RESUMO
KEY POINTS: Association of plasma membrane BKCa channels with BK-ß subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa ) by BK-ß subunits is not established. MitoBKCa -α and the regulatory BK-ß1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK-ß1 (left-shifted voltage dependence of activation, V1/2 = -55 mV, 12 µm matrix Ca2+ ). In BK-ß1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47 mV at 12 µm matrix Ca2+ ) Co-expression of BKCa with the BK-ß1 subunit in HeLa cells doubled the density of BKCa in mitochondria. The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK-ß1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling. ABSTRACT: Association of the plasma membrane BKCa channel with auxiliary BK-ß1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBKCa ) with regulatory subunits is unknown. We report that mitoBKCa functionally associates with its regulatory subunit BK-ß1 in adult rodent cardiomyocytes. Cardiac mitoBKCa is a calcium- and voltage-activated channel that is sensitive to paxilline with a large conductance for K+ of 300 pS. Additionally, mitoBKCa displays a high open probability (Po ) and voltage half-activation (V1/2 = -55 mV, n = 7) resembling that of plasma membrane BKCa when associated with its regulatory BK-ß1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa -α and its BK-ß1 subunit. Mitochondria from the BK-ß1 knockout (KO) mice showed sparse mitoBKCa currents (five patches with mitoBKCa activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V1/2 of activation (+47 mV in 12 µm matrix Ca2+ ). The reduced activity of mitoBKCa was accompanied by a high expression of BKCa transcript in the BK-ß1 KO, suggesting a lower abundance of mitoBKCa channels in this genotype. Accordingly, BK-ß1subunit increased the localization of BKDEC (i.e. the splice variant of BKCa that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-ß1 KO mitochondria displayed a more rapid Ca2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBKCa associates with its regulatory BK-ß1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBKCa channel that helps to maintain mitochondrial Ca2+ homeostasis.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Ativação do Canal Iônico , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Masculino , Miócitos Cardíacos/fisiologia , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
STED (stimulated emission depletion) is a popular super-resolution fluorescence microscopy technique. In this paper, we present a concise guide to building a resonant-scanning STED microscope with ultrafast photon-counting acquisition. The STED microscope has two channels, using a pulsed laser and a continuous-wave (CW) laser as the depletion laser source, respectively. The CW STED channel preforms time-gated detection to enhance optical resolution in this channel. We use a resonant mirror to attain high scanning speed and ultrafast photon counting acquisition to scan a large field of view, which help reduce photobleaching. We discuss some practical issues in building a STED microscope, including creating a hollow depletion beam profile, manipulating polarization, and monitoring optical aberration. We also demonstrate a STED image enhancement method using stationary wavelet expansion and image analysis methods to register objects and to quantify colocalization in STED microscopy.
Assuntos
Aumento da Imagem , Lasers , Microscopia de Fluorescência/instrumentação , FotodegradaçãoRESUMO
The large-conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca), MaxiK), which is encoded by the Kcnma1 gene, is generally expressed at the plasma membrane of excitable and nonexcitable cells. However, in adult cardiomyocytes, a BK(Ca)-like channel activity has been reported in the mitochondria but not at the plasma membrane. The putative opening of this channel with the BK(Ca) agonist, NS1619, protects the heart from ischemic insult. However, the molecular origin of mitochondrial BK(Ca) (mitoBK(Ca)) is unknown because its linkage to Kcnma1 has been questioned on biochemical and molecular grounds. Here, we unequivocally demonstrate that the molecular correlate of mitoBK(Ca) is the Kcnma1 gene, which produces a protein that migrates at â¼140 kDa and arranges in clusters of â¼50 nm in purified mitochondria. Physiological experiments further support the origin of mitoBK(Ca) as a Kcnma1 product because NS1619-mediated cardioprotection was absent in Kcnma1 knockout mice. Finally, BKCa transcript analysis and expression in adult cardiomyocytes led to the discovery of a 50-aa C-terminal splice insert as essential for the mitochondrial targeting of mitoBK(Ca).
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Mitocôndrias Cardíacas/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/deficiência , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/ultraestrutura , Dados de Sequência Molecular , Peso Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de AminoácidosRESUMO
Angiotensin II (ANG-II) and BK channels play important roles in the regulation of blood pressure. In arterial smooth muscle, ANG-II inhibits BK channels, but the underlying molecular mechanisms are unknown. Here, we first investigated whether ANG-II utilizes its type 1 receptor (AT1R) to modulate BK activity. Pharmacological, biochemical, and molecular evidence supports a role for AT1R. In renal arterial myocytes, the AT1R antagonist losartan (10 µM) abolished the ANG-II (1 µM)-induced reduction of whole cell BK currents, and BK channels and ANG-II receptors were found to co-localize at the cell periphery. We also found that BK inhibition via ANG-II-activated AT1R was independent of G-protein activation (assessed with 500 µM GDPßS). In BK-expressing HEK293T cells, ANG-II (1 µM) also induced a reduction of BK currents, which was contingent on AT1R expression. The molecular mechanisms of AT1R and BK channel coupling were investigated in co-transfected cells. Co-immunoprecipitation showed formation of a macromolecular complex, and live immunolabeling demonstrated that both proteins co-localized at the plasma membrane with high proximity indexes as in arterial myocytes. Consistent with a close association, we discovered that the sole AT1R expression could decrease BK channel voltage sensitivity. Truncated BK proteins revealed that the voltage-sensing conduction cassette is sufficient for BK-AT1R association. Finally, C-terminal yellow and cyan fluorescent fusion proteins, AT1R-YFP and BK-CFP, displayed robust co-localized Förster resonance energy transfer, demonstrating intermolecular interactions at their C termini. Overall, our results strongly suggest that AT1R regulates BK channels through a close protein-protein interaction involving multiple BK regions and independent of G-protein activation.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/administração & dosagem , Proteínas de Ligação ao GTP/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Angiotensina II/metabolismo , Animais , Sinalização do Cálcio , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Losartan/administração & dosagem , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Técnicas de Patch-Clamp , Ratos , Artéria Renal/efeitos dos fármacos , Artéria Renal/metabolismoRESUMO
To take full advantage of fast resonant scanning in super-resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue-to-digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (â¼50 × 50 µm) with fast scanning that reduces photobleaching, and advances the time-gated continuous wave STED technology to the usage of resonant scanning with hardware-based time-gating. The assembled system provides superb signal-to-noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant-scanning continuous wave STED microscopy with online time-gated detection.
Assuntos
Microscopia Confocal/métodos , Microscopia Confocal/normas , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/normas , Fótons , Desenho de Equipamento , Aumento da Imagem , Miócitos CardíacosRESUMO
BACKGROUND: Treatment-related mortality and abandonment of therapy are major barriers to successful treatment of childhood acute lymphoblastic leukemia (ALL) in the developing world. PROCEDURE: A collaboration was undertaken between Instituto Nacional de Cancerologia (Bogota, Colombia), which serves a poor patient population in an upper-middle income country, and Dana-Farber/Boston Children's Cancer and Blood Disorders Center (Boston, USA). Several interventions aimed at reducing toxic deaths and abandonment were implemented, including a reduced-intensity treatment regimen and a psychosocial effort targeting abandonment. We performed a cohort study to assess impact. RESULTS: The Study Population comprised 99 children with ALL diagnosed between 2007 and 2010, and the Historic Cohort comprised 181 children treated prior to the study interventions (1995-2004). Significant improvements were achieved in the rate of deaths in complete remission (13% to 3%; P = 0.005), abandonment (32% to 9%; P < 0.001), and event-free survival with abandonment considered an event (47% to 65% at 2 years; P = 0.016). However, relapse rate did not improve. Medically unnecessary treatment delays were common, and landmark analysis revealed that initiating the PIII phase of therapy ≥4 weeks delayed predicted markedly inferior disease-free survival (P = 0.016). Conversely, patients who received therapy without excessive delays had outcomes approaching those achieved in high-income countries. CONCLUSIONS: Implementation of a twinning program was followed by reductions in abandonment and toxic deaths, but relapse rate did not improve. Inappropriate treatment delays were common and strongly predicted treatment failure. These findings highlight the importance of adherence to treatment schedule for effective therapy of ALL.
Assuntos
Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Suspensão de Tratamento , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Pré-Escolar , Estudos de Coortes , Países em Desenvolvimento , Intervalo Livre de Doença , Feminino , Disparidades em Assistência à Saúde , Humanos , Lactente , Masculino , Recidiva Local de NeoplasiaRESUMO
The large conductance voltage- and Ca(2+)-activated K(+) channel (MaxiK, BK(Ca), BK) is composed of four pore-forming α-subunits and can be associated with regulatory ß-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A(2) prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK ß1-subunit in TP-MaxiK association. We found that the ß1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that ß1 and TP are closely associated at the cell periphery. The molecular mechanism of ß1-TP interaction involves predominantly the ß1 extracellular loop. As reported previously, TP activation by the thromboxane A(2) analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP(o)) as a function of voltage. However, the positive shift of the FP(o) versus voltage curve by U46619 relative to the control was less prominent when ß1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, ß1 gene ablation reduced the EC(50) of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the ß1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Tromboxano A2/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Vasoconstrição/efeitos dos fármacosRESUMO
The large-conductance Ca2+- and voltage-activated K+ (MaxiK, BK, BKCa, Slo1, KCa1.1) channel role in cell signalling is becoming apparent as we learn how the channel interacts with a multiplicity of proteins not only at the plasma membrane but also in intracellular organelles including the endoplasmic reticulum, nucleus, and mitochondria. In this review, we focus on the interactions of MaxiK channels with seven-transmembrane G protein-coupled receptors and discuss information suggesting that, the channel big C-terminus may act as the nucleus of signalling molecules including kinases relevant for cell death and survival. Increasing evidence indicates that the channel is able to associate with a variety of receptors including ß-adrenergic receptors, G protein-coupled estrogen receptors, acetylcholine receptors, thromboxane A2 receptors, and angiotensin II receptors, which highlights the varied functions that the channel has (or may have) not only in regulating contraction/relaxation of muscle cells or neurotransmission in the brain but also in cell metabolism, proliferation, migration, and gene expression. In line with this view, MaxiK channels have been implicated in obesity and in brain, prostate, and mammary cancers. A better understanding on the molecular mechanisms underlying or triggered by MaxiK channel abnormalities like overexpression in certain cancers may lead to new therapeutics to prevent devastating diseases.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Proteoma/metabolismoRESUMO
Protein myristoylation is a means by which cells anchor proteins into membranes. The most common type of myristoylation occurs at an N-terminal glycine. However, myristoylation rarely occurs at an internal amino acid residue. Here we tested whether the α-subunit of the human large-conductance voltage- and Ca(2+)-activated K(+) channel (hSlo1) might undergo internal myristoylation. hSlo1 expressed in HEK293T cells incorporated [(3)H]myristic acid via a posttranslational mechanism, which is insensitive to cycloheximide, an inhibitor of protein biosynthesis. In-gel hydrolysis of [(3)H]myristoyl-hSlo1 with alkaline NH(2)OH (which cleaves hydroxyesters) but not neutral NH(2)OH (which cleaves thioesters) completely removed [(3)H]myristate from hSlo1, suggesting the involvement of a hydroxyester bond between hSlo1's hydroxyl-bearing serine, threonine, and/or tyrosine residues and myristic acid; this type of esterification was further confirmed by its resistance to alkaline Tris·HCl. Treatment of cells expressing hSlo1 with 100 µM myristic acid caused alteration of hSlo1 activation kinetics and a 40% decrease in hSlo1 current density from 20 to 12 nA*MΩ. Immunocytochemistry confirmed a decrease in hSlo1 plasmalemma localization by myristic acid. Replacement of the six serines or the seven threonines (but not of the single tyrosine) of hSlo1 intracellular loops 1 and 3 with alanines decreased hSlo1 direct myristoylation by 40-44%, whereas in combination decreased myristoylation by nearly 90% and abolished the myristic acid-induced change in current density. Our data demonstrate that an ion channel, hSlo1, is internally and posttranslationally myristoylated. Myristoylation occurs mainly at hSlo1 intracellular loop 1 or 3, and is an additional mechanism for channel surface expression regulation.
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Ácido Mirístico/metabolismo , Serina/metabolismo , Treonina/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Ésteres , Humanos , Imuno-Histoquímica , Cinética , 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/genética , Mutação , Técnicas de Patch-Clamp , Processamento de Proteína Pós-Traducional , Treonina/químicaRESUMO
Large conductance voltage- and calcium-activated potassium channels (MaxiK, BK(Ca)) are well known for sustaining cerebral and coronary arterial tone and for their linkage to vasodilator ß-adrenergic receptors. However, how MaxiK channels are linked to counterbalancing vasoconstrictor receptors is unknown. Here, we show that vasopressive thromboxane A2 receptors (TP) can intimately couple with and inhibit MaxiK channels. Activation of the receptor with its agonist trans-inhibits MaxiK independently of G-protein activation. This unconventional mechanism is supported by independent lines of evidence: (i) inhibition of MaxiK current by thromboxane A2 mimetic, U46619, occurs even when G-protein activity is suppressed; (ii) MaxiK and TP physically associate and display a high degree of proximity; and (iii) Förster resonance energy transfer occurs between fluorescently labeled MaxiK and TP, supporting a direct interaction. The molecular mechanism of MaxiK-TP intimate interaction involves the receptor's first intracellular loop and C terminus, and it entails the voltage-sensing conduction cassette of MaxiK channel. Further, physiological evidence of MaxiK-TP physical interaction is given in human coronaries and rat aorta, and by confirming TP role (with antagonist SQ29,548) in the U46619-induced MaxiK inhibition in human coronaries. We propose that vasoconstrictor TP receptor and MaxiK-channel direct interaction facilitates G-protein-independent TP to MaxiK trans-inhibition, which would promote vasoconstriction.
Assuntos
Aorta/metabolismo , Vasos Coronários/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes , Ativação Enzimática , Ácidos Graxos Insaturados , Proteínas de Ligação ao GTP/genética , Humanos , Hidrazinas/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Receptores de Tromboxano A2 e Prostaglandina H2/agonistas , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Vasoconstritores/farmacologiaRESUMO
The large conductance calcium- and voltage-activated potassium channel (BK(Ca)) is widely expressed at the plasma membrane. This channel is involved in a variety of fundamental cellular functions including excitability, smooth muscle contractility, and Ca(2+) homeostasis, as well as in pathological situations like proinflammatory responses in rheumatoid arthritis, and cancer cell proliferation. Immunochemical, biochemical and pharmacological studies from over a decade have intermittently shown the presence of BK(Ca) in intracellular organelles. To date, intracellular BK(Ca) (iBK(Ca)) has been localized in the mitochondria, endoplasmic reticulum, nucleus and Golgi apparatus but its functional role remains largely unknown except for the mitochondrial BK(Ca) whose opening is thought to play a role in protecting the heart from ischaemic injury. In the nucleus, pharmacology suggests a role in regulating nuclear Ca(2+), membrane potential and eNOS expression. Establishing the molecular correlates of iBK(Ca), the mechanisms defining iBK(Ca) organelle-specific targeting, and their modulation are challenging questions. This review summarizes iBK(Ca) channels, their possible functions, and efforts to identify their molecular correlates.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Animais , Humanos , Organelas/fisiologiaRESUMO
Estrogen receptor α (ERα) regulates gene transcription via "genomic" (binding directly or indirectly, typically via Sp1 or AP-1 sites, to target genes) and/or "nongenomic" (signaling) mechanisms. ERα activation by estrogen up-regulates the murine Ca(2+)-activated K(+) channel α subunit gene (mSlo1) via genomic mechanisms. Here, we investigated whether ERα also drives transcription of the human (hSlo1) gene. Consistent with this view, estrogen increased hSlo1 transcript levels in primary human smooth muscle cells. Promoter studies revealed that estrogen/hERα-mediated hSlo1 transcription was nearly 6-fold more efficient than for mSlo1 (EC(50), 0.07 versus 0.4 nM). Unlike the genomic transcriptional mechanism employed by mSlo1, hSlo1 exhibits a nongenomic hERα-mediated regulatory mechanism. This is supported by the following: 1) efficient hSlo1 transcription after disruption of the DNA-binding domain of hERα or knockdown of Sp1, and 2) lack of AP-1 sites in the hSlo1 promoter. Three nongenomic signaling pathways were explored: Src, Rho, and PI3K. Inhibition of Src with 10 µM PP2, and reported downstream ERK with 25 µM PD98059 did not prevent estrogen action but caused an increase in hSlo1 basal transcription; conversely, constitutively active c-Src (Y527F) decreased hSlo1 basal transcription even preventing its estrogen/hERα-mediated transcriptional activation. Rho inhibition by coexpressed Clostridium botulinum C3 transferase did not alter estrogen action. In contrast, inhibition of PI3K activity with 10 µM LY294002 decreased estrogen-stimulated hSlo1 transcription by â¼40%. These results indicate that the nongenomic PI3K signaling pathway plays a role in estrogen/hERα-stimulated hSlo1 gene expression; whereas c-Src activity leads to hSlo1 gene tonic repression independently of estrogen, likely through ERK activation.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Proteínas Tirosina Quinases/metabolismo , Transcrição Gênica , Adolescente , Proteína Tirosina Quinase CSK , Células Cultivadas , Criança , MAP Quinases Reguladas por Sinal Extracelular , Feminino , Humanos , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , Fosfatidilinositol 3-Quinases , Quinases da Família srcRESUMO
RATIONALE: Fast-transient outward K(+) (I(to,f)) and ultrarapid delayed rectifier K(+) currents (I(K,slow), also known as I(Kur)) contribute to mouse cardiac repolarization. Gender studies on these currents have reported conflicting results. OBJECTIVE: Key missing information in these studies is the estral stage of the animals. We revisited gender-related differences in K(+) currents, taking into consideration the females' estral stage. We hypothesized that changes in estrogen levels during the estral cycle could play a role in determining the densities of K(+) currents underlying ventricular repolarization. METHODS AND RESULTS: Peak total K(+) current (I(K,total)) densities (pA/pF, at +40 mV) were much higher in males (48.6+/-3.0) versus females at estrus (27.2+/-2.3) but not at diestrus-2 (39.1+/-3.4). Underlying this change, I(to,f) and I(K,slow) were lower in females at estrus versus males and diestrus-2 (I(K,slow): male 21.9+/-1.8, estrus 14.6+/-0.6, diestrus-2 20.3+/-1.4; I(to,f): male 26.8+/-1.9, estrus 14.9+/-1.6, diestrus-2 22.1+/-2.1). Lower I(K,slow) in estrus was attributable to only I(K,slow)(1) reduction, without changes in I(K,slow)(2). Estrogen treatment of ovariectomized mice decreased I(K,total) (46.4+/-3.0 to 28.4+/-1.6), I(to,f) (26.6+/-1.6 to 12.8+/-1.0) and I(K,slow) (22.2+/-1.6 to 17.2+/-1.4). Transcript levels of Kv4.3 and Kv1.5 (underlying I(to,f) and I(K,slow), respectively) were lower in estrus versus diestrus-2 and male. In ovariectomized mice, estrogen treatment resulted in downregulation of Kv4.3 and Kv1.5 but not Kv4.2, KChIP2, or Kv2.1 transcripts. K(+) current reduction in high estrogenic conditions were associated with prolongation of the action potential duration and corrected QT interval. CONCLUSION: Downregulation of Kv4.3 and Kv1.5 transcripts by estrogen are one mechanism defining gender-related differences in mouse ventricular repolarization.
Assuntos
Potenciais de Ação/fisiologia , Estrogênios/farmacologia , Ventrículos do Coração/metabolismo , Potássio/metabolismo , Caracteres Sexuais , Potenciais de Ação/efeitos dos fármacos , Animais , Estrogênios/metabolismo , Ciclo Estral/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Ventrículos do Coração/citologia , Proteínas Interatuantes com Canais de Kv/biossíntese , Masculino , Camundongos , Superfamília Shaker de Canais de Potássio/biossínteseRESUMO
The voltage-dependent anion channel (VDAC) constitutes the major pathway for the entry and exit of metabolites across the outer membrane of the mitochondria and can serve as a scaffold for molecules that modulate the organelle. We report the crystal structure of a beta-barrel eukaryotic membrane protein, the murine VDAC1 (mVDAC1) at 2.3 A resolution, revealing a high-resolution image of its architecture formed by 19 beta-strands. Unlike the recent NMR structure of human VDAC1, the position of the voltage-sensing N-terminal segment is clearly resolved. The alpha-helix of the N-terminal segment is oriented against the interior wall, causing a partial narrowing at the center of the pore. This segment is ideally positioned to regulate the conductance of ions and metabolites passing through the VDAC pore.
Assuntos
Ativação do Canal Iônico , Canal de Ânion 1 Dependente de Voltagem/química , Animais , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Porosidade , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Solubilidade , Eletricidade EstáticaRESUMO
Introduction: Cancer patients have significant levels of emotional distress. The National Comprehensive Cancer Network (NCCN) developed the distress management tool to quickly assess significant distress in oncological patients who require intervention. For its use in Colombia, we made its cross-cultural adaptation and validation. Objective: To determine the operative characteristics of the distress management tool, version 2.2018, in patients seen at the Instituto Nacional de Cancerología (INC) in Colombia. Materials and methods: Counting with the authorization from the NCCN, we translated, made the cross-cultural adaptation, and evaluated the operational characteristics of the tool. We included 343 cancer patients seen at the INC, who filled out the cross-culturally adapted instrument. A diagnostic test study was carried out with a semi-structured interview as a reference. Results: The patients had an average age of 49.7 years (SD=15) and the majority were women (67%). The instrument had an area under the ROC curve of 0.81 (95% CI: 0.77 -0.86); its optimal cut-off point was 3.5 approached to 4 when using integers on the scale; its sensitivity was 0.81 (95% CI: 0.76 - 0.85), and its specificity, 0.69 (95% CI: 0.64 - 0.74). The agreement percentage between the result of the interview and the instrument was 73% (kappa = 0.64; p< 0.001). Conclusions: The distress management tool allowed for the detection of moderate to severe distress requiring intervention and management. This instrument was adapted and validated in cancer patients in Colombia keeping the cutoff point at ≥ 4 as in the original version.
Introducción. Los pacientes con cáncer presentan niveles significativos de malestar emocional. La National Comprehensive Cancer Network (NCCN) desarrolló un instrumento (Distress Management) para evaluarlo de forma rápida en pacientes oncológicos. Para su utilización en Colombia, se hizo la adaptación transcultural y se validó. Objetivo. Determinar las características operativas del instrumento de malestar emocional, versión 2.2018, en pacientes atendidos en el Instituto Nacional de Cancerología. Materiales y métodos. Previa autorización de la NCCN, se procedió a la traducción, adaptación transcultural y evaluación de las características operativas del instrumento. Se incluyeron 343 pacientes con diagnóstico de cáncer atendidos en el Instituto Nacional de Cancerología, quienes diligenciaron el instrumento adaptado transculturalmente. Se efectuó un estudio de prueba diagnóstica como patrón de referencia mediante una entrevista semiestructurada. Resultados. Los pacientes tenían una edad promedio de 49,7 años (DE=15) y la mayoría (67 %) eran mujeres. El instrumento tuvo un área bajo la curva ROC de 0,81 (IC95% 0,77-0,86); el punto de corte óptimo fue de 3,5, el cual se aproximó a 4; la sensibilidad fue de 0,81 (IC95% 0,76-0,85) y la especificidad de 0,69 (IC95% 0,64-0,74). El porcentaje de acuerdo entre el resultado de la entrevista y el instrumento fue de 73 % (kappa=0,64; p<0,001). Conclusiones. El instrumento de malestar emocional permitió detectar el malestar emocional moderado a grave que requiere intervención y manejo. Este instrumento fue adaptado y validado en pacientes con cáncer en Colombia, conservándose el punto de corte en ≥4 como en la versión original.
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Neoplasias , Colômbia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
To quantify spatial protein-protein proximity (colocalization) in paired microscopic images of two sets of proteins labeled by distinct fluorophores, we showed that the cross-correlation and the autocorrelation functions of image intensity consisted of fast and slowly decaying components. The fast component resulted from clusters of proteins specifically labeled, and the slow component resulted from image heterogeneity and a broadly-distributed background. To better evaluate spatial proximity between the two specifically labeled proteins, we extracted the fast-decaying component by fitting the sharp peak in correlation functions to a Gaussian function, which was then used to obtain protein-protein proximity index and the Pearson's correlation coefficient. We also employed the median-filter method as a universal approach for background reduction to minimize nonspecific fluorescence. We illustrated our method by analyzing computer-simulated images and biological images.
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Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Proteínas/química , Algoritmos , Animais , Linhagem Celular , Células Cultivadas , Simulação por Computador , Humanos , Camundongos , Modelos Químicos , Dinâmica não Linear , Distribuição Normal , Fatores de TempoRESUMO
Several studies have recently demonstrated that G protein-coupled receptor 30 (GPER) can directly bind to estrogen and mediate its action. We investigated the role and the mechanism of estrogen-induced cardioprotection after ischemia-reperfusion using a specific GPER agonist G1. Isolated hearts from male mice were perfused using Langendorff technique with oxygenated (95% O(2) and 5% CO(2)) Krebs Henseleit buffer (control), with G1 (1 microM), and G1 (1 microM) together with extracellular signal-regulated kinase (Erk) inhibitor PD-98059 (5 microM). After 20 min of perfusion, hearts were subjected to 20 min global normothermic (37 degrees C) ischemia followed by 40 min reperfusion. Cardiac function was measured, and myocardial necrosis was evaluated by triphenyltetrazolium chloride staining at the end of the reperfusion. Mitochondria were isolated after 10 min of reperfusion to assess the Ca(2+) load required to induce mitochondria permeability transition pore (mPTP) opening. G1-treated hearts developed better functional recovery with higher rate pressure product (RPP, 6140 +/- 264 vs. 2,640 +/- 334 beats mmHg(-1) min(-1), P < 0.05). The infarct size decreased significantly in G1-treated hearts (21 +/- 2 vs. 46 +/- 3%, P < 0.001), and the Ca(2+) load required to induce mPTP opening increased (2.4 +/- 0.06 vs. 1.6 +/- 0.11 microM/mg mitochondrial protein, P < 0.05) compared with the controls. The protective effect of G1 was abolished in the presence of PD-98059 [RPP: 4,120 +/- 46 beats mmHg(-1) min(-1), infarct size: 53 +/- 2%, and Ca(2+) retention capacity: 1.4 +/- 0.11 microM/mg mitochondrial protein (P < 0.05)]. These results suggest that GPER activation provides a cardioprotective effect after ischemia-reperfusion by inhibiting the mPTP opening, and this effect is mediated by the Erk pathway.
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Mitocôndrias Cardíacas/fisiologia , Miocárdio/patologia , Traumatismo por Reperfusão/patologia , Trifosfato de Adenosina/fisiologia , Animais , Western Blotting , Inibidores Enzimáticos/uso terapêutico , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Flavonoides/uso terapêutico , Cardiopatias/prevenção & controle , Hemodinâmica/fisiologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Estresse Oxidativo/fisiologia , Permeabilidade , Substâncias Protetoras , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The process of parturition involves the complex interplay of factors that change the excitability and contractile activity of the uterus. We have compared the relative gene expression profile of myometrium from rats before parturition (21 days pregnant) and during delivery, using high-density DNA microarray. Of 8,740 sequences available in the array, a total of 3,782 were detected as present. From the sequences that were significantly altered, 59 genes were upregulated and 82 genes were downregulated. We were able to detect changes in genes described to have altered expression level at term, including connexin 43 and 26, cyclooxygenase 2, and oxytocin receptor, as well as novel genes that have been not previously associated with parturition. Quantitative real-time PCR on selected genes further confirmed the microarray data. Here we report for the first time that aquaporin5 (AQP5), a member of the aquaporin water channel family, was dramatically downregulated during parturition (approximately 100-fold by microarray and approximately 50-fold by real-time PCR). The emerging profile highlights biochemical cascades occurring in a period of approximately 36 h that trigger parturition and the initiation of myometrium reverse remodeling postpartum. The microarray analysis uncovered genes that were previously suspected to play a role in parturition. This regulation involves genes from immune/inflammatory response, steroid/lipid metabolism, calcium homeostasis, cell volume regulation, cell signaling, cell division, and tissue remodeling, suggesting the presence of multiple and redundant mechanisms altered in the process of birth.
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Expressão Gênica , Miométrio/metabolismo , Parto/genética , Prenhez/genética , Animais , Aquaporina 5/genética , Aquaporina 5/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Feminino , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Parto/metabolismo , Gravidez , Ratos , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismoRESUMO
Voltage-gated K(+) (Kv) channels are key determinants of cardiac and neuronal excitability. A substantial body of evidence has accumulated in support of a role for Src family tyrosine kinases in the regulation of Kv channels. In this study, we examined the possibility that c-Src tyrosine kinase participates in the modulation of the transient voltage-dependent K(+) channel Kv4.3. Supporting a mechanistic link between Kv4.3 and c-Src, confocal microscopy analysis of HEK293 cells stably transfected with Kv4.3 showed high degree of co-localization of the two proteins at the plasma membrane. Our results further demonstrate an association between Kv4.3 and c-Src by co-immunoprecipitation and GST pull-down assays, this interaction being mediated by the SH2 and SH3 domains of c-Src. Furthermore, we show that Kv4.3 is tyrosine phosphorylated under basal conditions. The functional relevance of the observed interaction between Kv4.3 and c-Src was established in patch-clamp experiments, where application of the Src inhibitor PP2 caused a decrease in Kv4.3 peak current amplitude, but not the inactive structural analogue PP3. Conversely, intracellular application of recombinant c-Src kinase or the protein tyrosine phosphatase inhibitor bpV(phen) increased Kv4.3 peak current amplitude. In conclusion, our findings provide evidence that c-Src-induced Kv4.3 channel activation involves their association in a macromolecular complex and suggest a role for c-Src-Kv4.3 pathway in regulating cardiac and neuronal excitability.
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Canais de Potássio Shal/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Eletrofisiologia , Ativação Enzimática , Humanos , Técnicas de Patch-Clamp , Fosfotirosina/metabolismo , Ligação Proteica , Transporte Proteico , Canais de Potássio Shal/genética , Regulação para CimaRESUMO
Sepiapterin reductase (SPR) catalyzes the final step of tetrahydrobiopterin (H(4)B) biosynthesis and the first step of H(4)B regeneration from an exogenous precursor sepiapterin. Despite the potential significance of SPR in regulating H(4)B-dependent nitric oxide (NO(*)) production, the endothelium-specific sequence and functions of SPR remain elusive. We first cloned endothelial SPR cDNA from bovine aortic endothelial cells (Genebank: DQ978331). In cells transiently transfected with SPR gene, SPR activity (HPLC) was dramatically increased by 19-fold, corresponding to a significant increase in endothelial H(4)B content (HPLC) and NO(*) production (electron spin resonance). In vivo delivery of SPR gene significantly increased vascular SPR protein expression (mouse vs. bovine antibodies to differentiate endogenous vs. exogenous), activity, H(4)B content, and NO(*) production, as well as NO(*)-dependent vasorelaxation. In endothelial cells transfected with small interfering RNA specific for SPR, approximately 87% of mRNA were attenuated (real-time quantitative RT-PCR), corresponding to a significant reduction in SPR protein expression and activity, which was associated with decreases in both intracellular H(4)B content and NO(*) level. Exogenous administration of sepiapterin to endothelial cells significantly upregulated H(4)B and NO(*) levels, which were attenuated by SPR RNA interference (RNAi). H(4)B-stimulated increase in NO(*) production, however, was SPR RNAi independent. GTP cyclohydrolase 1 expression and activity, as well as dihydrofolate reductase expression, were not affected by SPR RNAi, whereas dihydrofolate reductase activity was significantly downregulated. These data represent the first to study endothelial SPR functionally and clearly demonstrate an important role of endothelial SPR in modulating H(4)B and NO(*) bioavailability.