Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 56
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Int J Mol Sci ; 24(21)2023 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-37958713

RESUMO

Reperfusion after ischemia would cause massive myocardial injury, which leads to oxidative stress (OS). Calcium homeostasis imbalance plays an essential role in myocardial OS injury. CaV1.2 calcium channel mediates calcium influx into cardiomyocytes, and its activity is modulated by a region of calpastatin (CAST) domain L, CSL54-64. In this study, the effect of Ahf-caltide, derived from CSL54-64, on myocardial OS injury was investigated. Ahf-caltide decreased the levels of LDH, MDA and ROS and increased heart rate, coronary flow, cell survival and SOD activity during OS. In addition, Ahf-caltide permeated into H9c2 cells and increased CaV1.2, CaVß2 and CAST levels by inhibiting protein degradation. At different Ca2+ concentrations (25 nM, 10 µM, 1 mM), the binding of CSL to the IQ motif in the C terminus of the CaV1.2 channel was increased in a H2O2 concentration-dependent manner. CSL54-64 was predicted to be responsible for the binding of CSL to CaV1.2. In conclusion, Ahf-caltide exerted a cardioprotective effect on myocardial OS injury by stabilizing CaV1.2 protein expression. Our study, for the first time, proposed that restoring calcium homeostasis by targeting the CaV1.2 calcium channel and its regulating factor CAST could be a novel treatment for myocardial OS injury.


Assuntos
Cálcio , Peróxido de Hidrogênio , Cálcio/metabolismo , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Miócitos Cardíacos/metabolismo , Peptídeos/farmacologia , Estresse Oxidativo
2.
Int J Mol Sci ; 24(7)2023 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-37047381

RESUMO

Cav1.2 Ca2+ channels, a type of voltage-gated L-type Ca2+ channel, are ubiquitously expressed, and the predominant Ca2+ channel type, in working cardiac myocytes. Cav1.2 channels are regulated by the direct interactions with calmodulin (CaM), a Ca2+-binding protein that causes Ca2+-dependent facilitation (CDF) and inactivation (CDI). Ca2+-free CaM (apoCaM) also contributes to the regulation of Cav1.2 channels. Furthermore, CaM indirectly affects channel activity by activating CaM-dependent enzymes, such as CaM-dependent protein kinase II and calcineurin (a CaM-dependent protein phosphatase). In this article, we review the recent progress in identifying the role of apoCaM in the channel 'rundown' phenomena and related repriming of channels, and CDF, as well as the role of Ca2+/CaM in CDI. In addition, the role of CaM in channel clustering is reviewed.


Assuntos
Canais de Cálcio Tipo L , Calmodulina , Calmodulina/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo
3.
Front Cell Neurosci ; 16: 867385, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35496903

RESUMO

Parkinson's disease (PD), a common neurodegenerative disease characterized by motor dysfunction, results from the death of dopaminergic neurons in the substantia nigra pars compacta (SNc). Although the precise causes of PD are still unknown, several risk factors for PD have been determined, including aging, genetic mutations, environmental factors, and gender. Currently, the molecular mechanisms underlying risk factor-related neurodegeneration in PD remain elusive. Endoplasmic reticulum stress, excessive reactive oxygen species production, and impaired autophagy have been implicated in neuronal death in the SNc in PD. Considering that these pathological processes are tightly associated with intracellular Ca2+, it is reasonable to hypothesize that dysregulation of Ca2+ handling may mediate risk factors-related PD pathogenesis. We review the recent findings on how risk factors cause Ca2+ dyshomeostasis and how aberrant Ca2+ handling triggers dopaminergic neurodegeneration in the SNc in PD, thus putting forward the possibility that manipulation of specific Ca2+ handling proteins and subcellular Ca2+ homeostasis may lead to new promising strategies for PD treatment.

4.
Neurochem Res ; 46(3): 523-534, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33394222

RESUMO

Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca2+/calmodulin (CaM) binds to VGSC type II (NaV1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in NaV1.2 IQ motif, Arg1902Cys (R1902C), has been reported to affect the combination between CaM and the IQ motif compared to that of the wild type IQ motif. However, the detailed properties for the Ca2+-regulated binding of CaM to NaV1.2 IQ (1901Lys-1927Lys, IQwt) and mutant IQ motif (IQR1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of NaV1.2 and its mutant was investigated by protein pull-down experiments. We discovered that the combination between CaM and the IQ motif was U-shaped with the highest at [Ca2+] ≈ free and the lowest at 100 nM [Ca2+]. In the IQR1902C mutant, Ca2+-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQR1902C at 100 and 500 nM [Ca2+] was increased compared to that of IQwt. Both N- and C lobe of CaM could bind with NaV1.2 IQ motif and IQR1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and NaV1.2 IQ motif. This research offers novel insight to the regulation of NaV1.2 IQwt and IQR1902C motif, an autism-associated mutation, by CaM.


Assuntos
Calmodulina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Transtorno Autístico/genética , Calmodulina/química , Humanos , Simulação de Acoplamento Molecular , Mutação , Canal de Sódio Disparado por Voltagem NAV1.2/química , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Ligação Proteica
5.
Pflugers Arch ; 472(7): 911-922, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32472332

RESUMO

TWIK-related acid-sensitive K+ (TASK) channels contribute to the resting membrane potential in various kinds of cells, such as brain neurons, smooth muscle cells, and endocrine cells. Loss-of-function mutations at multiple sites in the KCNK3 gene encoding for TASK1 channels are one of the causes of pulmonary arterial hypertension in humans, whereas a mutation at only one site is reported for TASK3 channels, resulting in a syndrome of mental retardation, hypotonia, and facial dysmorphism. TASK channels are subject to regulation by G protein-coupled receptors (GPCRs). Two mechanisms have been proposed for the GPCR-mediated inhibition of TASK channels: a change in gating and channel endocytosis. The most feasible mechanism for altered gating is diacylglycerol binding to a site in the C-terminus, which is shared by TASK1 and TASK3. The inhibition of channel function by endocytosis requires the presence of a tyrosine residue subjected to phosphorylation by the non-receptor tyrosine kinase Src and a dileucine motif in the C-terminus of TASK1. Therefore, homomeric TASK1 and heteromeric TASK1-TASK3 channels, but not homomeric TASK3, are internalized by GPCR stimulation. Tyrosine phosphorylation by Src is expected to result in a conformational change in the C-terminus, allowing for AP-2, an adaptor protein for clathrin, to bind to the dileucine motif. It is likely that a raft membrane domain is a platform where TASK1 is located and the signaling molecules protein kinase C, Pyk2, and Src are recruited in sequence in response to GPCR stimulation.


Assuntos
Canalopatias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Transporte Proteico/fisiologia , Animais , Humanos , Fosforilação/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia
6.
Am J Physiol Cell Physiol ; 318(5): C991-C1004, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32186935

RESUMO

Calmodulin (CaM) mutations are associated with congenital long QT (LQT) syndrome (LQTS), which may be related to the dysregulation of the cardiac-predominant Ca2+ channel isoform CaV1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, the interaction of this CaM mutant with the CaV1.2 channel has not been determined. In this study, by utilizing a semiquantitative pull-down assay, we explored the interaction of CaM-E141G with CaM-binding peptide fragments of the CaV1.2 channel. Using the patch-clamp technique, we also investigated the electrophysiological effects of the mutant on CaV1.2 channel activity. We found that the maximum binding (Bmax) of CaM-E141G to the proximal COOH-terminal region, PreIQ-IQ, PreIQ, IQ, and NT (an NH2-terminal peptide) was decreased (by 17.71-59.26%) compared with that of wild-type CaM (CaM-WT). In particular, the Ca2+-dependent increase in Bmax became slower with the combination of CaM-E141G + PreIQ and IQ but faster in the case of NT. Functionally, CaM-WT and CaM-E141G at 500 nM Ca2+ decreased CaV1.2 channel activity to 24.88% and 55.99%, respectively, compared with 100 nM Ca2+, showing that the inhibitory effect was attenuated in CaM-E141G. The mean open time of the CaV1.2 channel was increased, and the number of blank traces with no channel opening was significantly decreased. Overall, CaM-E141G exhibits disrupted binding with the CaV1.2 channel and induces a flickering gating mode, which may result in the dysfunction of the CaV1.2 channel and, thus, the development of LQTS. The present study is the first to investigate the detailed binding properties and single-channel gating mode induced by the interaction of CaM-E141G with the CaV1.2 channel.


Assuntos
Canais de Cálcio Tipo L/genética , Calmodulina/genética , Síndrome do QT Longo/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Cobaias , Humanos , Ativação do Canal Iônico/genética , Cinética , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/fisiopatologia , Mutação de Sentido Incorreto/genética , Técnicas de Patch-Clamp , Peptídeos/genética , Ligação Proteica/genética , Isoformas de Proteínas/genética
7.
Protein Expr Purif ; 160: 7-10, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30926461

RESUMO

Both recombinant glutathione-S-transferase (GST)-fused and GST-cleaved fragments of an L-type voltage-gated Ca2+ channel (Cav1.2) are used frequently in GST pull-down assays to investigate the interactions between regulatory proteins and the Cav1.2 channel. However, GST-fused and GST-cleaved proximal C-terminal fragments of the guinea-pig cardiac Cav1.2 channel (CT1, amino acids 1509-1791) heterologously expressed in Escherichia coli (E. coli) are difficult to be recovered in a bioactive form because they are only poorly soluble. In this study, we developed a new method to solubilize and purify CT1. GST-CT1 expressed in E. coli was extracted and treated with an inclusion body solubilization and renaturation kit. Then, after adsorption to glutathione Sepharose beads, GST-CT1 was treated with protease to release CT1. However, the cleaved CT1 was insoluble and remained attached to the beads. Therefore, CT1 was treated again with the inclusion body solubilization and renaturation kit. Using this method, GST-CT1 and CT1 were purified with a high yield. GST pull-down experiments showed a dose-dependent interaction between GST-CT1 and calmodulin (CaM), and between GST-CaM and CT1, suggesting recovered bioactivity of GST-CT1 and CT1. This protocol may also be applied to purify other insoluble GST-fused proteins.


Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/isolamento & purificação , Glutationa Transferase/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glutationa Transferase/química , Glutationa Transferase/genética , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
8.
Int J Mol Sci ; 19(9)2018 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-30142967

RESUMO

Calmodulin (CaM) is well known as an activator of calcium/calmodulin-dependent protein kinase II (CaMKII). Voltage-gated sodium channels (VGSCs) are basic signaling molecules in excitable cells and are crucial molecular targets for nervous system agents. However, the way in which Ca2+/CaM/CaMKII cascade modulates NaV1.1 IQ (isoleucine and glutamine) domain of VGSCs remains obscure. In this study, the binding of CaM, its mutants at calcium binding sites (CaM12, CaM34, and CaM1234), and truncated proteins (N-lobe and C-lobe) to NaV1.1 IQ domain were detected by pull-down assay. Our data showed that the binding of Ca2+/CaM to the NaV1.1 IQ was concentration-dependent. ApoCaM (Ca2+-free form of calmodulin) bound to NaV1.1 IQ domain preferentially more than Ca2+/CaM. Additionally, the C-lobe of CaM was the predominant domain involved in apoCaM binding to NaV1.1 IQ domain. By contrast, the N-lobe of CaM was predominant in the binding of Ca2+/CaM to NaV1.1 IQ domain. Moreover, CaMKII-mediated phosphorylation increased the binding of Ca2+/CaM to NaV1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of NaV1.1 IQ domain. This study provides novel mechanisms for the modulation of NaV1.1 by the Ca2+/CaM/CaMKII axis. For the first time, we uncover the effect of Ca2+, lobe-specificity and CaMKII on CaM binding to NaV1.1.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Cálcio/química , Calmodulina/química , Canal de Sódio Disparado por Voltagem NAV1.1/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Cinética , Simulação de Acoplamento Molecular , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Termodinâmica
9.
J Pharmacol Sci ; 137(2): 187-194, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30042022

RESUMO

Activity of cardiac Cav1.2 channels is enhanced by cyclic AMP-PKA signaling. In this study, we studied the effects of PKA phosphorylation on the binding of calmodulin to the fragment peptide of the proximal C-terminal tail of α1C subunit (CT1, a.a. 1509-1789 of guinea-pig). In the pull-down assay, in vitro PKA phosphorylation significantly decreased calmodulin binding to CT1 (61%) at high [Ca2+]. The phosphoresistant (CT1SA) and phosphomimetic (CT1SD) CT1 mutants, in which three PKA sites (Ser1574, 1626, 1699) were mutated to Ala and Asp, respectively, bound with calmodulin with 99% and 65% amount, respectively, compared to that of wild-type CT1. In contrast, at low [Ca2+], calmodulin-binding to CT1SD was higher (33-35%) than that to CT1SA. The distal C-terminal region of α1C (CT3, a.a. 1942-2169) is known to interact with CT1 and inhibit channel activity. CT3 bound to CT1SD was also significantly less than that to CT1SA. In inside-out patch, PKA catalytic subunit (PKAc) facilitated Ca2+ channel activity at both high and low Ca2+ condition. Altogether, these results support the hypothesis that PKA phosphorylation may enhance channel activity and attenuate the Ca2+-dependent inactivation, at least partially, by modulating calmodulin-CT1 interaction both directly and indirectly via CT3-CT1 interaction.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Domínio Catalítico , Células Cultivadas , AMP Cíclico/metabolismo , Cobaias , Fosforilação , Ligação Proteica
10.
Eur J Pharmacol ; 824: 99-107, 2018 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-29438706

RESUMO

Tricyclodecan-9-yl-xanthogenate (D609) is widely known for its antitumor and antiviral properties via the inhibition of phosphatidylcholine-specific phospholipase C and sphingomyelin synthase. Previously, we found that chronic application of D609 suppressed the K+ channel, KCNQ1/KCNE1, more drastically than expected from its actions on the enzymes, suggesting a direct action of D609 on the channel. Here, we aimed to test this possibility by studying the affinity, specificity, and mechanisms of D609 on KCNQ1/KCNE1. The effect of D609 on KCNQ1/KCNE1 was studied using an in vitro expression system and in native cells, using electrophysiological techniques. We found that D609 rapidly and reversibly inhibited KCNQ1/KCNE1 channels expressed in human embryonic kidney 293 T (HEK293T) cells, in a concentration-dependent manner with a high affinity. D609 neither suppressed endogenous K+ currents in HEK293T cells, nor inhibited the sustained and transient K+ currents of mouse neostriatal neurons, but blocked a KCNQ1/KCNE1-like current in neostriatal neurons. D609 potently blocked IKs, the cardiac KCNQ1/KCNE1 channel, in guinea pig cardiac muscle cells. The action of D609 on KCNQ1/KCNE1 depended on the usage of the channel, suggesting that D609 binds to the channel in the open state. We identified D609 as a potent and specific open channel blocker of KCNQ1/KCNE1. Because KCNQ1/KCNE1 is highly expressed in the heart, the inner ear and the pancreas, D609, when used as an antitumor or antiviral drug, may affect the function of a number of organs in vivo even when used at low concentrations.


Assuntos
Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canal de Potássio KCNQ1/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Tionas/farmacologia , Antineoplásicos/química , Hidrocarbonetos Aromáticos com Pontes/química , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Canal de Potássio KCNQ1/metabolismo , Norbornanos , Bloqueadores dos Canais de Potássio/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Solubilidade , Tiocarbamatos , Tionas/química , Água/química
11.
J Pharmacol Sci ; 133(4): 240-246, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28391995

RESUMO

Cardiac Cav1.2 channels, coupling membrane stimulation to intracellular Ca2+ signaling, are regulated by multiple cytoplasmic factors, such as calmodulin (CaM), phosphorylation, Ca2+, ATP and intramolecular fragments of the channel. The interaction between distal and proximal C-terminal regulatory domains (DCRD and PCRD) of Cav1.2 channel is suggested to inhibit the channel activity, while PKA-mediated phosphorylation facilitates Cav1.2 channel by releasing such an interaction. Here, we report that the interaction between the distal C-terminus (CT3) and the proximal C-terminus (CT1) are inhibited by CaM in a Ca2+-dependent manner. Furthermore, CT3D (a short CT3 with DCRD truncated) interacts with CT1B (a short CT1 with EF-hand and PCRD truncated), revealing a new interaction between distal and proximal C-terminus. Ca2+/CaM inhibited the binding of CT3D to CT1B more strongly than the binding between CT3 and CT1, implying that the interaction of DCRD/PCRD (in CT3/CT1) might cooperate with the binding of CT3D to CT1B. We name the new CT1B-binding region of CT3D as CaM-competitive domain (CCD). The electrophysiological experiments show that CT3D inhibits while CT1B facilitates Cav1.2 channel activity in inside-out patches in guinea-pig ventricular myocytes. These results suggest that distal C-terminus inhibits Cav1.2 channel through modulation of the CaM-binding property of the channels.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/química , Calmodulina/metabolismo , Calmodulina/fisiologia , Fenômenos Eletrofisiológicos , Cobaias , Ventrículos do Coração/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Fosforilação/fisiologia , Ligação Proteica , Domínios Proteicos/fisiologia
12.
J Physiol ; 595(8): 2465-2477, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28130847

RESUMO

KEY POINTS: Cav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant-derivatives, C-terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside-out mode. α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C-tail. ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation. CaM-linked channels are useful tools for investigating Cav1.2 channels in the inside-out mode; the fast run-down is prevented by only ATP and the slow run-down is nearly absent. ABSTRACT: Calmodulin (CaM) plays a critical role in regulation of Cav1.2 Ca2+ channels. CaM binds to the channel directly, maintaining channel activity and regulating it in a Ca2+ -dependent manner. To explore the molecular mechanisms involved, we compared the activity of the wildtype channel (α1C) and mutant derivatives, C-terminal deleted (α1C∆) and α1C∆ linked to CaM (α1C∆CaM). These were co-expressed with ß2a and α2δ subunits in HEK293 cells. In the inside-out mode, α1C and α1C∆ showed minimal open-probabilities in a basic internal solution (run-down), whereas α1C∆ with CaM and α1C∆CaM maintained detectable channel activity, confirming that CaM was necessary, but not sufficient, for channel activity. Previously, we reported that ATP was required to maintain channel activity of α1C. Unlike α1C, the mutant channels did not require ATP for activation in the early phase (3-5 min). However, α1C∆ with CaM + ATP and α1C∆CaM with ATP maintained activity, even in the late phase (after 7-9 min). These results suggested that CaM and ATP interacted dynamically with the proximal C-terminal tail of the channel and, thereby, produced channel activity. In addition, okadaic acid, a protein phosphatase inhibitor, could substitute for the effects of ATP on α1C but not on the mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels, further indicating that ATP has dual effects. One maintains phosphorylation of the channel and the other becomes apparent when the distal carboxyl-terminal tail is removed.


Assuntos
Trifosfato de Adenosina/metabolismo , Canais de Cálcio Tipo L/metabolismo , Calmodulina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Calmodulina/farmacologia , Células HEK293 , Humanos , Ligação Proteica/fisiologia , Coelhos , Ratos
13.
Am J Physiol Cell Physiol ; 310(10): C773-9, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26739491

RESUMO

This study aimed to investigate protein phosphatases involved in the run down of Cav1.2 Ca(2+) channels. Single ventricular myocytes obtained from adult guinea pig hearts were used to record Ca(2+) channel currents with the patch-clamp technique. Calmodulin (CaM) and ATP were used to restore channel activity in inside-out patches. Inhibitors of protein phosphatases were applied to investigate the role of phosphatases. The specific protein phosphatase type 1 (PP1) inhibitor (PP1 inhibitor-2) and protein phosphatase type 2A (PP2A) inhibitor (fostriecin) abolished the slow run down of Cav1.2 Ca(2+) channels, which was evident as the time-dependent attenuation of the reversing effect of CaM/ATP on the run down. However, protein phosphatase type 2B (PP2B, calcineurin) inhibitor cyclosporine A together with cyclophilin A had no effect on the channel run down. Furthermore, PP1 inhibitor-2 mainly prolonged the open time constants of the channel, specifically, the slow open time. Fostriecin primarily shortened the slow close time constants. Our data suggest that PP1 and PP2A were involved in the slow phase of Cav1.2 Ca(2+) channel run down. In addition, they exerted different effects on the open-close kinetics of the channel. All above support the view that PP1 and PP2A may dephosphorylate distinct phosphorylation sites on the Cav1.2 Ca(2+) channel.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Miócitos Cardíacos/fisiologia , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Animais , Células Cultivadas , Feminino , Cobaias , Cinética , Potenciais da Membrana/fisiologia
14.
Am J Physiol Cell Physiol ; 310(2): C136-41, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26561637

RESUMO

Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca(2+) channels (Ca(V)1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the Ca(V)1.2 channel activity is regulated by additional cytoplasmic factors. To test this hypothesis, we examined the role of cAMP-dependent protein kinase A (PKA) and protein phosphatases in the regulation of Ca(V)1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. Ca(V)1.2 channel activity quickly disappeared after the patch was excised from the cell and recovered to only 9% of that in the cell-attached mode on application of CaM + ATP at 10 min after the inside out. However, immediate exposure of the excised patch to the catalytic subunit of PKA + ATP or the nonspecific phosphatase inhibitor okadaic acid significantly increased the Ca(V)1.2 channel activity recovery by CaM + ATP (114 and 96%, respectively) at 10 min. Interestingly, incubation of the excised patches with cAMP + ATP also increased CaM/ATP-induced Ca(V)1.2 channel activity recovery (108%), and this effect was blocked by the nonspecific protein kinase inhibitor K252a. The channel activity in the inside-out mode was not maintained by either catalytic subunit of PKA or cAMP + ATP in the absence of CaM, but was stably maintained in the presence of CaM for more than 40 min. These results suggest that PKA and phosphatase(s) attached on or near the Ca(V)1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miócitos Cardíacos/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sistema Livre de Células , Células Cultivadas , Cobaias , Ativação do Canal Iônico/fisiologia
15.
J Pharmacol Sci ; 129(3): 143-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26422671

RESUMO

This study aimed to investigate the intracellular Mg(2+) regulation of the L-type Ca(2+) channels in guinea pig ventricular myocytes. By adopting the inside-out configuration of the patch clamp technique, single channel currents of the L-type Ca(2+) channels were recorded at different intracellular Mg(2+) concentrations ([Mg(2+)]i). At free [Mg(2+)]i of 0, 10(-9), 10(-7), 10(-5), 10(-3), and 10(-1) M, 1.4 µM CaM + 3 mM ATP induced channel activities of 44%, 117%, 202%, 181%, 147%, and 20% of the control activity in cell-attached mode, respectively, showing a bell-shaped concentration-response relationship. Moreover, the intracellular Mg(2+) modulated the Ca(2+) channel gating properties, accounting for alterations in channel activities. These results imply that Mg(2+) has a dual effect on the L-type Ca(2+) channels: facilitation and inhibition. Lower [Mg(2+)]i maintains and enhances the basal activity of Ca(2+) channels, whereas higher [Mg(2+)]i inhibits channel activity. Taken together, our data from the application of an [Mg(2+)]i series suggest that the dual effect of Mg(2+) upon the L-type Ca(2+) channels exhibits long open-time dependence.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Magnésio/fisiologia , Células Musculares/metabolismo , Animais , Células Cultivadas , Cobaias , Ventrículos do Coração/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Magnésio/farmacologia , Técnicas de Patch-Clamp/métodos
16.
J Pharmacol Sci ; 128(3): 137-43, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26169579

RESUMO

Although it has been well documented that redox can modulate Cav1.2 channel activity, the underlying mechanisms are not fully understood. In our study, we examined the effects of redox on Cav1.2 channel activity and on CaM interaction with the Cav1.2 α1 subunit. Dithiothreitol (DTT, 1 mM) in the cell-attached mode decreased, while hydrogen peroxide (H2O2, 1 mM) increased channel activity to 72 and 303%, respectively. The effects of redox were maintained in the inside-out mode where channel activity was induced by CaM + ATP: DTT (1 mM) decreased, while H2O2 (1 mM) increased the channel activity. These results were mimicked by the thioredoxin and oxidized glutathione system. To test whether the redox state might determine channel activity by affecting the CaM interaction with the channel, we examined the effects of DTT and H2O2 on CaM binding to the N- and C-terminal fragments of the channel. We found that DTT concentration-dependently inhibited CaM binding to the C-terminus (IC50 37 µM), but H2O2 had no effect. Neither DTT nor H2O2 had an effect on CaM interaction with the N-terminus. These results suggest that redox-mediated regulation of the Cav1.2 channel is governed, at least partially, by modulation of the CaM interaction with the channel.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Calmodulina/fisiologia , Miócitos Cardíacos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Canais de Cálcio Tipo L/metabolismo , Calmodulina/metabolismo , Células Cultivadas , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Cobaias , Ventrículos do Coração/citologia , Peróxido de Hidrogênio/farmacologia , Oxirredução
17.
Biochem Biophys Res Commun ; 460(3): 813-8, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25824040

RESUMO

The activity of Cav1.2 Ca(2+) channels is maintained in the presence of calmodulin and ATP, even in cell-free patches, and thus a channel ATP-binding site has been suggested. In this study, we examined whether other nucleotides, such as GTP, UTP, CTP, ADP and AMP, could be substituted for ATP in guinea-pig ventricular myocytes. We found that all the nucleotides tested could re-prime the Ca(2+) channels in the presence of 1 µM calmodulin in the inside-out mode. The order of efficacy was ATP > GTP > UTP > ADP > CTP ≈ AMP. Thus, the presumed nucleotide-binding site in the channel seemed to favor a purine rather than pyrimidine base and a triphosphate rather than a di- or mono-phosphate group. Furthermore, a high concentration (10 mM) of GTP, UTP, CTP, ADP and AMP had inhibitory effects on the channel activity. These results provide information on the putative nucleotide-binding site(s) in Cav1.2 Ca(2+) channels.


Assuntos
Canais de Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Músculo Liso Vascular/metabolismo , Nucleotídeos/fisiologia , Animais , Cobaias , Ventrículos do Coração/citologia , Músculo Liso Vascular/citologia
19.
FEBS Lett ; 588(21): 3855-61, 2014 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-25268113

RESUMO

The present study examined the binding of the individual N- and C-lobes of calmodulin (CaM) to Cav1.2 at different Ca(2+) concentration ([Ca(2+)]) from ≈ free to 2mM, and found that they may bind to Cav1.2 Ca(2+)-dependently. In particular, using the patch-clamp technique, we confirmed that the N- or C-lobes can rescue the basal activity of Cav1.2 from run-down, demonstrating the functional relevance of the individual lobes. The data imply that at resting [Ca(2+)], CaM may tether to the channel with its single lobe, leading to multiple CaM molecule binding to increase the grade of Ca(2+)-dependent regulation of Cav1.2.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Ventrículos do Coração/citologia , Miócitos Cardíacos/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Calmodulina/genética , Fenômenos Eletrofisiológicos , Cobaias , Células HEK293 , Ventrículos do Coração/fisiopatologia , Humanos , Mutagênese Sítio-Dirigida , Mutação
20.
Am J Physiol Cell Physiol ; 307(11): C999-C1009, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25209265

RESUMO

Cardiac L-type Ca(2+) channels are modulated by phosphorylation by protein kinase A (PKA). To explore the PKA-targeted phosphorylation site(s), five potential phosphorylation sites in the carboxyl (COOH) terminal region of the α1C-subunit of the guinea pig Cav1.2 Ca(2+) channel were mutated by replacing serine (S) or threonine (T) residues with alanine (A): S1574A (C1 site), S1626A (C2), S1699A (C3), T1908A, (C4), S1927A (C5), and their various combinations. The wild-type Ca(2+) channel activity was enhanced three- to fourfold by the adenylyl cyclase activator forskolin (Fsk, 5 µM), and that of mutants at C3, C4, C5, and combination of these sites was also significantly increased by Fsk. However, Fsk did not modulate the activity of the C1 and C2 mutants and mutants of combined sites involving the C1 site. Three peptides of the COOH-terminal tail of α1C, termed CT1 [corresponding to amino acids (aa) 1509-1789, containing sites C1-3], CT2 (aa 1778-2003, containing sites C4 and C5), and CT3 (aa 1942-2169), were constructed, and their phosphorylation by PKA was examined. CT1 and CT2, but not CT3, were phosphorylated in vitro by PKA. Three CT1 mutants at two sites of C1-C3 were also phosphorylated by PKA, but the mutant at all three sites was not. The CT2 mutant at the C4 site was phosphorylated by PKA, but the mutant at C5 sites was not. These results suggest that Ser(1574) (C1 site) may be a potential site for the channel modulation mediated by PKA.


Assuntos
Canais de Cálcio Tipo L/metabolismo , AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Domínio Catalítico , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Mucosa Intestinal/citologia , Leucotrieno D4/farmacologia , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Ratos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...