RESUMO
The interaction of the porphyrin derivative H2TCPPSpm4, having spermine pendants in the four meso positions, with the G-quadruplex (GQ) structure formed by the DNA aptamer TGGGAG has been investigated by means of UV, electronic circular dichroism and PAGE studies. The results reported here demonstrate that the porphyrin derivative is capable of stabilizing or destabilizing the higher-ordered structures of parallel GQs, depending on the method used to reach their relative stoichiometry (titration vs. single addition). Noteworthily, when two equivalents of H2TCPPSpm4 were mixed directly with one equivalent of the (TGGGAG)4 GQ to reach a 2 : 1 H2TCPPSpm4 : GQ ratio T1/2 higher than 80 °C was also observed confirming the presence of higher-ordered GQ structures.
Assuntos
Quadruplex G , Porfirinas/química , Espermina/química , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Dicroísmo Circular , Eletroforese em Gel de Campo Pulsado , Oligonucleotídeos/química , Transição de Fase , Espectrofotometria UltravioletaRESUMO
AIMS/HYPOTHESIS: To validate the reported association between rs10494366 in NOS1AP (the gene encoding nitric oxide synthase-1 adaptor protein) and the incidence of type 2 diabetes in calcium channel blocker (CCB) users and to identify additional NOS1AP variants associated with type 2 diabetes risk. METHODS: Data from 9 years of follow-up in 9,221 middle-aged white and 2,724 African-American adults free of diabetes at baseline from the Atherosclerosis Risk in Communities study were analysed. Nineteen NOS1AP variants were examined for associations with incident diabetes and fasting glucose levels stratified by baseline CCB use. RESULTS: Prevalence of CCB use at baseline was 2.7% (n = 247) in whites and 2.3% (n = 72) in African-Americans. Among white CCB users, the G allele of rs10494366 was associated with lower diabetes incidence (HR 0.57, 95% CI 0.35-0.92, p = 0.016). The association was marginally significant after adjusting for age, sex, obesity, smoking, alcohol use, physical activity, hypertension, heart rate and electrocardiographic QT interval (HR 0.63, 95% CI 0.38-1.04, p = 0.052). rs10494366 was associated with lower average fasting glucose during follow-up (p = 0.037). No other variants were associated with diabetes risk in CCB users after multiple-testing correction. No associations were observed between any NOS1AP variant and diabetes development in non-CCB users. NOS1AP variants were not associated with diabetes risk in either African-American CCB users or non-CCB users. CONCLUSIONS/INTERPRETATION: We have independently replicated the association between rs10494366 in NOS1AP and incident diabetes among white CCB users. Further exploration of NOS1AP variants and type 2 diabetes and functional studies of NOS1AP in type 2 diabetes pathology is warranted.
Assuntos
Aterosclerose/genética , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Adulto , Negro ou Afro-Americano , População Negra , Glicemia/metabolismo , Eletrocardiografia/métodos , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Risco , População BrancaRESUMO
We describe here the occurrence of a serotonin syndrome (SS) in a 64-year-old depressed female patient with alcoholic hepatic cirrhosis after treatment with SSRIs. Two weeks after the increase of the dosage of sertraline, the patient developed a full-blown SS, which resolved completely after the discontinuation of the drug. The therapy with citalopram led again to development of milder SS, this time immediately after the increase of the dosage. Our case illustrates the variability of the clinical presentation and the temporal evolution of SS in a patient with preexisting medical illness affecting hepatic metabolism. Reexposure of patients with a history of SS to another serotoninergic drug should be avoided; if necessary, it must be carried out with the utmost caution.
Assuntos
Citalopram/efeitos adversos , Transtorno Depressivo Maior/tratamento farmacológico , Inibidores Seletivos de Recaptação de Serotonina/efeitos adversos , Síndrome da Serotonina/induzido quimicamente , Sertralina/efeitos adversos , Citalopram/uso terapêutico , Feminino , Humanos , Pessoa de Meia-Idade , Recidiva , Sertralina/uso terapêuticoRESUMO
PURPOSE: Patients resected for colorectal cancer are at increased risk for an anastomotic recurrence, for adenomatous polyps and for a metachronous cancer. A regular colonoscopic surveillance in these patients is justified for early detection and potential resection of anastomotic recurrences, new primary cancer and adenomatous polyps. PATIENTS AND METHODS: 322 patients were observed and resected for colorectal cancer between 1970 and 1988, with complete staging agreed to be included in a follow-up program (median follow-up: 105 months). To December 1993 all the patients were submitted to colonoscopy once yearly for the first 5 years and then every 2 years. RESULTS: Anastomotic recurrence was observed in 22 of the 253 patients who underwent resection for rectal or sigmoid adenocarcinoma (8.7%). Sixteen of these patients were submitted to a second curative resection with a median survival of 35 months; the median survival was 6 months in the 6 patients who could not undergo this operation (p = 0.0018). Metachronous adenomas of the residual colon were found in 24 patients (7.4%) and metachronous cancers in 5 (1.5%) at Stage A, according to Dukes' classification. CONCLUSIONS: In patients resected for rectal or sigmoid carcinoma, a sigmoidoscopy should be performed every 6 months for the first 2 years for the early detection of anastomotic recurrences. In all cases, a colonoscopy should be performed every 5 years after surgery to detect metachronous lesions at early stage. Before surgery, a "clean colon" should always be established to detect possible synchronous lesions.
Assuntos
Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Colectomia , Colonoscopia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Adenocarcinoma/prevenção & controle , Adulto , Idoso , Neoplasias Colorretais/prevenção & controle , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Segunda Neoplasia Primária/prevenção & controle , Valor Preditivo dos Testes , Fatores de Tempo , Resultado do TratamentoRESUMO
The transient outward potassium current (I(to)) is an important repolarizing current in the mammalian heart. I(to) is regulated by adrenergic stimulation; however, the effect of agonists on this current, and consequently the action potential duration and profile, is variable. An important source of the variability is the difference in the channel genes that underlie I(to). There are two subfamilies of candidate genes that are likely to encode I(to) in the mammalian heart: Kv4 and Kv1.4; the predominance of either gene is a function of the species, stage of development, and region of the heart. The existence of different isoforms of the Kv4 family (principally Kv4.2 or Kv4.3) further complicates the effect of alpha-adrenergic modulation of cardiac I(to). In the human ventricle, hKv4.3 is the predominant gene underlying I(to). Two splice variants of human Kv4.3 (hKv4.3) are present in the human ventricle; the longer splice variant contains a 19-amino acid insert in the COOH-terminus with a consensus protein kinase C (PKC) site. We used heterologous expression of hKv4.3 splice variants and studies of human ventricular myocytes to demonstrate that alpha-adrenergic modulation of I(to) occurs through a PKC signaling pathway and that only the long splice variant (hKv4.3-L) is modulated via this pathway. Only a single hKv4.3-L monomer in the tetrameric I(to) channel is required to confer sensitivity to phenylephrine (PE). Mutation of the PKC site in hKv4.3-L eliminates alpha-adrenergic modulation of the hKv4.3-encoded current. The similar, albeit less robust, modulation of human ventricular I(to) by PE suggests that hKv4.3-L is expressed in a functional form in the human heart.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Canais de Potássio/metabolismo , Receptores Adrenérgicos alfa 1/genética , Agonistas alfa-Adrenérgicos/farmacologia , Alcaloides , Processamento Alternativo/fisiologia , Benzofenantridinas , Carcinógenos/farmacologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica/fisiologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mutagênese Sítio-Dirigida/fisiologia , Miocárdio/citologia , Fenantridinas/farmacologia , Fenilefrina/farmacologia , Ésteres de Forbol/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Canais de Potássio ShalRESUMO
The permeation pathway of the Na channel is formed by asymmetric loops (P segments) contributed by each of the four domains of the protein. In contrast to the analogous region of K channels, previously we (Yamagishi, T., M. Janecki, E. Marban, and G. Tomaselli. 1997. Biophys. J. 73:195-204) have shown that the P segments do not span the selectivity region, that is, they are accessible only from the extracellular surface. The portion of the P-segment NH(2)-terminal to the selectivity region is referred to as SS1. To explore further the topology and functional role of the SS1 region, 40 amino acids NH(2)-terminal to the selectivity ring (10 in each of the P segments) of the rat skeletal muscle Na channel were substituted by cysteine and expressed in tsA-201 cells. Selected mutants in each domain could be blocked with high affinity by externally applied Cd(2)+ and were resistant to tetrodotoxin as compared with the wild-type channel. None of the externally applied sulfhydryl-specific methanethiosulfonate reagents modified the current through any of the mutant channels. Both R395C and R750C altered ionic selectivity, producing significant increases in K(+) and NH(4)(+) currents. The pattern of side chain accessibility is consistent with a pore helix like that observed in the crystal structure of the bacterial K channel, KcsA. Structure prediction of the Na channel using the program PHDhtm suggests an alpha helix in the SS1 region of each domain channel. We conclude that each of the P segments undergoes a hairpin turn in the permeation pathway, such that amino acids on both sides of the putative selectivity filter line the outer mouth of the pore. Evolutionary conservation of the pore helix motif from bacterial K channels to mammalian Na channels identifies this structure as a critical feature in the architecture of ion selective pores.
Assuntos
Canais de Sódio/genética , Canais de Sódio/fisiologia , Sequência de Aminoácidos/genética , Animais , Cádmio/farmacologia , Eletrofisiologia , Previsões , Humanos , Mesilatos/farmacologia , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Ratos , Canais de Sódio/efeitos dos fármacos , Tetrodotoxina/farmacologiaRESUMO
Recent studies implicate increased cGMP synthesis as a postreceptor contributor to reduced cardiac sympathetic responsiveness. Here we provide the first evidence that modulation of this interaction by cGMP-specific phosphodiesterase PDE5A is also diminished in failing hearts, providing a novel mechanism for blunted beta-adrenergic signaling in this disorder. In normal conscious dogs chronically instrumented for left ventricular pressure-dimension analysis, PDE5A inhibition by EMD82639 had modest basal effects but markedly blunted dobutamine-enhanced systolic and diastolic function. In failing hearts (tachypacing model), however, EMD82639 had negligible effects on either basal or dobutamine-stimulated function. Whole myocardium from failing hearts had 50% lower PDE5A protein expression and 30% less total and EMD92639-inhibitable cGMP-PDE activity. Although corresponding myocyte protein and enzyme activity was similar among groups, the proportion of EMD82639-inhibitable activity was significantly lower in failure cells. Immunohistochemistry confirmed PDE5A expression in both the vasculature and myocytes of normal and failing hearts, but there was loss of z-band localization in failing myocytes that suggested altered intracellular localization. Thus, PDE5A regulation of cGMP in the heart can potently modulate beta-adrenergic stimulation, and alterations in enzyme localization and reduced synthesis may blunt this pathway in cardiac failure, contributing to dampening of the beta-adrenergic response.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/fisiologia , Baixo Débito Cardíaco/enzimologia , Receptores Adrenérgicos beta/fisiologia , Transdução de Sinais , 3',5'-GMP Cíclico Fosfodiesterases/análise , 3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Adenilil Ciclases/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Pressão Sanguínea , Colforsina/farmacologia , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Dobutamina/farmacologia , Cães , Feminino , Coração/efeitos dos fármacos , Hemodinâmica , Imuno-Histoquímica , Masculino , Contração Miocárdica/efeitos dos fármacos , Miocárdio/enzimologia , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/farmacologia , Purinas , Purinonas/farmacologia , Pirazóis/farmacologia , Citrato de Sildenafila , SulfonasRESUMO
mu-Conotoxins (mu-CTX) are potent oligopeptide blockers of sodium channels. The best characterized forms of mu-CTX, GIIIA and GIIIB, have similar primary and three-dimensional structures and comparable potencies (IC(50) approximately 30 nM) for block of wild-type skeletal muscle Na(+) channels. The two toxins are thus considered to be indistinguishable by their target channels. We have found mutations in the domain II pore region (D762K and E765K) that decrease GIIIB blocking affinity approximately 200-fold, but reduce GIIIA affinity by only approximately 4-fold, compared with wild-type channels. Synthetic mu-CTX GIIIA mutants reveal that the critical residue for differential recognition is at position 14, the site of the only charge difference between the two toxin isoforms. Therefore, engineered Na(+) channels, but not wild-type channels, can discriminate between two highly homologous conotoxins. Latent specificity of toxin-channel interactions, such as that revealed here, is a principle worthy of exploitation in the design and construction of improved biosensors.
Assuntos
Conotoxinas/genética , Conotoxinas/farmacologia , Venenos de Moluscos/genética , Venenos de Moluscos/farmacologia , Mutagênese Sítio-Dirigida , Bloqueadores dos Canais de Sódio , Canais de Sódio/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Arginina/genética , Ácido Aspártico/genética , Conotoxinas/biossíntese , Conotoxinas/metabolismo , Ácido Glutâmico/genética , Glutamina/genética , Lisina/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Dados de Sequência Molecular , Venenos de Moluscos/biossíntese , Venenos de Moluscos/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica/genética , Ratos , Canais de Sódio/biossíntese , Canais de Sódio/metabolismo , TermodinâmicaRESUMO
BACKGROUND: Colorectal cancer is one of the leading causes of death from cancer in Western countries. Removal of adenomas is based on the assumption that it could lead to a reduction in the incidence of colorectal cancer, as demonstrated by the National Polyp Study in the USA. A critical issue is whether the benefit observed in clinical trials can also be observed in standard clinical practice. To address the issue, a multicentre Italian collaborative study was organised. METHODS: The study cohort comprised 1693 subjects of both sexes, aged 40-69 years, enrolled between 1980 and 1987 following a total colon examination (TCE) (that is, total colonoscopy or colonoscopy and double contrast barium enema), with removal of at least one adenoma larger than 5 mm in diameter. Exclusion criteria were genetic syndromes, previous adenomas or colorectal cancer, previous colonic resection, inflammatory bowel disease, or sessile adenomas more than 3 cm in diameter. Follow up ended in December 1996 by TCE or telephone interview, and review of the medical records, clinical files, or death certificates. Incidence ratios for colorectal cancer were compared with expected age and sex specific incidences in the Italian general population. RESULTS: Follow up data were obtained for 97.3% of cases for a total of 14 211 person/years. Mean follow up was 10.5 years. Six colorectal cancer cases (four in males, two in females) at various stages were ascertained (one at 29 months, two at five years, one at seven years, one at eight years, and one at 10 years from the index examination). The number of cancers expected in the reference population was 17.7 for an incidence ratio of 0.34 (confidence interval 0.23-0.63; p<0.01). CONCLUSIONS: Colonoscopic polypectomy substantially reduced the incidence of colorectal cancer in the cohort compared with that expected in the general population. These results are of particular relevance considering that those with adenomas are at increased risk of colorectal cancer and that this retrospective study was performed on data obtained in standard clinical practice. This observation strengthens the concept of effective population screening in view of the fact that adenomatous polyps are the most frequent neoplastic outcome of screening and their removal is associated with a decrease in the incidence of colorectal cancer.
Assuntos
Adenoma/cirurgia , Pólipos do Colo/cirurgia , Colonoscopia/métodos , Neoplasias Colorretais/prevenção & controle , Programas de Rastreamento/métodos , Adulto , Idoso , Estudos de Coortes , Colonoscopia/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The purpose of this prospective randomized study was to compare the safety and efficacy of the cephalic approach versus a contrast-guided extrathoracic approach for placement of endocardial leads. Despite an increased incidence of lead fracture, the intrathoracic subclavian approach remains the dominant approach for placement of pacemaker and implantable defibrillator leads. Although this complication can be prevented by lead placement in the cephalic vein or by lead placement in the extrathoracic subclavian or axillary vein, these approaches have not gained acceptance. A total of 200 patients were randomized to undergo placement of pacemaker or implantable defibrillator leads via the contrast-guided extrathoracic subclavian vein approach or the cephalic approach. Lead placement was accomplished in 99 of the 100 patients randomized to the extrathoracic subclavian vein approach as compared to 64 of 100 patients using the cephalic approach. In addition to a higher initial success rate, the extrathoracic subclavian vein medial approach was determined to be preferable as evidenced by a shorter procedure time and less blood loss. There was no difference in the incidence of complications. In conclusion, these results demonstrate that lead placement in the extrathoracic subclavian vein guided by contrast venography is effective and safe. It was also associated with no increased risk of complications as compared with the cephalic approach. These findings suggest that the contrast-guided approach to the extrathoracic portion of the subclavian vein should be considered as an alternative to the cephalic approach.
Assuntos
Desfibriladores Implantáveis , Eletrodos Implantados , Marca-Passo Artificial , Flebografia , Idoso , Idoso de 80 Anos ou mais , Análise de Falha de Equipamento , Segurança de Equipamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculos Peitorais/irrigação sanguínea , Estudos Prospectivos , Veia Subclávia/diagnóstico por imagem , Resultado do TratamentoRESUMO
Saxitoxin (STX) and its derivatives are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. During algal blooms ("red tides"), shellfish accumulate saxitoxins leading to paralytic shellfish poisoning (PSP) in human consumers. PSP is a consequence of the high-affinity block of voltage-dependent Na channels in neuronal and muscle cells. PSP poses a significant public health threat and an enormous economic challenge to the shellfish industry worldwide. The standard screening method for marine toxins is the mouse mortality bioassay that is ethically problematic, costly and time-consuming. We report here an alternative, functional assay based on electrical recordings in cultured cells stably expressing a PSP target molecule, the STX-sensitive skeletal muscle Na channel. STX-equivalent concentration in the extracts was calibrated by comparison with purified STX, yielding a highly significant correlation (R=0.95; N=30) between electrophysiological determinations and the values obtained by conventional methods. This simple, economical, and reproducible assay obviates the need to sacrifice millions of animals in mandatory paralytic shellfish toxin screening programs.
Assuntos
Toxinas Marinhas/toxicidade , Paralisia/induzido quimicamente , Saxitoxina/toxicidade , Frutos do Mar/análise , Bloqueadores dos Canais de Sódio , Animais , Ligação Competitiva/efeitos dos fármacos , Linhagem Celular , Eletrofisiologia , Humanos , Camundongos , Técnicas de Patch-Clamp , Proteínas Recombinantes , Reprodutibilidade dos Testes , Canais de Sódio/genéticaRESUMO
mu-Conotoxins (mu-CTXs) specifically inhibit Na(+) flux by occluding the pore of voltage-gated Na(+) channels. Although the three-dimensional structures of mu-CTXs are well defined, the molecular configuration of the channel receptor is much less certain; even the fundamental question of whether the four homologous Na(+) channel domains are arranged in a clockwise or counter-clockwise configuration remains unanswered. Residues Asp(762) and Glu(765) from domain II and Asp(1241) from domain III of rat skeletal muscle Na(+) channels are known to be critical for mu-CTX binding. We probed toxin-channel interactions by determining the potency of block of wild-type, D762K, E765K, and D1241C channels by wild-type and point-mutated mu-CTXs (R1A, Q14D, K11A, K16A, and R19A). Individual interaction energies for different toxin-channel pairs were quantified from the half-blocking concentrations using mutant cycle analysis. We find that Asp(762) and Glu(765) interact strongly with Gln(14) and Arg(19) but not Arg(1) and that Asp(1241) is tightly coupled to Lys(16) but not Arg(1) or Lys(11). These newly identified toxin-channel interactions within adjacent domains, interpreted in light of the known asymmetric toxin structure, fix the orientation of the toxin with respect to the channel and reveal that the four internal domains of Na(+) channels are arranged in a clockwise configuration as viewed from the extracellular surface.
Assuntos
Conotoxinas , Canais de Sódio/química , Animais , Linhagem Celular , Mutação Puntual , Conformação Proteica , Canais de Sódio/genética , Canais de Sódio/metabolismoRESUMO
Heart failure (HF) is a complex disease that presents a major public health challenge to Western society. The prevalence of HF increases with age in the elderly population, and the societal disease burden will increase with prolongation of life expectancy. HF is initially characterized by an adaptive increase of neurohumoral activation to compensate for reduction of cardiac output. This leads to a combination of neurohumoral activation and mechanical stress in the failing heart that trigger a cascade of maladaptive electrical and structural events that impair both the systolic and diastolic function of the heart.
Assuntos
Insuficiência Cardíaca/fisiopatologia , Canais Iônicos/fisiologia , Disfunção Ventricular Esquerda/fisiopatologia , Remodelação Ventricular/fisiologia , Arritmias Cardíacas/fisiopatologia , Eletrofisiologia , Humanos , Transdução de SinaisRESUMO
The KvLQT1 gene encodes a voltage-gated potassium channel. Mutations in KvLQT1 underlie the dominantly transmitted Ward-Romano long QT syndrome, which causes cardiac arrhythmia, and the recessively transmitted Jervell and Lange-Nielsen syndrome, which causes both cardiac arrhythmia and congenital deafness. KvLQT1 is also disrupted by balanced germline chromosomal rearrangements in patients with Beckwith-Wiedemann syndrome (BWS), which causes prenatal overgrowth and cancer. Because of the diverse human disorders and organ systems affected by this gene, we developed an animal model by inactivating the murine Kvlqt1. No electrocardiographic abnormalities were observed. However, homozygous mice exhibited complete deafness, as well as circular movement and repetitive falling, suggesting imbalance. Histochemical study revealed severe anatomic disruption of the cochlear and vestibular end organs, suggesting that Kvlqt1 is essential for normal development of the inner ear. Surprisingly, homozygous mice also displayed threefold enlargement by weight of the stomach resulting from mucous neck cell hyperplasia. Finally, there were no features of BWS, suggesting that Kvlqt1 is not responsible for BWS.
Assuntos
Surdez/genética , Hiperplasia/genética , Síndrome do QT Longo/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/deficiência , Canais de Potássio/metabolismo , Estômago/patologia , Animais , Tronco Encefálico/fisiologia , Cóclea/patologia , Cóclea/fisiopatologia , Surdez/fisiopatologia , Modelos Animais de Doenças , Orelha Interna/patologia , Orelha Interna/fisiopatologia , Eletrocardiografia , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Histocitoquímica , Humanos , Hiperplasia/patologia , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Tamanho do Órgão , Fenótipo , Canais de Potássio/genéticaRESUMO
Potassium channels encoded by HERG underlie I:(Kr), a sensitive target for most class III antiarrhythmic drugs, including methanesulfonanilides such as Dd-sotalol. Recently it was shown that these drugs are trapped in the channel as it closes during hyperpolarization. At the same time, HERG channels rapidly open and inactivate when depolarized, and methanesulfonanilide block is known to develop in a use-dependent manner, suggesting a potential role for inactivation in drug binding. However, the role of HERG inactivation in class III drug action is uncertain: pore mutations that remove inactivation reduce block, yet many of these mutations also modify the channel permeation properties and could alter drug affinity through gating-independent mechanisms. In the present study, we identify a definitive role for inactivation gating in Dd-sotalol block of HERG, using interventions complementary to mutagenesis. These interventions (addition of extracellular Cd(2+), removal of extracellular Na(+)) modify the voltage dependence of inactivation but not activation. In normal extracellular solutions, block of HERG current by 300 micromol/L Dd-sotalol reached 80% after a 10-minute period of repetitive depolarization to +20 mV. Maneuvers that impeded steady-state inactivation also reduced Dd-sotalol block of HERG: 100 micromol/L Cd(2+) reduced steady-state block to 55% at +20 mV (P:<0.05); removing extracellular Na(+) reduced block to 44% (P:<0.05). An inactivation-disabling mutation (G628C-S631C) reduced Dd-sotalol block to only 11% (P:<0.05 versus wild type). However, increasing the rate of channel inactivation by depolarizing to +60 mV reduced Dd-sotalol block to 49% (P:<0.05 versus +20 mV), suggesting that the drug does not primarily bind to the inactivated state. Coexpression of MiRP1 with HERG had no effect on inactivation gating and did not modify Dd-sotalol block. We postulate that Dd-sotalol accesses its receptor in the open pore, and the drug-receptor interaction is then stabilized by inactivation. Whereas deactivation traps the bound methanesulfonanilide during hyperpolarization, we propose that HERG inactivation stabilizes the drug-receptor interaction during membrane depolarization.
Assuntos
Antiarrítmicos/farmacologia , Proteínas de Transporte de Cátions , Ativação do Canal Iônico/efeitos dos fármacos , Síndrome do QT Longo/metabolismo , Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Sotalol/farmacologia , Animais , Células CHO , Cádmio/farmacologia , Cricetinae , Canais de Potássio Éter-A-Go-Go , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Sódio/metabolismo , TransfecçãoRESUMO
The Kv4.3-encoded current (I:(Kv4.3)) has been identified as the major component of the voltage-dependent Ca(2+)-independent transient outward current (I:(to1)) in human and canine ventricular cells. Experimental evidence supports a correlation between I:(to1) density and prominence of the phase 1 notch; however, the role of I:(to1) in modulating action potential duration (APD) remains unclear. To help resolve this role, Markov state models of the human and canine Kv4.3- and Kv1.4-encoded currents at 35 degrees C are developed on the basis of experimental measurements. A model of canine I:(to1) is formulated as the combination of these Kv4.3 and Kv1.4 currents and is incorporated into an existing canine ventricular myocyte model. Simulations demonstrate strong coupling between L-type Ca(2+) current and I:(Kv4.3) and predict a bimodal relationship between I:(Kv4.3) density and APD whereby perturbations in I:(Kv4.3) density may produce either prolongation or shortening of APD, depending on baseline I:(to1) current level.
Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Modelos Cardiovasculares , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Cães , Genes Reporter , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Humanos , Internet , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Canal de Potássio Kv1.4 , Cadeias de Markov , Miocárdio/citologia , Miocárdio/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/genética , Tempo de Reação , Canais de Potássio Shal , Interface Usuário-ComputadorRESUMO
Voltage-gated sodium (Na(+)) channels are a fundamental target for modulating excitability in neuronal and muscle cells. When depolarized, Na(+) channels may gradually enter long-lived, slow-inactivated conformational states, causing a cumulative loss of function. Although the structural motifs that underlie transient, depolarization-induced Na(+) channel conformational states are increasingly recognized, the conformational changes responsible for more sustained forms of inactivation are unresolved. Recent studies have shown that slow inactivation components exhibiting a range of kinetic behavior (from tens of milliseconds to seconds) are modified by mutations in the outer pore P-segments. We examined the state-dependent accessibility of an engineered cysteine in the domain III, P-segment (F1236C; rat skeletal muscle) to methanethiosulfonate-ethylammonium (MTSEA) using whole-cell current recordings in HEK 293 cells. F1236C was reactive with MTSEA applied from outside, but not inside the cell, and modification was markedly increased by depolarization. Depolarized F1236C channels exhibited both intermediate (I(M); tau approximately 30 ms) and slower (I(S); tau approximately 2 s) kinetic components of slow inactivation. Trains of brief, 5-ms depolarizations, which did not induce slow inactivation, produced more rapid modification than did longer (100 ms or 6 s) pulse widths, suggesting both the I(M) and I(S) kinetic components inhibit depolarization-induced MTSEA accessibility of the cysteine side chain. Lidocaine inhibited the depolarization-dependent sulfhydryl modification induced by sustained (100 ms) depolarizations, but not by brief (5 ms) depolarizations. We conclude that competing forces influence the depolarization-dependent modification of the cysteine side chain: conformational changes associated with brief periods of depolarization enhance accessibility, whereas slow inactivation tends to inhibit the side chain accessibility. The findings suggest that slow Na(+) channel inactivation and use-dependent lidocaine action are linked to a structural rearrangement in the outer pore.
Assuntos
Anestésicos Locais/farmacologia , Lidocaína/farmacologia , Canais de Sódio/fisiologia , Animais , Cisteína/genética , Eletrofisiologia , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/farmacologia , Indicadores e Reagentes/farmacologia , Cinética , Potenciais da Membrana/fisiologia , Músculo Esquelético/fisiologia , Mutagênese Sítio-Dirigida , RatosRESUMO
The increased risk of sudden cardiac death in patients with myocardial hypertrophy and heart failure is the result of remodeling that occurs in both the myocyte and interstitial compartments of the heart. Action potential prolongation is a hallmark of hypertrophied and failing myocardium and is a consequence of differential expression and function of membrane currents and transporters. Functional downregulation of K currents in the ventricle is a recurring theme in hypertrophy and failure; the reduction in the density of the transient outward current (I(to)) is the most consistent observation, whereas data on the density of the inward (I(K1)) and the delayed rectifier (I(K)) currents are more contradictory. The altered intracellular Ca handling of the myopathic hearts prolongs the decay of the L-type Ca current and favors extrusion of cytosolic Ca2+ via the Na+-Ca2+ exchanger. The interaction between such altered membrane currents and a changed neurohumoral milieu creates a substrate that is highly susceptible to potentially lethal ventricular arrhythmias.
Assuntos
Arritmias Cardíacas/etiologia , Cardiomiopatias/complicações , Cardiomiopatias/metabolismo , Sistema de Condução Cardíaco/fisiologia , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/metabolismo , Biologia Molecular , Potenciais de Ação , Canais de Cálcio/fisiologia , Morte Súbita Cardíaca/etiologia , Eletrofisiologia , Homeostase , Humanos , Fatores de Risco , Remodelação Ventricular/fisiologiaRESUMO
INTRODUCTION: The purpose of this study was to analyze the pattern of initiation of sustained ventricular arrhythmias in patients with varying types of underlying structural heart disease. METHODS AND RESULTS: The study group consisted of 90 patients with an implantable cardioverter defibrillator. Cardiovascular diagnoses included coronary artery disease in 64 patients (71%). The patients were divided into four groups based on the type and severity of structural heart disease. Two hundred sixty episodes of sustained ventricular arrhythmias were analyzed. The mean coupling interval of the initiating beat of all ventricular arrhythmias was 523 +/- 171 msec. The coupling interval of the initiating beat was longer in patients with impaired ventricular function, particularly those with nonischemic dilated cardiomyopathy. The prematurity index was similar regardless of the type of underlying structural heart disease. However, the prematurity index was shorter in patients with polymorphic ventricular tachycardia (VT) compared to those with monomorphic VT. A pause was observed more commonly before the onset of polymorphic VT/ventricular fibrillation than sustained monomorphic VT. Two hundred twenty-two (85%) of the arrhythmia episodes were initiated by a late-coupled premature beat, 33 (13%) were initiated by an early-coupled premature beat, and 5 episodes (2%) were initiated with a short-long-short sequence. The pattern of initiation of the ventricular arrhythmias was similar in all patient groups and for both monomorphic and polymorphic tachycardias. CONCLUSION: These findings demonstrate that sustained ventricular arrhythmias typically are initiated by late-coupled ventricular premature depolarizations, regardless of the type or severity of underlying structural heart disease or resultant arrhythmia.