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1.
JCI Insight ; 9(11)2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38855866

RESUMO

TANGO2-deficiency disorder (TDD) is an autosomal-recessive genetic disease caused by biallelic loss-of-function variants in the TANGO2 gene. TDD-associated cardiac arrhythmias are recalcitrant to standard antiarrhythmic medications and constitute the leading cause of death. Disease modeling for TDD has been primarily carried out using human dermal fibroblast and, more recently, in Drosophila by multiple research groups. No human cardiomyocyte system has been reported, which greatly hinders the investigation and understanding of TDD-associated arrhythmias. Here, we established potentially novel patient-derived induced pluripotent stem cell differentiated cardiomyocyte (iPSC-CM) models that recapitulate key electrophysiological abnormalities in TDD. These electrophysiological abnormalities were rescued in iPSC-CMs with either adenoviral expression of WT-TANGO2 or correction of the pathogenic variant using CRISPR editing. Our natural history study in patients with TDD suggests that the intake of multivitamin/B complex greatly diminished the risk of cardiac crises in patients with TDD. In agreement with the clinical findings, we demonstrated that high-dose folate (vitamin B9) virtually abolishes arrhythmias in TDD iPSC-CMs and that folate's effect was blocked by the dihydrofolate reductase inhibitor methotrexate, supporting the need for intracellular folate to mediate antiarrhythmic effects. In summary, data from TDD iPSC-CM models together with clinical observations support the use of B vitamins to mitigate cardiac crises in patients with TDD, providing potentially life-saving treatment strategies during life-threatening events.


Assuntos
Arritmias Cardíacas , Ácido Fólico , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ácido Fólico/metabolismo , Ácido Fólico/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/genética , Masculino , Feminino , Criança
2.
Circ Res ; 133(1): e1-e16, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37154033

RESUMO

BACKGROUND: Atrial fibrillation (AF), the most common arrhythmia, is associated with the downregulation of FKBP5 (encoding FKBP5 [FK506 binding protein 5]). However, the function of FKBP5 in the heart remains unknown. Here, we elucidate the consequences of cardiomyocyte-restricted loss of FKBP5 on cardiac function and AF development and study the underlying mechanisms. METHODS: Right atrial samples from patients with AF were used to assess the protein levels of FKBP5. A cardiomyocyte-specific FKBP5 knockdown mouse model was established by crossbreeding Fkbp5flox/flox mice with Myh6MerCreMer/+ mice. Cardiac function and AF inducibility were assessed by echocardiography and programmed intracardiac stimulation. Histology, optical mapping, cellular electrophysiology, and biochemistry were employed to elucidate the proarrhythmic mechanisms due to loss of cardiomyocyte FKBP5. RESULTS: FKBP5 protein levels were lower in the atrial lysates of patients with paroxysmal AF or long-lasting persistent (chronic) AF. Cardiomyocyte-specific knockdown mice exhibited increased AF inducibility and duration compared with control mice. Enhanced AF susceptibility in cardiomyocyte-specific knockdown mice was associated with the development of action potential alternans and spontaneous Ca2+ waves, and increased protein levels and activity of the NCX1 (Na+/Ca2+-exchanger 1), mimicking the cellular phenotype of chronic AF patients. FKBP5-deficiency enhanced transcription of Slc8a1 (encoding NCX1) via transcription factor hypoxia-inducible factor 1α. In vitro studies revealed that FKBP5 negatively modulated the protein levels of hypoxia-inducible factor 1α by competitively interacting with heat-shock protein 90. Injections of the heat-shock protein 90 inhibitor 17-AAG normalized protein levels of hypoxia-inducible factor 1α and NCX1 and reduced AF susceptibility in cardiomyocyte-specific knockdown mice. Furthermore, the atrial cardiomyocyte-selective knockdown of FKBP5 was sufficient to enhance AF arrhythmogenesis. CONCLUSIONS: This is the first study to demonstrate a role for the FKBP5-deficiency in atrial arrhythmogenesis and to establish FKBP5 as a negative regulator of hypoxia-inducible factor 1α in cardiomyocytes. Our results identify a potential molecular mechanism for the proarrhythmic NCX1 upregulation in chronic AF patients.


Assuntos
Fibrilação Atrial , Camundongos , Animais , Fibrilação Atrial/metabolismo , Regulação para Baixo , Miócitos Cardíacos/metabolismo , Hipóxia/metabolismo , Proteínas de Choque Térmico/metabolismo
3.
Am J Physiol Lung Cell Mol Physiol ; 324(4): L468-L479, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36809074

RESUMO

Propylene glycol (PG) is a common delivery vehicle for nicotine and flavorings in e-cigarette (e-cig) liquids and is largely considered safe for ingestion. However, little is known about its effects as an e-cig aerosol on the airway. Here, we investigated whether pure PG e-cig aerosols in realistic daily amounts impact parameters of mucociliary function and airway inflammation in a large animal model (sheep) in vivo and primary human bronchial epithelial cells (HBECs) in vitro. Five-day exposure of sheep to e-cig aerosols of 100% PG increased mucus concentrations (% mucus solids) of tracheal secretions. PG e-cig aerosols further increased the activity of matrix metalloproteinase-9 (MMP-9) in tracheal secretions. In vitro exposure of HBECs to e-cig aerosols of 100% PG decreased ciliary beating and increased mucus concentrations. PG e-cig aerosols further reduced the activity of large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. We show here for the first time that PG can be metabolized to methylglyoxal (MGO) in airway epithelia. PG e-cig aerosols increased levels of MGO and MGO alone reduced BK activity. Patch-clamp experiments suggest that MGO can disrupt the interaction between the major pore-forming BK subunit human Slo1 (hSlo1) and the gamma regulatory subunit LRRC26. PG exposures also caused a significant increase in mRNA expression levels of MMP9 and interleukin 1 beta (IL1B). Taken together, these data show that PG e-cig aerosols cause mucus hyperconcentration in sheep in vivo and HBECs in vitro, likely by disrupting the function of BK channels important for airway hydration.


Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Humanos , Animais , Ovinos , Canais de Potássio Ativados por Cálcio de Condutância Alta , Óxido de Magnésio , Aerossóis , Propilenoglicóis
4.
Sci Adv ; 8(50): eabq5772, 2022 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-36516264

RESUMO

BK channels uniquely integrate voltage and calcium signaling in diverse cell types through allosteric activation of their K+-conducting pore by structurally distinct V and Ca2+ sensor domains. Here, we define mechanisms and interaction pathways that link V sensors to the pore by analyzing effects on allosteric coupling of point mutations in the context of Slo1 BK channel structure. A gating lever, mediated by S4/S5 segment interaction within the transmembrane domain, rotates to engage and stabilize the open conformation of the S6 inner pore helix upon V sensor activation. In addition, an indirect pathway, mediated by the carboxyl-terminal cytosolic domain (CTD) and C-linker that connects the CTD to S6, stabilizes the closed conformation when V sensors are at rest. Unexpectedly, this mechanism, which bypasses the covalent connections of C-linker to CTD and pore, also transduces Ca2+-dependent coupling in a manner that is completely nonadditive with voltage, analogous to the function of a digital logic (OR) gate.

5.
Sci Rep ; 10(1): 4922, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188864

RESUMO

Early work in rodents highlighted the gut microbiota's importance in metabolic disease, including Type II Diabetes Mellitus (T2DM) and obesity. Glucagon-like peptide-1 (GLP-1), an incretin secreted by L-cells lining the gastrointestinal epithelium, has important functions: promoting insulin secretion, insulin sensitivity, and ß-cell mass, while inhibiting gastric emptying and appetite. We set out to identify microbial strains with GLP-1 stimulatory activity as potential metabolic disease therapeutics. Over 1500 human-derived strains were isolated from healthy individuals and screened for GLP-1 modulation by incubating bacterial cell-free supernatants with NCI H716 L-cells. Approximately 45 strains capable of increasing GLP-1 were discovered. All GLP-1 positive strains were identified as Staphylococcus epidermidis by 16S rRNA sequencing. Mass spectrometry analysis identified a 3 kDa peptide, Hld (delta-toxin), present in GLP-1 positive supernatants but absent in GLP-1 neutral supernatants. Studies in NCI-H716 cells and human jejunal enteroids engineered to make more enteroendocrine cells demonstrated that Hld alone is sufficient to enhance GLP-1 secretion. When administered in high-fat-fed mice, Hld-producing S. epidermidis significantly reduced markers associated with obesity and T2DM. Further characterization of Hld suggests GLP-1 stimulatory action of Hld occurs via calcium signaling. The presented results identify a novel host-microbe interaction which may ultimately lead to the development of a microbial peptide-based therapeutic for metabolic disease.


Assuntos
Proteínas de Bactérias , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Doenças Metabólicas/metabolismo , Peptídeos/farmacologia , Animais , Proteínas de Bactérias/química , Biomarcadores , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Relação Dose-Resposta a Droga , Feminino , Peptídeo 1 Semelhante ao Glucagon/genética , Humanos , Espaço Intracelular , Doenças Metabólicas/etiologia , Camundongos , Peptídeos/química
6.
Am J Respir Crit Care Med ; 201(3): 313-324, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31613648

RESUMO

Rationale: Despite therapeutic progress in treating cystic fibrosis (CF) airway disease, airway inflammation with associated mucociliary dysfunction remains largely unaddressed. Inflammation reduces the activity of apically expressed large-conductance Ca2+-activated and voltage-dependent K+ (BK) channels, critical for mucociliary function in the absence of CFTR (CF transmembrane conductance regulator).Objectives: To test losartan as an antiinflammatory therapy in CF using CF human bronchial epithelial cells and an ovine model of CF-like airway disease.Methods: Losartan's antiinflammatory effectiveness to rescue BK activity and thus mucociliary function was tested in vitro using primary, fully redifferentiated human airway epithelial cells homozygous for F508del and in vivo using a previously validated, now expanded pharmacologic sheep model of CF-like, inflammation-associated mucociliary dysfunction.Measurements and Main Results: Nasal scrapings from patients with CF showed that neutrophilic inflammation correlated with reduced expression of LRRC26 (leucine rich repeat containing 26), the γ subunit mandatory for BK function in the airways. TGF-ß1 (transforming growth factor ß1), downstream of neutrophil elastase, decreased mucociliary parameters in vitro. These were rescued by losartan at concentrations achieved by nebulization in the airway and oral application in the bloodstream: BK dysfunction recovered acutely and over time (the latter via an increase in LRRC26 expression), ciliary beat frequency and airway surface liquid volume improved, and mucus hyperconcentration and cellular inflammation decreased. These effects did not depend on angiotensin receptor blockade. Expanding on a validated and published nongenetic, CF-like sheep model, ewes inhaled CFTRinh172 and neutrophil elastase for 3 days, which resulted in prolonged tracheal mucus velocity reduction, mucus hyperconcentration, and increased TGF-ß1. Nebulized losartan rescued both mucus transport and mucus hyperconcentration and reduced TGF-ß1.Conclusions: Losartan effectively reversed CF- and inflammation-associated mucociliary dysfunction, independent of its angiotensin receptor blockade.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Fibrose Cística/fisiopatologia , Losartan/farmacologia , Depuração Mucociliar/efeitos dos fármacos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Brônquios/citologia , Células Cultivadas , Fibrose Cística/tratamento farmacológico , Modelos Animais de Doenças , Células Epiteliais , Feminino , Humanos , Inflamação/fisiopatologia , Losartan/uso terapêutico , Ovinos
7.
Cell Death Dis ; 7(10): e2426, 2016 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-27763639

RESUMO

Myoblasts are mononucleated precursors of myofibers; they persist in mature skeletal muscles for growth and regeneration post injury. During myotonic dystrophy type 1 (DM1), a complex autosomal-dominant neuromuscular disease, the differentiation of skeletal myoblasts into functional myotubes is impaired, resulting in muscle wasting and weakness. The mechanisms leading to this altered differentiation are not fully understood. Here, we demonstrate that the calcium- and voltage-dependent potassium channel, KCa1.1 (BK, Slo1, KCNMA1), regulates myoblast proliferation, migration, and fusion. We also show a loss of plasma membrane expression of the pore-forming α subunit of KCa1.1 in DM1 myoblasts. Inhibiting the function of KCa1.1 in healthy myoblasts induced an increase in cytosolic calcium levels and altered nuclear factor kappa B (NFκB) levels without affecting cell survival. In these normal cells, KCa1.1 block resulted in enhanced proliferation and decreased matrix metalloproteinase secretion, migration, and myotube fusion, phenotypes all observed in DM1 myoblasts and associated with disease pathogenesis. In contrast, introducing functional KCa1.1 α-subunits into DM1 myoblasts normalized their proliferation and rescued expression of the late myogenic marker Mef2. Our results identify KCa1.1 channels as crucial regulators of skeletal myogenesis and suggest these channels as novel therapeutic targets in DM1.


Assuntos
Diferenciação Celular , Movimento Celular , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Cálcio/metabolismo , Fusão Celular , Membrana Celular/metabolismo , Proliferação de Células , Células Cultivadas , Humanos , Espaço Intracelular/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Distrofia Miotônica/patologia , NF-kappa B/metabolismo
8.
Arthritis Rheumatol ; 67(1): 96-106, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25252152

RESUMO

OBJECTIVE: Fibroblast-like synoviocytes (FLS) participate in joint inflammation and damage in rheumatoid arthritis (RA) and its animal models. The purpose of this study was to define the importance of KCa1.1 (BK, Maxi-K, Slo1, KCNMA1) channel expression and function in FLS and to establish these channels as potential new targets for RA therapy. METHODS: We compared KCa1.1 expression levels in FLS from rats with pristane-induced arthritis (PIA) and in FLS from healthy rats. We then used ex vivo functional assays combined with small interfering RNA-induced knockdown, overexpression, and functional modulation of KCa1.1 in PIA FLS. Finally, we determined the effectiveness of modulating KCa1.1 in 2 rat models of RA, moderate PIA and severe collagen-induced arthritis (CIA). RESULTS: We found that PIA FLS expressed the KCa1.1 channel as their major potassium channel, as has been found in FLS from patients with RA. In contrast, FLS from healthy rats expressed fewer of these channels. Inhibiting the function or expression of KCa1.1 ex vivo reduced proliferation and invasive properties of, as well as protease production by, PIA FLS, whereas opening native KCa1.1 or overexpressing the channel enhanced the invasiveness of both FLS from rats with PIA and FLS from healthy rats. Treatment with a KCa1.1 channel blocker at the onset of clinical signs stopped disease progression in the PIA and CIA models, reduced joint and bone damage, and inhibited FLS invasiveness and proliferation. CONCLUSION: Our results demonstrate a critical role of KCa1.1 channels in the regulation of FLS invasiveness and suggest that KCa1.1 channels represent potential therapeutic targets in RA.


Assuntos
Artrite Reumatoide/patologia , Artrite Reumatoide/prevenção & controle , Movimento Celular/fisiologia , Fibroblastos/patologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Membrana Sinovial/patologia , Animais , Artrite Experimental/patologia , Artrite Experimental/fisiopatologia , Artrite Reumatoide/induzido quimicamente , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Feminino , Fibroblastos/fisiologia , Indóis/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Metaloproteinase 2 da Matriz/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Membrana Sinovial/fisiopatologia , Terpenos/efeitos adversos
9.
Circulation ; 129(12): 1276-1285, 2014 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-24398018

RESUMO

BACKGROUND: The progression of atrial fibrillation (AF) from paroxysmal to persistent forms remains a major clinical challenge. Abnormal sarcoplasmic reticulum (SR) Ca(2+) leak via the ryanodine receptor type 2 (RyR2) has been observed as a source of ectopic activity in various AF models. However, its potential role in progression to long-lasting spontaneous AF (sAF) has never been tested. This study was designed to test the hypothesis that enhanced RyR2-mediated Ca(2+) release underlies the development of a substrate for sAF and to elucidate the underlying mechanisms. METHODS AND RESULTS: CREM-IbΔC-X transgenic (CREM) mice developed age-dependent progression from spontaneous atrial ectopy to paroxysmal and eventually long-lasting AF. The development of sAF in CREM mice was preceded by enhanced diastolic Ca(2+) release, atrial enlargement, and marked conduction abnormalities. Genetic inhibition of Ca(2+)/calmodulin-dependent protein kinase II-mediated RyR2-S2814 phosphorylation in CREM mice normalized open probability of RyR2 channels and SR Ca(2+) release, delayed the development of spontaneous atrial ectopy, fully prevented sAF, suppressed atrial dilation, and forestalled atrial conduction abnormalities. Hyperactive RyR2 channels directly stimulated the Ca(2+)-dependent hypertrophic pathway nuclear factor of activated T cell/Rcan1-4, suggesting a role for the nuclear factor of activated T cell/Rcan1-4 system in the development of a substrate for long-lasting AF in CREM mice. CONCLUSIONS: RyR2-mediated SR Ca(2+) leak directly underlies the development of a substrate for sAF in CREM mice, the first demonstration of a molecular mechanism underlying AF progression and sAF substrate development in an experimental model. Our work demonstrates that the role of abnormal diastolic Ca(2+) release in AF may not be restricted to the generation of atrial ectopy but extends to the development of atrial remodeling underlying the AF substrate.


Assuntos
Fibrilação Atrial/metabolismo , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Fatores Etários , Animais , Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo
10.
J Gen Physiol ; 140(6): 625-34, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23183698

RESUMO

Large conductance calcium- and voltage-dependent BK potassium channels (aka BK(Ca), MaxiK, Slo1, KCa1.1, and KCNMA1) are expressed in a wide variety of tissues throughout the body and are activated by both intracellular Ca(2+) and membrane depolarization. Owing to these properties, BK channels participate in diverse physiological processes from electrical excitability in neurons and secretory cells, and regulation of smooth muscle tone to tuning of auditory hair cells (Vergara et al., 1998; Ghatta et al., 2006). The response to voltage and Ca(2+) allows BK channels to integrate electrical and calcium signaling, which is central to their physiological role. Understanding how BK and other multimodal channels are regulated by and integrate diverse stimuli is not only physiologically important but also relevant to the topic of conformational coupling. As a voltage- and ligand-dependent channel, BK channels contain both voltage-sensor and ligand-binding domains as well as a gate to regulate the flow of K(+) through the pore. Coupling of conformational changes in one domain to another provides the basis for transducing voltage and ligand binding into channel opening and, therefore, defines, together with the functional properties of the gate and sensors, the signal transduction properties of the channel. The goal of this perspective is to provide an overview on the role and molecular basis of conformational coupling between functional domains in BK channels and outline some of the questions that remain to be answered.


Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Potenciais da Membrana/fisiologia , Conformação Proteica
11.
J Biol Chem ; 287(6): 4014-22, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22074915

RESUMO

Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA). Potassium channels have regulatory roles in many cell functions. We have identified the calcium- and voltage-gated KCa1.1 channel (BK, Maxi-K, Slo1, KCNMA1) as the major potassium channel expressed at the plasma membrane of FLS isolated from patients with RA (RA-FLS). We further show that blocking this channel perturbs the calcium homeostasis of the cells and inhibits the proliferation, production of VEGF, IL-8, and pro-MMP-2, and migration and invasion of RA-FLS. Our findings indicate a regulatory role of KCa1.1 channels in RA-FLS function and suggest this channel as a potential target for the treatment of RA.


Assuntos
Membrana Celular/metabolismo , Regulação da Expressão Gênica , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/biossíntese , Febre Reumática/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Cálcio/metabolismo , Membrana Celular/patologia , Proliferação de Células , Precursores Enzimáticos/biossíntese , Feminino , Gelatinases/biossíntese , Células HEK293 , Homeostase , Humanos , Interleucina-8/biossíntese , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Febre Reumática/patologia , Membrana Sinovial/patologia , Fator A de Crescimento do Endotélio Vascular/biossíntese
12.
Channels (Austin) ; 3(4): 249-58, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19617704

RESUMO

Large-conductance Ca(2+)- and voltage-gated Slo1 BK channels are allosterically activated by depolarization and intracellular ligands such as Ca(2+). Of the two high-affinity Ca(2+) sensors present in the channel, the RCK1 sensor also mediates H(+)-dependent activation of the channel. In this study, we examined the comparative mechanisms of the channel activation by Ca(2+) and H(+). Steady-state macroscopic conductance-voltage measurements as well as single-channel openings at negative voltages where voltage-sensor activation is negligible showed that at respective saturating concentrations Ca(2+) is more effective in relative stabilization of the open conformation than H(+). Calculations using the Debye-Hückel formalism suggest that small structural changes in the RCK1 sensor, on the order of few angstroms, may accompany the H(+)-mediated opening of the channel. While the efficacy of H(+) in activation of the channel is less than that of Ca(2+), H(+) more effectively accelerates the activation kinetics when examined at the concentrations equipotent on macroscopic voltage-dependent activation. The RCK1 sensor therefore is capable of transducing the nature of the bound ligand and transmits qualitatively different information to the channel's permeation gate.


Assuntos
Cálcio/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Prótons , Linhagem Celular , Eletrofisiologia , Humanos , Cinética , Canal de Potássio Kv1.1/fisiologia , Ligantes , Conformação Proteica
13.
J Biol Chem ; 284(35): 23353-63, 2009 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-19561088

RESUMO

The structural basis underlying the gating of large conductance Ca(2+)-activated K(+) (BK) channels remains elusive. We found that substitution of Leu-312 in the S6 transmembrane segment of mSlo1 BK channels with hydrophilic amino acids of smaller side-chain volume favored the open state. The sensitivities of channels to calcium and voltage were modified by some mutations and completely abolished by others. Interpretation of the results in terms of an allosteric model suggests that the calcium-insensitive mutants greatly destabilize the closed relative to the open conformation and may also disrupt the allosteric coupling between Ca(2+) or voltage sensors and the gate. Some Phe-315 mutations also favor the open state, suggesting that Leu-312 and Phe-315 may interact in the closed state, forming a major energy barrier that the channel has to overcome to open. Homology modeling and molecular dynamic simulations further support that the side chain of Leu-312 can couple strongly with the aromatic ring of Phe-315 in neighboring subunits (L-F coupling) to maintain the channel closed. Additionally, single-channel recordings indicate that the calcium-insensitive mutants, whose kinetics can be approximately characterized by a two-state closed-open (C-O) model, exhibit nearly 100% open probability under physiological conditions without alterations in single-channel conductance. These findings provide a basis for understanding the structure and gating of the BK channel pore.


Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Substituição de Aminoácidos , Animais , Cálcio/metabolismo , Humanos , Ativação do Canal Iônico , Cinética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Camundongos , Conformação Molecular , Conformação Proteica
15.
J Gen Physiol ; 131(5): 483-502, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18443360

RESUMO

Copper is an essential trace element that may serve as a signaling molecule in the nervous system. Here we show that extracellular Cu2+ is a potent inhibitor of BK and Shaker K+ channels. At low micromolar concentrations, Cu2+ rapidly and reversibly reduces macrosocopic K+ conductance (G(K)) evoked from mSlo1 BK channels by membrane depolarization. GK is reduced in a dose-dependent manner with an IC50 and Hill coefficient of 2 microM and 1.0, respectively. Saturating 100 microM Cu2+ shifts the GK-V relation by +74 mV and reduces G(Kmax) by 27% without affecting single channel conductance. However, 100 microM Cu2+ fails to inhibit GK when applied during membrane depolarization, suggesting that Cu2+ interacts poorly with the activated channel. Of other transition metal ions tested, only Zn2+ and Cd2+ had significant effects at 100 microM with IC(50)s > 0.5 mM, suggesting the binding site is Cu2+ selective. Mutation of external Cys or His residues did not alter Cu2+ sensitivity. However, four putative Cu2+-coordinating residues were identified (D133, Q151, D153, and R207) in transmembrane segments S1, S2, and S4 of the mSlo1 voltage sensor, based on the ability of substitutions at these positions to alter Cu2+ and/or Cd2+ sensitivity. Consistent with the presence of acidic residues in the binding site, Cu2+ sensitivity was reduced at low extracellular pH. The three charged positions in S1, S2, and S4 are highly conserved among voltage-gated channels and could play a general role in metal sensitivity. We demonstrate that Shaker, like mSlo1, is much more sensitive to Cu2+ than Zn2+ and that sensitivity to these metals is altered by mutating the conserved positions in S1 or S4 or reducing pH. Our results suggest that the voltage sensor forms a state- and pH-dependent, metal-selective binding pocket that may be occupied by Cu2+ at physiologically relevant concentrations to inhibit activation of BK and other channels.


Assuntos
Cobre/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/fisiologia , Superfamília Shaker de Canais de Potássio/química , Superfamília Shaker de Canais de Potássio/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cádmio/farmacologia , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Espaço Extracelular , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Potenciais da Membrana , Oócitos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Eletricidade Estática , Relação Estrutura-Atividade , Xenopus laevis , Zinco/farmacologia
16.
J Gen Physiol ; 131(1): 13-32, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18166624

RESUMO

BK (Slo1) potassium channels are activated by millimolar intracellular Mg(2+) as well as micromolar Ca(2+) and membrane depolarization. Mg(2+) and Ca(2+) act in an approximately additive manner at different binding sites to shift the conductance-voltage (G(K)-V) relation, suggesting that these ligands might work through functionally similar but independent mechanisms. However, we find that the mechanism of Mg(2+) action is highly dependent on voltage sensor activation and therefore differs fundamentally from that of Ca(2+). Evidence that Ca(2+) acts independently of voltage sensor activation includes an ability to increase open probability (P(O)) at extreme negative voltages where voltage sensors are in the resting state; 2 microM Ca(2+) increases P(O) more than 15-fold at -120 mV. However 10 mM Mg(2+), which has an effect on the G(K)-V relation similar to 2 microM Ca(2+), has no detectable effect on P(O) when voltage sensors are in the resting state. Gating currents are only slightly altered by Mg(2+) when channels are closed, indicating that Mg(2+) does not act merely to promote voltage sensor activation. Indeed, channel opening is facilitated in a voltage-independent manner by Mg(2+) in a mutant (R210C) whose voltage sensors are constitutively activated. Thus, 10 mM Mg(2+) increases P(O) only when voltage sensors are activated, effectively strengthening the allosteric coupling of voltage sensor activation to channel opening. Increasing Mg(2+) from 10 to 100 mM, to occupy very low affinity binding sites, has additional effects on gating that more closely resemble those of Ca(2+). The effects of Mg(2+) on steady-state activation and I(K) kinetics are discussed in terms of an allosteric gating scheme and the state-dependent interactions between Mg(2+) and voltage sensor that may underlie this mechanism.


Assuntos
Eletrofisiologia , Ativação do Canal Iônico/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Magnésio/fisiologia , Regulação Alostérica/fisiologia , Animais , Cálcio/fisiologia , Membrana Celular/fisiologia , Feminino , Modelos Biológicos , Oócitos/citologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Transdução de Sinais/fisiologia , Eletricidade Estática , Xenopus laevis
17.
Proc Natl Acad Sci U S A ; 104(46): 18270-5, 2007 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-17984060

RESUMO

The voltage-sensor domain (VSD) of voltage-dependent ion channels and enzymes is critical for cellular responses to membrane potential. The VSD can also be regulated by interaction with intracellular proteins and ligands, but how this occurs is poorly understood. Here, we show that the VSD of the BK-type K(+) channel is regulated by a state-dependent interaction with its own tethered cytosolic domain that depends on both intracellular Mg(2+) and the open state of the channel pore. Mg(2+) bound to the cytosolic RCK1 domain enhances VSD activation by electrostatic interaction with Arg-213 in transmembrane segment S4. Our results demonstrate that a cytosolic domain can come close enough to the VSD to regulate its activity electrostatically, thereby elucidating a mechanism of Mg(2+)-dependent activation in BK channels and suggesting a general pathway by which intracellular factors can modulate the function of voltage-dependent proteins.


Assuntos
Citosol/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Magnésio/metabolismo , Canais de Potássio/fisiologia , Animais , Camundongos , Eletricidade Estática
18.
Acc Chem Res ; 39(12): 918-24, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17176030

RESUMO

Heme plays critical roles in numerous biological phenomena. Recent evidence has uncovered a new role of heme in cellular signal transduction, and its mechanism involves reversible binding of heme to proteins. This Account highlights the novel function of heme as an intracellular messenger in the regulation of gene expression and ion channel function.


Assuntos
Heme/metabolismo , Transdução de Sinais , Animais , Regulação da Expressão Gênica , Heme/biossíntese , Heme/química , Hemeproteínas/química , Hemeproteínas/metabolismo , Canais Iônicos/metabolismo , Ligação Proteica
19.
J Gen Physiol ; 127(3): 309-28, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16505150

RESUMO

The activation of large conductance Ca(2+)-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K(+) (K(V)) channels. Yet BK and K(V) channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (P(o)) and I(K) kinetics (tau(I(K))) over an extended voltage range in 0-50 microM [Ca(2+)](i). mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of P(O). The voltage dependence of P(O) and tau(I(K)) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and K(V) channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to K(V) channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol(-1), indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.


Assuntos
Aminoácidos/metabolismo , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Potenciais da Membrana/fisiologia , Oócitos/fisiologia , Sódio/metabolismo , Animais , Células Cultivadas , Simulação por Computador , Modelos Biológicos , Estrutura Terciária de Proteína , Eletricidade Estática , Relação Estrutura-Atividade , Xenopus laevis
20.
J Gen Physiol ; 126(1): 7-21, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15955873

RESUMO

Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531-535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G-V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G-V simulated by weakening the coupling of both Ca(2+) binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca(2+)- and voltage-dependent gating as well as intrinsic stability of the open state.


Assuntos
Membrana Celular/fisiologia , Heme/metabolismo , Ativação do Canal Iônico/fisiologia , Rim/metabolismo , Modelos Biológicos , Canais de Potássio Cálcio-Ativados/fisiologia , Linhagem Celular , Membrana Celular/química , Células Cultivadas , Heme/química , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/fisiologia , Canais de Potássio Cálcio-Ativados/química , Ligação Proteica
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