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1.
J Biol Chem ; 295(14): 4723-4732, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32122972

RESUMO

The voltage-gated potassium channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1-S2 linker of the Kv1.5 channel. The low osmolarity-induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5 Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5 Our results further showed that the extracellular S1-S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1-S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1-S2 linker and made it no longer susceptible to proteinase K-mediated cleavage. In summary, the findings of our study indicate that the S1-S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this channel. By targeting the S1-S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic channel inhibition and results in an increase in IKv1.5.


Assuntos
Canal de Potássio Kv1.5/metabolismo , Potenciais da Membrana/fisiologia , Estresse Mecânico , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação , Células HEK293 , Humanos , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/genética , Células Musculares/citologia , Células Musculares/metabolismo , Pressão Osmótica , Domínios Proteicos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
2.
Cell Physiol Biochem ; 52(2): 302-314, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30816676

RESUMO

BACKGROUND/AIMS: The phenylalkylamine class of L-type Ca2+ channel antagonist verapamil prolongs the effective refractory period (ERP) of human atrium, which appears to contribute to the efficacy of verapamil in preventing reentrant-based atrial arrhythmias including atrial fibrillation. This study was designed to investigate the molecular and electrophysiological mechanism underlying the action of verapamil on human Kv1.5 (hKv1.5) channel that determines action potential duration and ERP in human atrium. METHODS: Site-directed mutagenesis created 10 single-point mutations within pore region of hKv1.5 channel. Wholecell patch-clamp method investigated the effect of verapamil on wild-type and mutant hKv1.5 channels heterologously expressed in Chinese hamster ovary cells. Docking simulation was conducted using open-state homology model of hKv1.5 channel pore. RESULTS: Verapamil preferentially blocked hKv1.5 channel in its open state with IC50 of 2.4±0.6 µM (n = 6). The blocking effect of verapamil was significantly attenuated in T479A, T480A, I502A, V505A, I508A, L510A, V512A and V516A mutants, compared with wild-type hKv1.5 channel. Computer docking simulation predicted that verapamil is positioned within central cavity of channel pore and has contact with Thr479, Thr480, Val505, Ile508, Ala509, Val512, Pro513 and Val516. CONCLUSION: Verapamil acts as an open-channel blocker of hKv1.5 channel, presumably due to direct binding to specific amino acids within pore region of hKv1.5 channel, such as Thr479, Thr480, Val505, Ile508, Val512 and Val516. This blocking effect of verapamil on hKv1.5 channel appears to contribute at least partly to prolongation of atrial ERP and resultant antiarrhythmic action on atrial fibrillation in humans.


Assuntos
Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/química , Simulação de Acoplamento Molecular , Mutação Puntual , Bloqueadores dos Canais de Potássio/química , Verapamil/química , Substituição de Aminoácidos , Animais , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Sítios de Ligação , Células CHO , Cricetulus , Humanos , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Verapamil/farmacologia
3.
J Biol Chem ; 293(40): 15347-15358, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30121572

RESUMO

The voltage-gated potassium channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N terminus, six transmembrane segments (S1-S6), and the C terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus). Interestingly, the cleavage does not impair channel function. Here, we investigated the role of the N terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments, including those that mimic PK-cleaved products. Our results disclosed that although expression of the pore-containing fragment (Frag(304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) was required for Frag(304-613) to traffic to the plasma membrane. Biochemical analysis revealed that the two fragments interacted throughout channel trafficking and maturation. In Frag(1-303)+(304-613)-coassembled channels, which lack a covalent linkage between S1 and S2, amino acid residues 1-209 were important for association with Frag(304-613), and residues 210-303 were necessary for mediating trafficking of coassembled channels to the plasma membrane. We conclude that the N terminus and S1 of Kv1.5 can attract and coassemble with the rest of the channel (i.e. Frag(304-613)) to form a functional channel independently of the S1-S2 linkage.


Assuntos
Canal de Potássio Kv1.5/química , Potenciais da Membrana/fisiologia , Fragmentos de Peptídeos/química , Subunidades Proteicas/química , Endopeptidase K/farmacologia , Expressão Gênica , Células HEK293 , Humanos , Transporte de Íons/efeitos dos fármacos , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Domínios Proteicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Relação Estrutura-Atividade , Transformação Genética
4.
Biochemistry ; 57(18): 2704-2710, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29652491

RESUMO

Molecular dynamics simulations are employed to determine the inhibitory mechanisms of three drugs, 5-(4-phenoxybutoxy)psoralen (PAP-1), vernakalant, and flecainide, on the voltage-gated K+ channel Kv1.5, a target for the treatment of cardiac arrhythmia. At neutral pH, PAP-1 is neutral, whereas the other two molecules carry one positive charge. We show that PAP-1 forms stable dimers in water, primarily through hydrophobic interactions between aromatic rings. All three molecules bind to the cavity between the Ile508 and Val512 residues from the four subunits of the channel. Once bound, the drug molecules are flexible, with the average root-mean-square fluctuation being between 2 and 3 Å, which is larger than the radius of gyration of a bulky amino acid. The presence of a monomeric PAP-1 causes the permeating K+ ion to dehydrate, thereby creating a significant energy barrier. In contrast, vernakalant blocks the ion permeation primarily via an electrostatic mechanism and, therefore, must be in the protonated and charged form to be effective.


Assuntos
Antiarrítmicos/química , Arritmias Cardíacas/tratamento farmacológico , Canal de Potássio Kv1.5/química , Sequência de Aminoácidos/genética , Anisóis/química , Anisóis/farmacologia , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/genética , Sítios de Ligação , Cristalografia por Raios X , Ficusina/química , Ficusina/uso terapêutico , Flecainida/química , Flecainida/uso terapêutico , Humanos , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/genética , Simulação de Dinâmica Molecular , Conformação Proteica/efeitos dos fármacos , Pirrolidinas/química , Pirrolidinas/farmacologia , Homologia de Sequência de Aminoácidos
5.
J Cardiovasc Pharmacol ; 71(1): 10-18, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29283926

RESUMO

Propofol blocks the voltage-gated human Kv1.5 (hKv1.5) channel by preferentially affecting in its open state. A previous mutational study suggested that several amino acids within the pore region of the hKv1.5 channel are involved in mediating the blocking action of propofol. The present investigation was undertaken to elucidate the predicted binding modes of propofol within the pore cavity of the open-state hKv1.5 channel, using computational docking and mutagenesis approaches. The docking simulation using a homology model of the hKv1.5 channel, constructed based on the crystal structure of the Kv1.2 channel, predicted that propofol was positioned at the base of the pore cavity of hKv1.5 channel, adjacent to 4 amino acids Thr479, Thr480, Val505, and Ile508, and formed arene-H interactions with Val505. The patch-clamp experiments on wild-type and mutant hKv1.5 channels constructed by site-directed mutagenesis revealed that the blocking potency of propofol was significantly reduced in T480A, V505A, and I508A but not in T479A mutants compared with wild-type hKv1.5 channel. These computational docking and experimental mutational analyses suggest that propofol is positioned at the base of the pore cavity and forms functional contact with Thr480, Val505, and Ile508 to directly block the hKv1.5 channel.


Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Canal de Potássio Kv1.5/antagonistas & inibidores , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Bloqueadores dos Canais de Potássio/farmacologia , Propofol/farmacologia , Animais , Sítios de Ligação , Células CHO , Cricetulus , Humanos , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mutação , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/metabolismo , Propofol/química , Propofol/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
6.
Eur Biophys J ; 46(6): 549-559, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28314880

RESUMO

The hERG channel is a voltage-gated potassium channel found in cardiomyocytes that contributes to the repolarization of the cell membrane following the cardiac action potential, an important step in the regulation of the cardiac cycle. The lipids surrounding K+ channels have been shown to play a key role in their regulation, with anionic lipids shown to alter gating properties. In this study, we investigate how anionic lipids interact with the pore helix of hERG and compare the results with those from Kv1.5, which possesses a pore helix more typical of K+ channels. Circular dichroism studies of the pore helix secondary structure reveal that the presence of the anionic lipid DMPS within the bilayer results in a slight unfolding of the pore helices from both hERG and Kv1.5, albeit to a lesser extent for Kv1.5. In the presence of anionic lipids, the two pore helices exhibit significantly different interactions with the lipid bilayer. We demonstrate that the pore helix from hERG causes significant perturbation to the order in lipid bicelles, which contrasts with only small changes observed for Kv1.5. These observations suggest that the atypical sequence of the pore helix of hERG may play a key role in determining how anionic lipids influence its gating.


Assuntos
Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Sequência de Aminoácidos , Humanos , Ativação do Canal Iônico , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estrutura Secundária de Proteína
7.
J Biomol Struct Dyn ; 35(2): 380-398, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26786269

RESUMO

The voltage gated Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) and play critical role in repolarization of action potential duration. It is the most rapidly activated channel and has very little or no inactivated states. In human cardiac cells, these channels are expressed more extensively in atrial myocytes than ventricle. From the evidences of its localization and functions, Kv1.5 has been declared a selective drug target for the treatment of atrial fibrillation (AF). In this present study, we have tried to identify the rapidly activating property of Kv1.5 and studied its mode of inhibition using molecular modeling, docking, and simulation techniques. Channel in open conformation is found to be stabilized quickly within the dipalmitoylphosphatidylcholine membrane, whereas most of the secondary structure elements were lost in closed state conformation. The obvious reason behind its ultra-rapid property is possibly due to the amino acid alteration in S4-S5 linker; the replacement of Lysine by Glutamine and vice versa. The popular published drugs as well as newly identified lead molecules were able to inhibit the Kv1.5 in a very similar pattern, mainly through the nonpolar interactions, and formed sable complexes. V512 is found as the main contributor for the interaction along with the other important residues such as V505, I508, A509, V512, P513, and V516. Furthermore, two screened novel compounds show surprisingly better inhibitory potency and can be considered for the future perspective of antiarrhythmic survey.


Assuntos
Canal de Potássio Kv1.5/química , Modelos Moleculares , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Ativação do Canal Iônico , Canal de Potássio Kv1.5/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Conformação Proteica , Estabilidade Proteica , Relação Estrutura-Atividade
8.
Biochim Biophys Acta ; 1858(6): 1082-90, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26874203

RESUMO

Kv1.5 channels mediate the ultra-rapidly activating delayed rectifier potassium current (IKur), which is important for atrial repolarization. It has been shown that cell-surface Kv1.5 channels are sensitive to cleavage by the extracellular serine protease, proteinase K (PK). Here, we investigated the effects of extracellular proteolytic digestion on the function of Kv1.5 channels stably expressed in HEK 293 cells. Our data demonstrate that PK treatment cleaved mature membrane-bound (75kDa) Kv1.5 channels at a single locus in the S1-S2 linker, producing 42-kDa N-terminal fragments and 33-kDa C-terminal fragments. Interestingly, such PK treatment did not affect the Kv1.5 current (IKv1.5) recorded using the whole-cell patch clamp technique. Analysis of cell-surface proteins isolated using biotinylation indicated that the PK-generated N- and C-terminal fragments were both present in the plasma membrane. Co-immunoprecipitation (co-IP) experiments indicated that the N- and C-terminal fragments are no longer associated after cleavage. Furthermore, following PK digestion, the N- and C-fragments degraded at different rates. PK is frequently used as a tool to analyze cell-surface localization of membrane proteins, and cleavage of cell-surface channels has been shown to abolish channel function (e.g. hERG). Our data, for the first time, demonstrate that cleavage of cell-surface channels assessed by Western blot analysis does not necessarily correlate with an elimination of the channel activities.


Assuntos
Canal de Potássio Kv1.5/fisiologia , Células HEK293 , Humanos , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/genética , Microscopia de Fluorescência , Técnicas de Patch-Clamp , Proteólise
9.
J Biol Chem ; 291(7): 3569-80, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26655221

RESUMO

Changes in voltage-dependent potassium channels (Kv channels) associate to proliferation in many cell types, including transfected HEK293 cells. In this system Kv1.5 overexpression decreases proliferation, whereas Kv1.3 expression increases it independently of K(+) fluxes. To identify Kv1.3 domains involved in a proliferation-associated signaling mechanism(s), we constructed chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels, which were expressed as GFP- or cherry-fusion proteins. We studied their trafficking and functional expression, combining immunocytochemical and electrophysiological methods, and their impact on cell proliferation. We found that the C terminus is necessary for Kv1.3-induced proliferation. We distinguished two residues (Tyr-447 and Ser-459) whose mutation to alanine abolished proliferation. The insertion into Kv1.5 of a sequence comprising these two residues increased proliferation rate. Moreover, Kv1.3 voltage-dependent transitions from closed to open conformation induced MEK-ERK1/2-dependent Tyr-447 phosphorylation. We conclude that the mechanisms for Kv1.3-induced proliferation involve the accessibility of key docking sites at the C terminus. For one of these sites (Tyr-447) we demonstrated the contribution of MEK/ERK-dependent phosphorylation, which is regulated by voltage-induced conformational changes.


Assuntos
Canal de Potássio Kv1.3/agonistas , Sistema de Sinalização das MAP Quinases , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Proliferação de Células , Células HEK293 , Humanos , Canal de Potássio Kv1.3/química , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.5/agonistas , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Mutagênese Insercional , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Mutação Puntual , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tirosina/metabolismo
10.
Nat Chem Biol ; 9(8): 473-4, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23868316

RESUMO

Drug design for voltage-gated ion channels has long been hampered by the absence of crystal structures and the challenge of achieving subtype selectivity. A combination of mutagenesis, electrophysiology and molecular modeling has led to the identification of a new side pocket binding site for the small molecule Psora-4 between the pore and the voltage-sensor domain of Kv1.5, offering opportunities to design allosteric ion channel modulators.


Assuntos
Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/química
11.
Cell Physiol Biochem ; 31(6): 968-80, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23839156

RESUMO

BACKGROUND/AIMS: Potassium channels are tetrameric proteins providing potassium selective passage through lipid embedded proteinaceous pores with highest fidelity. The selectivity results from binding to discrete potassium binding sites and stabilization of a hydrated potassium ion in a central internal cavity. The four potassium binding sites, generated by the conserved TTxGYGD signature sequence are formed by the backbone carbonyls of the amino acids TXGYG. Residues KV1.5-Val481, KV4.3-Leu368 and KV7.1- Ile 313 represent the amino acids in the X position of the respective channels. METHODS: Here, we study the impact of these residues on ion selectivity, permeation and inactivation kinetics as well as the modulation by ß-subunits using site-specific mutagenesis, electrophysiological analyses and molecular dynamics simulations. RESULTS: We identify this position as key in modulation of slow inactivation by structurally dissimilar ß-subunits in different KV channels. CONCLUSION: We propose a model in which structural changes accompanying activation and ß-subunit modulation allosterically constrain the backbone carbonyl oxygen atoms via the side chain of the respective X-residue in the signature sequence to reduce conductance during slow inactivation.


Assuntos
Canal de Potássio KCNQ1/metabolismo , Canal de Potássio Kv1.5/metabolismo , Canais de Potássio Shal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Potássio/metabolismo , Estrutura Quaternária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Canais de Potássio Shal/química , Canais de Potássio Shal/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
12.
Nat Chem Biol ; 9(8): 507-13, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23728494

RESUMO

Most known small-molecule inhibitors of voltage-gated ion channels have poor subtype specificity because they interact with a highly conserved binding site in the central cavity. Using alanine-scanning mutagenesis, electrophysiological recordings and molecular modeling, we have identified a new drug-binding site in Kv1.x channels. We report that Psora-4 can discriminate between related Kv channel subtypes because, in addition to binding the central pore cavity, it binds a second, less conserved site located in side pockets formed by the backsides of S5 and S6, the S4-S5 linker, part of the voltage sensor and the pore helix. Simultaneous drug occupation of both binding sites results in an extremely stable nonconducting state that confers high affinity, cooperativity, use-dependence and selectivity to Psora-4 inhibition of Kv1.x channels. This new mechanism of inhibition represents a molecular basis for the development of a new class of allosteric and selective voltage-gated channel inhibitors.


Assuntos
Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/química , Ficusina/química , Ficusina/farmacologia , Canal de Potássio Kv1.5/metabolismo , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Especificidade por Substrato
13.
Circ Res ; 111(7): 842-53, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22843785

RESUMO

RATIONALE: Kv1.5 (KCNA5) is expressed in the heart, where it underlies the I(Kur) current that controls atrial repolarization, and in the pulmonary vasculature, where it regulates vessel contractility in response to changes in oxygen tension. Atrial fibrillation and hypoxic pulmonary hypertension are characterized by downregulation of Kv1.5 protein expression, as well as with oxidative stress. Formation of sulfenic acid on cysteine residues of proteins is an important, dynamic mechanism for protein regulation under oxidative stress. Kv1.5 is widely reported to be redox-sensitive, and the channel possesses 6 potentially redox-sensitive intracellular cysteines. We therefore hypothesized that sulfenic acid modification of the channel itself may regulate Kv1.5 in response to oxidative stress. OBJECTIVE: To investigate how oxidative stress, via redox-sensitive modification of the channel with sulfenic acid, regulates trafficking and expression of Kv1.5. METHODS AND RESULTS: Labeling studies with the sulfenic acid-specific probe DAz and horseradish peroxidase-streptavidin Western blotting demonstrated a global increase in sulfenic acid-modified proteins in human patients with atrial fibrillation, as well as sulfenic acid modification to Kv1.5 in the heart. Further studies showed that Kv1.5 is modified with sulfenic acid on a single COOH-terminal cysteine (C581), and the level of sulfenic acid increases in response to oxidant exposure. Using live-cell immunofluorescence and whole-cell voltage-clamping, we found that modification of this cysteine is necessary and sufficient to reduce channel surface expression, promote its internalization, and block channel recycling back to the cell surface. Moreover, Western blotting demonstrated that sulfenic acid modification is a trigger for channel degradation under prolonged oxidative stress. CONCLUSIONS: Sulfenic acid modification to proteins, which is elevated in diseased human heart, regulates Kv1.5 channel surface expression and stability under oxidative stress and diverts channel from a recycling pathway to degradation. This provides a molecular mechanism linking oxidative stress and downregulation of channel expression observed in cardiovascular diseases.


Assuntos
Fibrilação Atrial/metabolismo , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/metabolismo , Miocárdio/metabolismo , Ácidos Sulfênicos/metabolismo , Sequência de Aminoácidos , Animais , Fibrilação Atrial/patologia , Estudos de Casos e Controles , Linhagem Celular , Células Cultivadas , Humanos , Camundongos , Modelos Animais , Dados de Sequência Molecular , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxirredução , Estresse Oxidativo/fisiologia , Ratos , Espécies Reativas de Oxigênio , Transdução de Sinais/fisiologia
14.
J Mol Cell Cardiol ; 51(6): 966-73, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21906601

RESUMO

We have demonstrated that the natural flavone acacetin selectively inhibits ultra-rapid delayed rectifier potassium current (I(Kur)) in human atria. However, molecular determinants of this ion channel blocker are unknown. The present study was designed to investigate the molecular determinants underlying the ability of acacetin to block hKv1.5 channels (coding I(Kur)) in human atrial myocytes using the whole-cell patch voltage-clamp technique to record membrane current in HEK 293 cells stably expressing the hKv1.5 gene or transiently expressing mutant hKv1.5 genes generated by site-directed mutagenesis. It was found that acacetin blocked hKv1.5 channels by binding to both closed and open channels. The blockade of hKv1.5 channels by acacetin was use- and frequency-dependent, and the IC(50) of acacetin for inhibiting hKv1.5 was 3.5, 3.1, 2.9, 2.1, and 1.7µM, respectively, at 0.2, 0.5, 1, 3, and 4Hz. The mutagenesis study showed that the hKv1.5 mutants V505A, I508A, and V512A in the S6-segment remarkably reduced the channel blocking properties by acacetin (IC(50), 29.5µM for V505A, 19.1µM for I508A, and 6.9µM for V512A). These results demonstrate the novel information that acacetin mainly blocks open hKv1.5 channels by binding to their S6 domain. The use- and rate-dependent blocking of hKv1.5 by acacetin is beneficial for anti-atrial fibrillation.


Assuntos
Flavonas/farmacologia , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/química , Bloqueadores dos Canais de Potássio/farmacologia , Substituição de Aminoácidos , Linhagem Celular , Flavonas/metabolismo , Células HEK293 , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Humanos , Canal de Potássio Kv1.5/genética , Potenciais da Membrana , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Bloqueadores dos Canais de Potássio/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína/genética
15.
Cardiovasc Res ; 89(4): 776-85, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21159668

RESUMO

The ultrarapid delayed rectifier channels have attracted considerable interest as targets for 'atrial-selective' antiarrhythmic drugs because they contribute to atrial but not to ventricular repolarization. Thus, I(Kur) channel blockers are expected to prolong selectively the atrial effective refractory period without inducing proarrhythmic effects due to excessive ventricular action potential prolongation. Here we provide an overview of the properties of I(Kur) channels in expression systems and native cardiomyocytes. The ion conducting pore of the channel is formed by four Kv1.5 α-subunits, whereas the ancillary ß-subunits Kvß1.2, Kvß1.3, and Kvß2.1 control channel trafficking and plasma membrane integration as well as activation and inactivation kinetics. Investigation of I(Kur) channel blockers in cardiomyocytes is complicated (i) by substantial overlap of I(Kur) with other currents, notably the transient outward current I(to), (ii) by lack of drug selectivity, and (iii) by disease-induced regulation of I(Kur). Some new compounds developed as I(Kur) blockers are described and their efficacy in treatment of atrial fibrillation (AF) is discussed. Current evidence suggests that pure I(Kur) channel block may not be sufficient to suppress AF.


Assuntos
Fibrilação Atrial/etiologia , Canal de Potássio Kv1.5/fisiologia , Potenciais de Ação , Animais , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/genética , Sítios de Ligação , Humanos , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/genética , Mutação , Miócitos Cardíacos/metabolismo , Fosforilação , Subunidades Proteicas
16.
Mini Rev Med Chem ; 10(7): 635-42, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20500153

RESUMO

Kv1.5 channels are homotetramers of alpha-pore subunits mainly present in human atrium and pulmonary vasculature. Thus, Kv1.5 is a pharmacological target for cardiovascular diseases. Kv beta 1.3 assemblies with Kv alpha 1.5 and modifies its gating and pharmacology. A further knowledge of alpha-beta interactions and pharmacology will lead a better design of new drugs.


Assuntos
Canal de Potássio Kv1.3/química , Canal de Potássio Kv1.5/química , Anestésicos Locais/química , Anestésicos Locais/farmacologia , Bupivacaína/química , Bupivacaína/farmacologia , Humanos , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.5/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína
17.
Am J Physiol Cell Physiol ; 298(3): C496-509, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20018952

RESUMO

The activity of voltage-gated K(+) (K(V)) channels plays an important role in regulating pulmonary artery smooth muscle cell (PASMC) contraction, proliferation, and apoptosis. The highly conserved NH(2)-terminal tetramerization domain (T1) of K(V) channels is important for proper channel assembly, association with regulatory K(V) beta-subunits, and localization of the channel to the plasma membrane. We recently reported two nonsynonymous mutations (G182R and E211D) in the KCNA5 gene of patients with idiopathic pulmonary arterial hypertension, which localize to the T1 domain of KCNA5. To study the electrophysiological properties and expression patterns of the mutants compared with the wild-type (WT) channel in vitro, we transfected HEK-293 cells with WT KCNA5, G182R, E211D, or the double mutant G182R/E211D channel. The mutants form functional channels; however, whole cell current kinetic differences between WT and mutant channels exist. Steady-state inactivation curves of the G182R and G182R/E211D channels reveal accelerated inactivation; the mutant channels inactivated at more hyperpolarized potentials compared with the WT channel. Channel protein expression was also decreased by the mutations. Compared with the WT channel, which was present in its mature glycosylated form, the mutant channels are present in greater proportion in their immature form in HEK-293 cells. Furthermore, G182R protein level is greatly reduced in COS-1 cells compared with WT. Immunostaining data support the hypothesis that, while WT protein localizes to the plasma membrane, mutant protein is mainly retained in intracellular packets. Overall, these data support a role for the T1 domain in channel kinetics as well as in KCNA5 channel subcellular localization.


Assuntos
Canal de Potássio Kv1.5/metabolismo , Potássio/metabolismo , 4-Aminopiridina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Glicosilação , Humanos , Cinética , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/genética , Potenciais da Membrana , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Polimorfismo de Nucleotídeo Único , Bloqueadores dos Canais de Potássio/farmacologia , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Transfecção
18.
Biochim Biophys Acta ; 1798(3): 367-75, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19961828

RESUMO

Nerve and muscle action potential repolarization are produced and modulated by the regulated expression and activity of several types of voltage-gated K(+) (K(v)) channels. Here, we show that sialylated N-glycans uniquely impact gating of a mammalian Shaker family K(v) channel isoform, K(v)1.5, but have no effect on gating of a second Shaker isoform, K(v)1.4. Each isoform contains one potential N-glycosylation site located along the S1-S2 linker; immunoblot analyses verified that K(v)1.4 and K(v)1.5 were N-glycosylated. The conductance-voltage (G-V) relationships and channel activation rates for two glycosylation-site deficient K(v)1.5 mutants, K(v)1.5(N290Q) and K(v)1.5(S292A), and for wild-type K(v)1.5 expressed under conditions of reduced sialylation, were each shifted linearly by a depolarizing approximately 18 mV compared to wild-type K(v)1.5 activation. External divalent cation screening experiments suggested that K(v)1.5 sialic acids contribute to an external surface potential that modulates K(v)1.5 activation. Channel availability was unaffected by changes in K(v)1.5 glycosylation or sialylation. The data indicate that sialic acid residues attached to N-glycans act through electrostatic mechanisms to modulate K(v)1.5 activation. The sialic acids fully account for effects of N-glycans on K(v)1.5 gating. Conversely, K(v)1.4 gating was unaffected by changes in channel sialylation or following mutagenesis to remove the N-glycosylation site. Each phenomenon is unique for K(v)1 channel isoforms, indicating that sialylated N-glycans modulate gating of homologous K(v)1 channels through isoform-specific mechanisms. Such modulation is relevant to changes in action potential repolarization that occur as ion channel expression and glycosylation are regulated.


Assuntos
Ativação do Canal Iônico/fisiologia , Canal de Potássio Kv1.4/metabolismo , Canal de Potássio Kv1.5/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Glicosilação , Humanos , Cinética , Canal de Potássio Kv1.4/química , Canal de Potássio Kv1.5/química , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Eletricidade Estática
19.
J Mol Graph Model ; 28(3): 226-35, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19713139

RESUMO

Vernakalant (RSD1235) is an investigational drug that converts atrial fibrillation rapidly and safely in patients intravenously [Roy et al., J. Am. Coll. Cardiol. 44 (2004) 2355-2361; Roy et al., Circulation 117 (2008) 1518-1525] and maintains sinus rhythm when given orally [Savelieva et al., Europace 10 (2008) 647-665]. Here, modeling using AutoDock4 allowed exploration of potential binding modes of vernakalant to the open-state of the Kv1.5 channel structure. Point mutations were made in the channel model based on earlier patch-clamp studies [Eldstrom et al., Mol. Pharmacol. 72 (2007) 1522-1534] and the docking simulations re-run to evaluate the ability of the docking software to predict changes in drug-channel interactions. Each AutoDock run predicted a binding conformation with an associated value for free energy of binding (FEB) in kcal/mol and an estimated inhibitory concentration (K(i)). The most favored conformation had a FEB of -7.12 kcal/mol and a predicted K(i) of 6.08 microM (the IC50 for vernakalant is 13.8 microM; [Eldstrom et al., Mol. Pharmacol. 72 (2007) 1522-1534]). This conformation makes contact with all four T480 residues and appears to be clearly positioned to block the channel pore.


Assuntos
Anisóis/química , Anisóis/metabolismo , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/metabolismo , Pirrolidinas/química , Pirrolidinas/metabolismo , Sequência de Aminoácidos , Antiarrítmicos/química , Antiarrítmicos/metabolismo , Sítios de Ligação , Humanos , Canal de Potássio Kv1.5/antagonistas & inibidores , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos
20.
J Hum Genet ; 54(5): 277-83, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19343045

RESUMO

Accumulating evidence reveals that genetic variants play pivotal roles in familial atrial fibrillation (AF). However, the molecular defects in most patients with AF remain to be identified. Here, we report on three novel KCNA5 mutations that were identified in 4 of 120 unrelated AF families. Among them, T527M was found in two AF families, and A576V and E610K in two other AF families, respectively. The mutations T527M and A576V were also detected in 2 and 1 of 256 patients with idiopathic AF, respectively. The same mutations were not observed in 200 secondary AF patients and 500 controls. Functional analyses revealed consistent loss-of-function effects of mutant KCNA5 proteins on the ultrarapidly activating delayed rectifier potassium currents. These findings expand the spectrum of mutations in KCNA5 linked to AF and provide new insight into the molecular mechanism involved in AF.


Assuntos
Fibrilação Atrial/genética , Canal de Potássio Kv1.5/genética , Mutação/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Fibrilação Atrial/fisiopatologia , Sequência de Bases , Fenômenos Eletrofisiológicos , Feminino , Testes Genéticos , Humanos , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/metabolismo , Masculino , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Linhagem , Fenótipo , Alinhamento de Sequência , Especificidade da Espécie
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