RESUMO
Impaired fertility is common among patients with chronic organ failure, including end-stage renal disease (ESRD). Women of childbearing age undergoing transplantation may experience rapid return of fertility. Pregnancy posttransplant presents numerous risks for the patient, fetus, and allograft. Maternal risks include hypertension and preeclampsia. Allograft risks include acute rejection and failure of the organ, and fetal risks include miscarriage, birth defects from immunosuppressants, premature delivery, and low birth weight. Belatacept, a selective T cell costimulation blocker, was approved for use in kidney transplant recipients in the United States in 2011. Little is known about the safety of belatacept during pregnancy in humans. We describe 2 cases of successful pregnancy and delivery with the use of belatacept-based immunosuppression. The Transplant Pregnancy Registry International (TPR) is a voluntary registry for transplant recipients who have had pregnancies or fathered a pregnancy posttransplant. To date, these 2 cases are the only known exposures to belatacept that have been reported to the TPR.
Assuntos
Abatacepte/uso terapêutico , Rejeição de Enxerto/tratamento farmacológico , Sobrevivência de Enxerto/efeitos dos fármacos , Falência Renal Crônica/cirurgia , Transplante de Rim/efeitos adversos , Adulto , Feminino , Idade Gestacional , Taxa de Filtração Glomerular , Rejeição de Enxerto/etiologia , Humanos , Imunossupressores/uso terapêutico , Recém-Nascido , Gravidez , Resultado da Gravidez , TransplantadosRESUMO
BACKGROUND: Nephrogenic systemic fibrosis (NSF) affects patients with impaired renal function who have received gadolinium-containing contrast agents (GCCAs). Increased dermal cellularity is a key diagnostic feature of NSF, however, the histologic findings can be subtle. OBJECTIVE: We sought to determine whether dermal cellularity in skin biopsy specimens from NSF cases: (1) differs significantly from that of controls; and (2) correlates with duration of the skin lesions, level of plasma creatinine, GCCA dose, or a combination of these. METHODS: Seventeen NSF skin biopsy specimens and age-, sex-, and site-matched controls were retrieved from the dermatopathology files of the Massachusetts General Hospital in Boston. Dermal cellularity was manually quantified on hematoxylin-eosin-stained sections and patient medical records were reviewed for demographic and clinical data. RESULTS: NSF cases showed a mean dermal cellularity of 70.8/high-power field (control mean: 14.4/high-power field, P < .001) and a cut-off range of 19 to 26/high-power field was established. No significant correlation was identified between dermal cellularity and demographic and clinical data. LIMITATIONS: In this retrospective analysis, duration of skin lesion was defined as the interval from most recent prior GCCA study, rather than the actual clinical onset, to time of skin biopsy, and the cumulative GCCA dose may reflect a minimum if GCCA was received at an outside institution. CONCLUSION: Enumeration of dermal cellularity on hematoxylin-eosin-stained sections can aid in the histologic diagnosis of NSF in the setting of chronic kidney disease and GCCA exposure and is independent of patient age, sex, plasma creatinine, time from last GCCA exposure, and GCCA dose.
Assuntos
Derme/patologia , Gadolínio/efeitos adversos , Nefropatias/diagnóstico , Dermopatia Fibrosante Nefrogênica/induzido quimicamente , Dermopatia Fibrosante Nefrogênica/patologia , Adulto , Idoso , Biópsia , Contagem de Células , Meios de Contraste/efeitos adversos , Creatinina/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
BACKGROUND: Orthostatic hypotension (OH) is a poorly understood complication of simultaneous pancreas-kidney (SPK) transplantation. We sought to determine the incidence, timing, and relationship of OH to rapid glycemic control in the early posttransplant period. METHODS: This was a nonrandomized retrospective single-center review of 75 SPK and 19 kidney-alone (KA) recipients with type 1 diabetes (DM). RESULTS: OH occurred in 57 (76%) SPK versus 2 (10%) KA recipients (odds ratio [OR] 61.72, 95% confidence interval [CI], 9.69-393.01; P < 0.001). The median onset of OH was 12 (interquartile range [IQR] 9-18) days posttransplant and resolved in 85% of SPK recipients after a median of 2.5 (IQR 1.2-6.3) months. Among SPK recipients, independent risk factors for OH were a shorter duration of DM (OR 0.85, 95% CI, 0.73-0.98; P = 0.03) and rapid glycemic control in the early posttransplant period (OR 1.13, 95% CI, 1.01-1.27; P = 0.04), as evidenced by a larger percent change in hemoglobin A1c (HbA1c) from transplant to month 3. OH patients had a higher median baseline HbA1c [8.3% (IQR 7.2-10.0) versus 7.1% (IQR 6.8-8.3); P = 0.07], lower median 3-month HbA1c [4.8% (IQR 4.6-5.2) versus 5.2% (IQR 5.0-5.4); P = 0.02], and a larger reduction in HbA1c over time as compared to recipients without OH (P < 0.01). CONCLUSIONS: Our results show that OH is more likely to occur following SPK versus KA transplantation and is strongly associated with rapid glucose normalization within the early posttransplant period.
RESUMO
BACKGROUND: Excessive weight (EW) gain is common after solid organ transplantation, but there is little information concerning obesity after pancreas transplantation. The study goal was to characterize EW gain after kidney-pancreas (KP) transplantation. METHODS: This was a retrospective single-center review of 100 KP recipients transplanted between September 2007 and June 2015. RESULTS: The median percent weight gain for all recipients at 1 year posttransplant was 10% (interquartile range, 2.7%-19.3%) of baseline weight. EW gain, defined as greater than or equal to a 19% 1-year increase in weight, included all recipients (n = 26) above the upper limit of interquartile range for weight gain at 1 year. In multivariate analysis, recipient age <40 years, the use of tacrolimus/mammalian target of rapamycin immunosuppression, and an acute rejection event were independent risk factors for EW gain. At a mean follow-up of 43±23 months, there was no difference in patient or graft survival between the EW and non-EW cohorts. Although mean hemoglobin A1c levels between groups were equivalent, the EW versus non-EW cohort displayed a significant increase in mean insulin levels and a trend towards higher C-peptide levels. Criteria for posttransplant metabolic syndrome was met in 34.6% of EW versus 17.6% of non-EW cohorts (P = 0.07). CONCLUSIONS: At intermediate-term follow-up, EW gain after KP transplantation was not associated with an increased risk of death or graft loss, although there was a trend toward a greater risk of posttransplant metabolic syndrome. There may be a metabolic consequence of successful pancreas transplantation that results in EW gain in a proportion of recipients, leading to an increased risk of long-term cardiovascular complications.
Assuntos
Diabetes Mellitus Tipo 2/cirurgia , Transplante de Rim/efeitos adversos , Síndrome Metabólica/epidemiologia , Obesidade/epidemiologia , Transplante de Pâncreas/efeitos adversos , Aumento de Peso , Adulto , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/mortalidade , Feminino , Seguimentos , Hemoglobinas Glicadas/análise , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Humanos , Imunossupressores/efeitos adversos , Insulina/sangue , Transplante de Rim/métodos , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/diagnóstico , Síndrome Metabólica/etiologia , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/diagnóstico , Obesidade/etiologia , Transplante de Pâncreas/métodos , Período Pós-Operatório , Estudos RetrospectivosRESUMO
PURPOSE: To retrospectively assess the association between gadopentetate dimeglumine exposure at magnetic resonance imaging and the development of nephrogenic systemic fibrosis (NSF). MATERIALS AND METHODS: This HIPAA-compliant study had institutional review board approval. Informed consent was waived. A search of medical and pathologic records was performed to identify patients with NSF that was diagnosed between January 1998 and December 2007. Patients with known exposure to gadolinium-based contrast agents other than gadopentetate dimeglumine were excluded. Medical records were then reviewed for gadopentetate dimeglumine exposure, renal status, concomitant diseases, timing of NSF symptom onset, date of NSF diagnosis, and clinical outcome. Skin gadolinium deposition was assessed for those patients with adequate available tissue. Spearman rank correlations were estimated to assess the relationship between the dose of gadopentetate dimeglumine and the time to onset of NSF. RESULTS: Thirty-six patients (mean age, 62.6 years; range, 30-83 years) had been exposed to gadopentetate dimeglumine prior to NSF onset. All had stage 5 chronic kidney disease and all but one were undergoing dialysis at the time of exposure. NSF developed within 3 months after the last gadopentetate dimeglumine exposure (range, 1-59 months) in 21 (66%) of 32 patients. The patients had been exposed to median cumulative gadopentetate dimeglumine volumes of 35, 40, 85, and 117.5 mL over the 3, 12, and 24 months and up to 11 years preceding the onset of NSF, respectively. Patients who received higher cumulative and total gadopentetate dimeglumine doses had a higher risk of developing NSF than did those who received lower doses (odds ratio = 1.2). Twenty (56%) of 36 patients died, with a median interval of 18 months between NSF symptom onset and death. CONCLUSION: NSF develops in patients with renal impairment after exposure to gadopentetate dimeglumine in a dose- and time-dependent manner. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.2531082160/-/DC1.
Assuntos
Meios de Contraste/efeitos adversos , Gadolínio DTPA/efeitos adversos , Imageamento por Ressonância Magnética , Dermopatia Fibrosante Nefrogênica/induzido quimicamente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Meios de Contraste/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Gadolínio DTPA/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Dermopatia Fibrosante Nefrogênica/epidemiologia , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
Potassium channels control the resting membrane potential and excitability of biological tissues. Many voltage-gated potassium channels are controlled through interactions with accessory subunits of the KCNE family through mechanisms still not known. Gating of mammalian channel KCNQ1 is dramatically regulated by KCNE subunits. We have found that multiple segments of the channel pore structure bind to the accessory protein KCNE1. The sites that confer KCNE1 binding are necessary for the functional interaction, and all sites must be present in the channel together for proper regulation by the accessory subunit. Specific gating control is localized to a single site of interaction between the ion channel and accessory subunit. Thus, direct physical interaction with the ion channel pore is the basis of KCNE1 regulation of K+ channels.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/química , Canais de Potássio/metabolismo , Animais , Western Blotting/métodos , Linhagem Celular , Cricetinae , Cricetulus , Condutividade Elétrica , Embrião de Mamíferos , Imunofluorescência/métodos , Humanos , Ativação do Canal Iônico , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Rim , Potenciais da Membrana/fisiologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp/métodos , Testes de Precipitina/métodos , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Transfecção/métodosRESUMO
IKs has been considered the potassium current most responsible for adrenergic/cAMP-mediated changes in cardiac repolarization during stress. Increasing biochemical, electrophysiological and genetic evidence however, points to a role for hERG/IKr in beta-adrenergic responses. Elevations of cAMP as seen in beta-adrenergic stimulation can result in PKA-dependent phosphorylation of hERG and direct binding of cAMP to the channel protein. Generally, there is a suppression of current density due to the channel phosphorylation. We recently identified a novel protein-protein interaction between hERG and the adaptor protein 14-3-3epsilon. Interaction sites exist on both N- and C-termini of hERG and the interaction is dynamic, requiring phosphorylation of the channel by PKA. When both sites bind to 14-3-3 proteins there is an acceleration and augmentation of current activation in contrast to the depression of current with phosphorylation alone. When sufficient 14-3-3 is available the phosphorylation state of the channel is stabilized and prolonged. Thus, 14-3-3 interactions with hERG provide a unique mechanism for plasticity in the autonomic control of stress-dependent regulation of cardiac membrane excitability. Here, we summarize our findings and report on our further efforts to analyse interactions between the native channel protein and 14-3-3 in cardiac myocytes.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Humanos , Síndrome do QT Longo , FosforilaçãoRESUMO
Acute kidney injury is a complication of open-heart surgery that carries a poor prognosis. Studies have shown that postoperative renal function deterioration in cardiovascular surgery patients increases in-hospital mortality and adversely affects long-term survival. Identifying individuals at risk for developing AKI and aggressive early intervention is extremely important to optimize outcomes. This paper provides an overview of the etiology, prognostic markers, risk factors, and prevention of AKI and treatments that may favorably affect outcomes.
Assuntos
Injúria Renal Aguda/etiologia , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Injúria Renal Aguda/epidemiologia , Saúde Global , Cardiopatias/cirurgia , Mortalidade Hospitalar/tendências , Humanos , Incidência , Complicações Pós-OperatóriasRESUMO
AIM: The objective of this study was to determine the effects of lysophosphatidic acid (LPA) on endothelial functions and molecular alternations in both porcine coronary arteries and human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: The vessel rings and HCAECs were treated with clinically relevant concentrations of LPA for different times. Vasomotor reactivity was studied with a myograph tension system. LPA (10 and 50 µM) treatment for the vessel rings significantly reduced endothelium-dependent vasorelaxation in response to bradykinin (×10(-5)M) by 32% and 49%, respectively, compared with the control (P<0.05). LPA decreased endothelial nitric oxide synthase (eNOS) mRNA and immunoreactivity levels in the vessel rings. In HCAECs, LPA reduced eNOS mRNA, phospho-eNOS and total eNOS protein levels. In addition, superoxide anion levels in LPA-treated vessel rings and HCAECs were significantly increased by lucegenin-enhanced chemiluminescence assay and dihydroethidium staining, respectively. Mitochondrial membrane potential and ATP content in LPA-treated HCAECs were substantially decreased. The mRNA levels of reactive oxygen species generating enzymes NOX4 and p40(phox) were increased, while endogenous antioxidant enzyme superoxide dismutase 1 was decreased in response to LPA treatment in HCAECs. Furthermore, exogenous antioxidant molecule selenomethionine (SeMet) effectively reversed these LPA-induced effects in both porcine coronary arteries and HCAECs. CONCLUSIONS: LPA causes endothelial dysfunction by a mechanism associated with decreased eNOS expression and increased oxidative stress in porcine coronary arteries and HCAECs.
Assuntos
Aterosclerose/fisiopatologia , Vasos Coronários/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Bradicinina/farmacologia , Regulação para Baixo , Endotélio Vascular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Selenometionina/farmacologia , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Superóxidos/metabolismo , SuínosRESUMO
INTRODUCTION: Vascular calcification in chronic kidney disease (CKD) is extremely common and contributes to significant morbidity and mortality among these patients. The pathogenesis is complex and involves multiple factors, including elevated calcium x phosphorus product as well as deficiencies in circulating or locally produced inhibitors of calcification, parathyroid hormone, hyperlipidemia and inflammation. Similarly, valvular heart calcifications as well as myocardial and pulmonary calcifications of fatal consequences can also occur, presumably related to the same pathogenetic factors (Figures 1, 2). Other forms of extraskeletal tissue calcification of nonfatal consequences but leading to incapacity can also develop in CKD patients (Figure 3). These complications may be prevented by awareness and early intervention directed towards correcting some of the aforementioned participating mechanisms.
Assuntos
Nefropatias/complicações , Calcificação Vascular/etiologia , Adolescente , Remodelação Óssea , Cálcio/metabolismo , Doença Crônica , Feminino , Humanos , Nefropatias/metabolismo , Nefropatias/terapia , Masculino , Hormônio Paratireóideo/metabolismo , Fósforo/metabolismo , Medição de Risco , Fatores de Risco , Calcificação Vascular/diagnóstico , Calcificação Vascular/metabolismo , Calcificação Vascular/prevenção & controleRESUMO
Contrast-induced nephropathy, also known as contrast-induced acute kidney injury, is associated with rapid and often irreversible decline in kidney function following the administration of iodinated contrast agents. Contrast-induced nephropathy is the third leading cause of acute kidney injury in hospitalized patients, and substantially increases mortality, morbidity, and length of hospitalization. Contrast-induced nephropathy follows a predictable time of onset and is potentially preventable. It has been the subject of numerous studies addressing characteristics of the populations at risk and prophylactic strategies. This evidence-based review summarizes recent literature and provides a nephrologists' perspective on contrast-induced nephropathy, focusing on: the pathophysiology of contrast-induced nephropathy; identification of populations at risk; correlation between contrast-induced nephropathy and the type of contrast agent used; and finally, measures to prevent contrast-induced nephropathy, including intravenous fluids, sodium bicarbonate, N-acetylcysteine, and hemofiltration/hemodialysis.
Assuntos
Meios de Contraste/efeitos adversos , Nefropatias/prevenção & controle , Acetilcisteína/uso terapêutico , Doença Aguda , Animais , Terapia Combinada , Citoproteção , Medicina Baseada em Evidências , Hidratação , Hemofiltração , Humanos , Nefropatias/induzido quimicamente , Nefropatias/fisiopatologia , Guias de Prática Clínica como Assunto , Diálise Renal , Medição de Risco , Fatores de Risco , Bicarbonato de Sódio/uso terapêutico , Resultado do TratamentoRESUMO
OBJECTIVE: Nephrogenic systemic fibrosis (NSF) is a rapidly progressive, debilitating condition that causes cutaneous and visceral fibrosis in patients with renal failure. Little is known about its prevalence or etiology. The aim of this study was to establish the prevalence of NSF and associated risk factors METHODS: Two cohorts of patients were recruited from 6 outpatient hemodialysis centers and examined for cutaneous changes of NSF, which were defined using a scoring system based on hyperpigmentation, hardening, and tethering of skin on the extremities. Demographic data were gathered, mortality was followed up prospectively for 24 months, and gadolinium exposure was ascertained for a subgroup of patients in the second cohort. RESULTS: Examination reproducibility was 97% in cohort 1. In cohort 2, 25 (13%) of 186 patients demonstrated cutaneous changes of NSF. Twenty-four-month mortality following examination was 48% and 20% in patients with and those without cutaneous changes of NSF, respectively (adjusted hazard ratio 2.9, 95% confidence interval [95% CI] 1.4-5.9). Cutaneous changes of NSF were observed in 16 (30%) of 54 patients with prior exposure to gadopentetate dimeglumine contrast during imaging studies. Exposure to gadolinium-containing contrast was associated with an increased risk of developing cutaneous changes of NSF (odds ratio 14.7, 95% CI 1.9-117.0) compared with nonexposed patients. CONCLUSION: In patients receiving hemodialysis, NSF is an underrecognized disorder that is associated with increased mortality. Exposure to gadolinium-containing contrast material appears to be a significant risk factor for the development of NSF.
Assuntos
Meios de Contraste/efeitos adversos , Fibrose/mortalidade , Gadolínio/efeitos adversos , Nefropatias/etiologia , Dermatopatias/induzido quimicamente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Fibrose/etiologia , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Fatores de Risco , Pele/efeitos dos fármacos , SíndromeAssuntos
Doenças Cardiovasculares/etiologia , Nefropatias Diabéticas/complicações , Coração/fisiopatologia , Anti-Hipertensivos/uso terapêutico , Glicemia/metabolismo , Pressão Sanguínea , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/fisiopatologia , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/fisiopatologia , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/fisiopatologia , Progressão da Doença , Hemoglobinas Glicadas/metabolismo , Coração/efeitos dos fármacos , Humanos , Hipoglicemiantes/uso terapêutico , Hipolipemiantes/uso terapêutico , Lipídeos/sangue , Guias de Prática Clínica como Assunto , Abandono do Hábito de FumarRESUMO
We previously reported that cloned human ether-a-go-go-related gene (HERG) K+ channels are regulated by changes in phosphatidylinositol 4,5-bisphosphate (PIP2) concentration. Here we investigated the molecular determinants of PIP2 interactions with HERG channel protein. To establish the molecular nature of the PIP2-HERG interaction, we examined a segment of the HERG COOH terminus with a high concentration of positively charged amino acids (nos. 883-894) as a possible site of interaction with negatively charged PIP2. When we excised deletion-HERG (D-HERG) or mutated methionine-substituted-HERG (M-HERG) this segment of HERG to neutralize the amino acid charge, the mutant channels produced current that was indistinguishable from wild-type HERG. Elevating internal PIP2, however, no longer accelerated the activation kinetics of the mutant HERG. Moreover, PIP2-dependent hyperpolarizing shifts in the voltage dependence of activation were abolished with both mutants. PIP2 effects on channel-inactivation kinetics remained intact, which suggests an uncoupling of inactivation and activation regulation by PIP2. The specific binding of radiolabeled PIP2 to both mutant channel proteins was nearly abolished. Stimulation of alpha1A-adrenergic receptors produced a reduction in current amplitude of the rapidly activating delayed rectifier K+ current (the current carried by ERG protein) from rabbit ventricular myocytes. The alpha-adrenergic-induced current reduction was accentuated by PKC blockers and also unmasked a depolarizing shift in the voltage dependence of activation, which supports the conclusion that receptor activation of PLC results in PIP2 consumption that alters channel activity. These results support a physiological role for PIP2 regulation of the rapidly activating delayed rectifier K+ current during autonomic stimulation and localize a site of interaction to the COOH-terminal tail of the HERG K+ channel.
Assuntos
Miócitos Cardíacos/metabolismo , Fosfatidilinositol 4,5-Difosfato/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Células Cultivadas , Canais de Potássio de Retificação Tardia , Canal de Potássio ERG1 , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go , Deleção de Genes , Humanos , Masculino , Mutação , Técnicas de Patch-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Isoformas de Proteínas/metabolismo , Coelhos , Receptores Adrenérgicos alfa 1/metabolismo , Fatores de TempoRESUMO
Acute stress provokes lethal cardiac arrhythmias in the hereditary long QT syndrome. Here we provide a novel molecular mechanism linking beta-adrenergic signaling and altered human ether-a-go-go related gene (HERG) channel activity. Stress stimulates beta-adrenergic receptors, leading to cAMP elevations that can regulate HERG K+ channels both directly and via phosphorylation by cAMP-dependent protein kinase (PKA). We show that HERG associates with 14-3-3epsilon to potentiate cAMP/PKA effects upon HERG. The binding of 14-3-3 occurs simultaneously at the N- and C-termini of the HERG channel. 14-3-3 accelerates and enhances HERG activation, an effect that requires PKA phosphorylation of HERG and dimerization of 14-3-3. The interaction also stabilizes the lifetime of the PKA-phosphorylated state of the channel by shielding the phosphates from cellular phosphatases. The net result is a prolongation of the effect of adrenergic stimulation upon HERG activity. Thus, 14-3-3 interactions with HERG may provide a unique mechanism for plasticity in the control of membrane excitability and cardiac rhythm.
Assuntos
Proteínas de Transporte de Cátions , Proteínas de Ligação a DNA , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transativadores , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetinae , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dimerização , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Humanos , Dados de Sequência Molecular , Fosforilação , Canais de Potássio/genética , Regulador Transcricional ERG , Técnicas do Sistema de Duplo-Híbrido , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
T-lymphocyte activation requires sustained Ca(2+) signaling dependent upon capacitative Ca(2+) entry (CCE). The protein(s) that forms the stores-operated Ca(2+) channel (SOCC) responsible for CCE has long been sought but has not been definitively identified. Members of the TRPV family (transient receptor potential superfamily-vanilloid receptor subfamily) of channel genes have been proposed to encode SOCCs responsible for CCE in non-excitable cells. Here we present evidence that a member of the TRPV group, CaT1, is involved in generating I(CRAC), the CCE current that is necessary for T-cell activation. CaT1 is expressed in Jurkat T-lymphocytes. When overexpressed in Jurkat cells, CaT1 produces a Ca(2+) entry current that mimics the endogenous I(CRAC) in its dependence on external Ca(2+), inactivation by elevated concentration of internal Ca(2+), and pharmacological block by capsaicin. Overexpressed CaT1 is partially regulated by the release of internal Ca(2+) stores via thapsigargin or receptor-mediated generation of inositol 1,4,5-trisphosphate. A pore-region mutant of CaT1, TRIA-CaT1, fails to carry Ca(2+) currents and associates with co-expressed wild type CaT1 to functionally suppress permeation of Ca(2+) ions. Expression of the TRIA-CaT1 mutant in Jurkat cells results in suppression of the endogenous I(CRAC). Taken together these results indicate that CaT1 is the channel protein that contributes to T-lymphocyte SOCCs either alone or as a subunit in a heterogeneous channel complex.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Animais , Células CHO , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Capsaicina/farmacologia , Clonagem Molecular , Cricetinae , DNA Complementar/metabolismo , Genes Dominantes , Humanos , Immunoblotting , Inositol 1,4,5-Trifosfato/metabolismo , Células Jurkat , Microscopia de Fluorescência , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV , Fatores de Tempo , TransfecçãoRESUMO
Cardiac I(Ks), the slowly activated delayed-rectifier K(+) current, is produced by the protein complex composed of alpha- and beta-subunits: KvLQT1 and minK. Mutations of genes encoding KvLQT1 and minK are responsible for the hereditary long QT syndrome (loci LQT1 and LQT5, respectively). MinK-L51H fails to traffic to the cell surface, thereby failing to produce effective I(Ks). We examined the effects that minK-L51H and an endoplasmic reticulum (ER)-targeted minK (minK-ER) exerted over the electrophysiology and biosynthesis of coexpressed KvLQT1. Both minK-L51H and minK-ER were sequestered primarily in the ER as confirmed by lack of plasma membrane expression. Glycosylation and immunofluorescence patterns of minK-L51H were qualitatively different for minK-ER, suggesting differences in trafficking. Cotransfection with the minK mutants resulted in reduced surface expression of KvLQT1 as assayed by whole cell voltage clamp and immunofluorescence. MinK-L51H reduced current amplitude by 91% compared with wild-type (WT) minK/KvLQT1, and the residual current was identical to KvLQT1 without minK. The phenotype of minK-L51H on I(Ks) was not dominant because coexpressed WT minK rescued the current and surface expression. Collectively, our data suggest that ER quality control prevents minK-L51H/KvLQT1 complexes from trafficking to the plasma membrane, resulting in decreased I(Ks). This is the first demonstration that a minK LQT mutation is capable of conferring trafficking defects onto its associated alpha-subunit.