos Renais/ultraestrutura , Masculino , Camundongos , Modelos Biológicos , Estresse Oxidativo , Podócitos/metabolismo , Sistema Renina-Angiotensina/fisiologia , Vitamina D/fisiologiaRESUMO
Despite belonging to a family of sugar cotransporters, human sodium/glucose transporter type 3 (hSGLT3) does not transport sugar, but it depolarizes the cell in the presence of extracellular sugar, and thus it has been suggested to work as a sugar sensor. In the human genome there is one SGLT3 gene, yet in mouse there are two. In this study we cloned one of them, mouse SGLT3b (mSGLT3b) and characterized the protein. We found that mSGLT3b has low affinity for sugars, as does hSGLT3, but surprisingly, mSGLT3b transports sugar, although the sugar transport is not as tightly coupled to cations as in SGLT1. Moreover, the sugar specificity of mSGLT3b has characteristics reminiscent of both SGLT1 and hSGLT3: mSGLT3b does not respond to galactose, similar to hSGLT3, but neither does it respond to 1-deoxynojirimycin, unlike hSGLT3 but similar to SGLT1. mSGLT3b has low apparent affinities for sugar and Na(+) and, furthermore, displays pre-steady-state currents, which in SGLT1 report on conformational changes in the protein. Finally, phlorizin, the typical inhibitor of SGLT proteins, also inhibits mSGLT3b. In summary, although mSGLT3b has some characteristics that resemble SGLT1 and others that are similar to hSGLT3, its low sugar affinity and uncoupled sugar transport lead us to conclude that mSGLT3b likely functions as a physiological glucose sensor similar to hSGLT3.
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Glucose/metabolismo , Proteínas de Transporte de Sódio-Glucose/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico , Relação Dose-Resposta a Droga , Galactose/metabolismo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucose/análogos & derivados , Cinética , Potenciais da Membrana , Camundongos , Oócitos , Florizina/farmacologia , Proteínas de Transporte de Sódio-Glucose/antagonistas & inibidores , Proteínas de Transporte de Sódio-Glucose/genética , Xenopus laevisRESUMO
In the past, only people diagnosed with celiac disease, approximately 1% of the population, avoided gluten consumption through all their meals. However, popular media often now mistakenly present gluten-free foods as being a healthier choice, and more people have now concluded that gluten is a harmful part of the diet. A review of literature on gluten-free diets, gluten sensitivity, celiac disease, and attitudes toward gluten consumption was undertaken to examine the prevalence and consequences of adopting a gluten-free diet and to provide guidance to healthcare practitioners whose patients are now often adopting this diet without medical input. Aside from celiac disease, nonceliac gluten sensitivity (NCGS) occurs in those persons in which gluten ingestion leads to symptomatic manifestations in the absence of celiac disease or wheat allergy but who report a remission of certain symptoms after removing gluten from their diet. However, it was been shown that a large percentage of people who claim NCGS do not feel those manifestations under a double-blind challenge to gluten. Moreover, some parents, believing that ingesting gluten is detrimental for their health, adopt gluten-free diets for their children. A review of existing data shows that there are detrimental effects to going gluten free, including loss of the dietary fiber, deficiencies in dietary minerals and vitamins, and potential heavy metal exposure. Healthcare practitioners should query patients about their dietary choices, and in cases of questionable adoption of gluten-free diet, patients and parents are educated about the detriments of a gluten-free diet, and in cases where patients continue to insist on gluten-free foods, referrals to nutritional counseling are warranted in order to minimize potential harm.
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As universities strive to raise their academic rank through the quality and quantity of scholarship in order to maintain their competitive edge and funding sources, faculty face pressure to increase number of publications and externally funded research (or project proposals). There are many challenges that make it difficult for faculty to meet a university's research demand, such as increased work load in academia, teaching large-size classes of students, and other strict university deadlines related to book ordering, scheduling classes, posting grades, etc. Faculty work group conflicts, faculty incivility, and dwindling grant/research funding add to faculty stress. In order to promote scholarship in academia, administrative support, collaborative work environments, mentoring, and appropriate appraisal systems are needed to enable faculty to be more productive and satisfied.
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Because of the many known maternal and neonatal health benefits of breastfeeding, there have been significant efforts to encourage exclusive breastfeeding, and many hospitals follow the guidelines of the Baby-Friendly Hospital Initiative. However, even with the right support, many women are unable to exclusively breastfeed, which may make them feel anxious and/or depressed. Psychological pressure to exclusively breastfeed has the potential to contribute to postpartum depression symptoms in new mothers who are unable to achieve their breastfeeding intentions. In this commentary, we focus on the well-being of the mother-infant dyad and argue for further research on maternal stress related to breastfeeding difficulties or pressure and the need to physically and psychologically assess and support women who are unable to breastfeed successfully or exclusively.
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Aleitamento Materno/psicologia , Comportamento de Escolha , Bem-Estar do Lactente/estatística & dados numéricos , Mães/psicologia , Adulto , Aleitamento Materno/estatística & dados numéricos , Feminino , Humanos , Lactente , Recém-Nascido , Mães/estatística & dados numéricosRESUMO
Podocytes are a major component of the glomerular filtration barrier, and their ability to sense insulin is essential to prevent proteinuria. Here we identify the insulin downstream effector GLUT4 as a key modulator of podocyte function in diabetic nephropathy (DN). Mice with a podocyte-specific deletion of GLUT4 (G4 KO) did not develop albuminuria despite having larger and fewer podocytes than wild-type (WT) mice. Glomeruli from G4 KO mice were protected from diabetes-induced hypertrophy, mesangial expansion, and albuminuria and failed to activate the mammalian target of rapamycin (mTOR) pathway. In order to investigate whether the protection observed in G4 KO mice was due to the failure to activate mTOR, we used three independent in vivo experiments. G4 KO mice did not develop lipopolysaccharide-induced albuminuria, which requires mTOR activation. On the contrary, G4 KO mice as well as WT mice treated with the mTOR inhibitor rapamycin developed worse adriamycin-induced nephropathy than WT mice, consistent with the fact that adriamycin toxicity is augmented by mTOR inhibition. In summary, GLUT4 deficiency in podocytes affects podocyte nutrient sensing, results in fewer and larger cells, and protects mice from the development of DN. This is the first evidence that podocyte hypertrophy concomitant with podocytopenia may be associated with protection from proteinuria.
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Regulação da Expressão Gênica/fisiologia , Transportador de Glucose Tipo 4/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Albuminúria , Animais , Tamanho Celular , Nefropatias Diabéticas , Doxorrubicina/toxicidade , Feminino , Barreira de Filtração Glomerular/citologia , Barreira de Filtração Glomerular/patologia , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/genética , Lipopolissacarídeos/toxicidade , CamundongosRESUMO
BACKGROUND: Sodium-glucose cotransporter proteins (SGLT) belong to the SLC5A family, characterized by the cotransport of Na(+) with solute. SGLT1 is responsible for intestinal glucose absorption. Until recently the only role described for SGLT proteins was to transport sugar with Na(+). However, human SGLT3 (hSGLT3) does not transport sugar but causes depolarization of the plasma membrane when expressed in Xenopus oocytes. For this reason SGLT3 was suggested to be a sugar sensor rather than a transporter. Despite 70% amino acid identity between hSGLT3 and hSGLT1, their sugar transport, apparent sugar affinities, and sugar specificity differ greatly. Residue 457 is important for the function of SGLT1 and mutation at this position in hSGLT1 causes glucose-galactose malabsorption. Moreover, the crystal structure of vibrio SGLT reveals that the residue corresponding to 457 interacts directly with the sugar molecule. We thus wondered if this residue could account for some of the functional differences between SGLT1 and SGLT3. METHODOLOGY/PRINCIPAL FINDINGS: We mutated the glutamate at position 457 in hSGLT3 to glutamine, the amino acid present in all SGLT1 proteins, and characterized the mutant. Surprisingly, we found that E457Q-hSGLT3 transported sugar, had the same stoichiometry as SGLT1, and that the sugar specificity and apparent affinities for most sugars were similar to hSGLT1. We also show that SGLT3 functions as a sugar sensor in a living organism. We expressed hSGLT3 and E457Q-hSGLT3 in C. elegans sensory neurons and found that animals sensed glucose in an hSGLT3-dependent manner. CONCLUSIONS/SIGNIFICANCE: In summary, we demonstrate that hSGLT3 functions as a sugar sensor in vivo and that mutating a single amino acid converts this sugar sensor into a sugar transporter similar to SGLT1.
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Mutação de Sentido Incorreto , Proteínas de Transporte de Sódio-Glucose/genética , Proteínas de Transporte de Sódio-Glucose/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Humanos , Proteínas de Transporte de Monossacarídeos , Proteínas Mutantes/fisiologia , Células Receptoras Sensoriais , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/fisiologia , Especificidade por Substrato/genéticaRESUMO
Human SGLT1 protein is an established sodium-glucose cotransporter. Despite widespread use of the mouse as a model organism, the mouse SGLT1 homologue has yet to be functionally characterized. Additionally, the crystal structure of a sugar transporter homologue, Vibrio SGLT, has recently been described, however, it offers limited information about the role of transmembrane segments outside of the core ligand binding domains. In particular, the amino acids in TM1 were not assigned in the structure. To examine the contribution of TM1 to the function of SGLT1, we have cloned and characterized the biophysical properties of SGLT1 from mouse, mSGLT1, and compared it to a clone containing an amino acid substitution in TM1, F36S. As predicted, both proteins formed functional Na+/sugar cotransporters, but F36S-mSGLT1 showed decreased rates of sugar uptake and decreased apparent affinities for both Na+ and sugar compared to mSGLT1. Analysis of pre-steady-state currents and comparison with the crystal structure of Vibrio SGLT provide plausible mechanisms to explain the differences in function of these two proteins. Our data suggest that amino acids in TM1, which are not involved in ligand binding and translocation pathways, significantly influence the functional properties of sodium-glucose carrier proteins.
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Glucose/metabolismo , Transportador 1 de Glucose-Sódio/fisiologia , Sódio/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Transporte Biológico Ativo , Humanos , Ativação do Canal Iônico , Camundongos , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Ratos , Transportador 1 de Glucose-Sódio/genéticaRESUMO
Imino sugars are used to treat type 2 diabetes mellitus [miglitol (Glyset)] and lysosomal storage disorders [miglustat (Zavesca)] based on the inhibition of alpha-glucosidases and glucosyltransferases. In this substrate specificity study, we examined the interactions of imino sugars with a novel human glucose sensor, sodium/glucose cotransporter type 3 (hSGLT3), using expression in Xenopus laevis oocytes and electrophysiology. The results for hSGLT3 are compared with those for alpha-glucosidases and human SGLT type 1 (hSGLT1), a well characterized sodium/glucose cotransporter of the SGLT family. In general, substrates have lower apparent affinities (K0.5) for hSGLT3 than hSGLT1 (D-glucose, alpha-methyl-D-glucose, 1-deoxy-D-glucose, and 4-deoxy-4-fluoro-D-glucose exhibit K0.5 values of 19, 21, 43, and 17 mM, respectively, for hSGLT3, and 0.5, 0.7, 10, and 0.07 mM, respectively, for hSGLT1). However, specificity of hSGLT3 binding is greater (D-galactose and 4-deoxy-4-fluoro-D-galactose are not hSGLT3 substrates, but have hSGLT1 K0.5 values of 0.6 and 1.3 mM). An important deviation from this trend is potent hSGLT3 activation by the imino sugars 1-deoxynojirimycin (DNJ), N-hydroxylethyl-1-deoxynojirimycin (miglitol), N-butyl-1-deoxynojirimycin (miglustat), N-ethyl-1-deoxynojirimycin, and 1-deoxynojirimycin-1-sulfonic acid, with K0.5 values of 0.5 to 9 microM. The diastereomer 1-deoxygalactonojirimycin activates hSGT3 with a K0.5 value of 11 mM, a 3000-fold less potent interaction than is observed for DNJ (4 microM). These imino sugar binding characteristics are similar to those for alpha-glucosidases, but there are no interactions with hSGLT1. This work provides insights into hSGLT3 and -1 substrate binding interactions, establishes a pharmacological profile to study endogenous hSGLT3, and may have important ramifications for the clinical application of imino sugars.
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Imino Açúcares/farmacologia , Proteínas de Transporte de Sódio-Glucose/agonistas , Animais , Técnicas Biossensoriais/métodos , Eletrofisiologia , Humanos , Oócitos , Ligação Proteica , Proteínas de Transporte de Sódio-Glucose/genética , Especificidade por Substrato , Transfecção , Xenopus laevisRESUMO
The sodium-dependent glucose cotransporter SGLT1 undergoes a series of voltage- and ligand-induced conformational changes that underlie the cotransport mechanism. In this study we describe how the binding of external Na changes the conformation of the sugar-binding domain, exposing residues that are involved in sugar recognition to the external environment. We constructed 15 individual Cys mutants in the four transmembrane helices (TMHs) that form the sugar binding and translocation domain. Each mutant was functionally characterized for transport kinetics and substrate specificity. Identification of interactions between mutated residues and hydroxyls on the pyranose ring was assessed by comparing the affinities of deoxy sugars to those of glucose. We determined conformation-dependent accessibility to the mutated residues by both a traditional substituted cysteine accessibility method (SCAM) and a new fluorescence binding assay. These data were integrated to orient the helices and construct a framework of residues that comprise the external sugar binding site. We present evidence that R499, Q457, and T460 play a direct role in sugar recognition and that five other residues are indirectly involved in transport. Arranging the four TMHs to account for Na-dependent accessibility and potential for sugar interaction allows us to propose a testable model for the SGLT1 sugar binding site.
Assuntos
Glucose/metabolismo , Transportador 1 de Glucose-Sódio/química , Transportador 1 de Glucose-Sódio/metabolismo , Sódio/metabolismo , Animais , Sítios de Ligação , Cisteína/genética , Cisteína/metabolismo , Humanos , Cinética , Modelos Biológicos , Modelos Moleculares , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Transportador 1 de Glucose-Sódio/genética , Especificidade por Substrato , Xenopus laevisRESUMO
A missense mutation (D434G) has recently been identified in the alpha subunit of the human large-conductance calcium-activated potassium (BK) channel. Interestingly, although the mutation causes an increase in open probability, individuals that carry the mutation have epilepsy and/or paroxysmal dyskinesia, disorders of increased brain excitability. To define the mechanisms of the mutation, we have used recordings from single channels and measurement of macroscopic conductances to examine the gating of the alpha subunit, modulation by the regulatory beta4 subunit, and the effect of Mg2+ on channel properties. Although there was relatively little difference in open dwell times for the mutant and wild-type alpha subunits, the mutant channel spent less time in a long-lived closed state. Co-expression of the beta4 subunit caused the wild-type channel to be less sensitive to calcium at low Ca2+ concentrations but had little effect on the mutant channel, further accentuating the difference between the wild-type and the mutant channels. In the absence of Ca2+, there was no difference in Mg2+ or voltage sensitivity of the mutant and wild-type channels, whereas in 2 mM Ca2+, the mutant channel had greater open probability at every Mg2+ concentration tested. We conclude that the D434G mutation modifies Ca2+ -dependent activation, but we find no evidence of a direct effect on activation by Mg2+ or voltage. The resulting enhancement of BK channel function leads to an increase in brain excitability, possibly due to more rapid repolarization of action potentials.
Assuntos
Encéfalo/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Mutação , Substituição de Aminoácidos , Animais , Células CHO , Cálcio/fisiologia , Clonagem Molecular , Cricetinae , Eletrofisiologia/métodos , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Potássio/fisiologia , Subunidades Proteicas/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Na(+)/glucose cotransport by SGLT1 is a tightly coupled process that is driven by the Na(+) electrochemical gradient across the plasma membrane. We have previously proposed that SGLT1 contains separate Na(+)- and glucose-binding domains, that A166 (in the Na(+) domain) is close to D454 (in the sugar domain), and that interactions between these residues influence sugar specificity and transport. We have now expressed the mutant D454C in Xenopus laevis oocytes and examined the role of charge on residue 454 by replacing the Asp with Cys or His, and by chemical mutation of D454C with alkylating reagents of different charge (MTSES(-), MTSET(+), MMTS(0), MTSHE(0), and iodoacetate(-)). Functional properties were examined by measuring sugar transport and cotransporter currents. In addition, D454C was labeled with fluorescent dyes and the fluorescence of the labeled transporter was recorded as a function of voltage and ligand concentration. The data shows that (1) aspartate 454 is critically important for the normal trafficking of the protein to the plasma membrane; (2) there were marked changes in the functional properties of D454C, i.e., a reduction in turnover number and a loss of voltage sensitivity, although there were no alterations in sugar selectivity or sugar and Na(+) affinity; (3) a negative charge on residue 454 increased Na(+) and sugar transport with a normal stoichiometry of 2 Na(+):1 sugar. A positive charge on residue 454, in contrast, uncoupled Na(+) and sugar transport, indicating the importance of the negative charge in the coordination of the cotransport mechanism.