Assuntos
Antraz/metabolismo , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Canais de Cálcio/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Fenômenos Eletrofisiológicos , Potenciais da Membrana , Ligação Proteica , Conformação Proteica , Transdução de SinaisRESUMO
The voltage-gated Cav1.2 calcium channels respond to membrane depolarization by increasing the membrane permeability to Ca(2+), a major signal for cardiac muscle contraction, regulation of vascular tone and CREB-dependent transcriptional activation. CACNB2 is one of the four homologous genes coding for the auxiliary Cavß subunits, which are important modulators of the Ca(2+) channel activity. Five serious mental disorders - autism spectrum disorder, attention deficit-hyperactivity disorder, bipolar disorder, major depressive disorder, and schizophrenia, - and three major cardiovascular diseases - hypertension, heart failure and sudden cardiac death, - have recently been linked to the CACNB2 gene coding for the Cavß2 subunits. Here I will focus on the Cavß2-specific molecular determinant ß2-CED as an emerging pharmacological target.
Assuntos
Arritmias Cardíacas/tratamento farmacológico , Canais de Cálcio Tipo L/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Hipertensão/tratamento farmacológico , Transtornos Mentais/tratamento farmacológico , Canais de Cálcio Tipo L/genética , HumanosRESUMO
Cav1.2 calcium channels are the principal proteins involved in electrical, mechanical, and/or signaling functions of the cell. Cav1.2 couples membrane depolarization to the transient increase in intracellular Ca(2+) concentration that is a trigger for muscle contraction and CREB-dependent transcriptional activation. The CACNA1C gene coding for the Cav1.2 pore-forming α 1C subunit is subject to extensive alternative splicing. This review is the first attempt to follow the association between cell proliferation, Cav1.2 expression and splice variation, and atherosclerosis. Based on insights into the association between the atherosclerosis-induced molecular remodeling of Cav1.2, proliferation of vascular smooth muscle cells, and CREB-dependent transcriptional signaling, this review will give a perspective outlook for the use of the CACNA1C exon skipping as a new potential gene therapy approach to atherosclerosis.
RESUMO
Fluorophore-assisted light inactivation (FALI) is an investigative tool to inactivate fluorescently labeled proteins by a mechanism of in situ photodestruction. We found that Ca(v)1.2 (L-type) and Ca(v)3.1 (T-type) calcium channels, labeled by genetic fusion with GFP derivatives, show differential sensitivity to FALI. Specifically, FALI silences Ca(v)1.2 calcium channels containing EYFP-labeled α(1C)subunits but does not affect the EYFP-α(1G) Ca(v)3.1 calcium channels or Ca(v)1.2 channels containing EYFP-labeled ß subunits. Our findings limit the applicability of acceptor photobleaching for the measurements of FRET but open an opportunity to combine the fluorescent imaging of the live cell expressing labeled calcium channels with selective functional inactivation of their specific subsets.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo T/metabolismo , Inativação Luminosa Assistida por Cromóforo , Animais , Células COS , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo T/genética , Chlorocebus aethiops , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ativação do Canal Iônico , Microscopia de Fluorescência/métodos , Fotodegradação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Raios UltravioletaRESUMO
One of the most consistent genetic findings to have emerged from bipolar disorder genome wide association studies (GWAS) is with CACNA1C, a gene that codes for the α(1C) subunit of the Ca(v)1.2 voltage-dependent L-type calcium channel (LTCC). Genetic variation in CACNA1C have also been associated with depression, schizophrenia, autism spectrum disorders, as well as changes in brain function and structure in control subjects who have no diagnosable psychiatric illness. These data are consistent with a continuum of shared neurobiological vulnerability between diverse-Diagnostic and Statistical Manual (DSM) defined-neuropsychiatric diseases. While involved in numerous cellular functions, Ca(v)1.2 is most frequently implicated in coupling of cell membrane depolarization to transient increase of the membrane permeability for calcium, leading to activation and, potentially, changes in intracellular signaling pathway activity, gene transcription, and synaptic plasticity. Ca(v)1.2 is involved in the proper function of numerous neurological circuits including those involving the hippocampus, amygdala, and mesolimbic reward system, which are strongly implicated in psychiatric disease pathophysiology. A number of behavioral effects of LTCC inhibitors have been described including antidepressant-like behavioral actions in rodent models. Clinical studies suggest possible treatment effects in a subset of patients with mood disorders. We review the genetic structure and variation of CACNA1C, discussing relevant human genetic and clinical findings, as well as the biological actions of Ca(v)1.2 that are most relevant to psychiatric illness.
Assuntos
Canais de Cálcio Tipo L/genética , Variação Genética/genética , Transtornos Mentais/genética , Transtornos Mentais/fisiopatologia , HumanosRESUMO
Voltage-gated L-type Cav1.2 calcium channels couple membrane depolarization to transient increase in cytoplasmic free Ca(2+) concentration that initiates a number of essential cellular functions including cardiac and vascular muscle contraction, gene expression, neuronal plasticity, and exocytosis. Inactivation or spontaneous termination of the calcium current through Cav1.2 is a critical step in regulation of these processes. The pathophysiological significance of this process is manifested in hypertension, heart failure, arrhythmia, and a number of other diseases where acceleration of the calcium current decay should present a benefit function. The central issue of this paper is the inactivation of the Cav1.2 calcium channel mediated by multiple determinants.
RESUMO
Voltage-gated Ca(v)1.2 calcium channels couple membrane depolarization to cAMP response-element-binding protein (CREB)-dependent transcriptional activation. To investigate the spatial and temporal organization of CREB-dependent transcriptional nuclear microdomains, we combined perforated patch-clamp technique and FRET microscopy for monitoring CREB and CREB-binding protein interaction in the nuclei of live cells. The experimental approach to the quantitative assessment of CREB-dependent transcriptional signaling evoked by cAMP- and Ca(v)1.2-dependent mechanisms was devised in COS1 cells expressing recombinant Ca(v)1.2 calcium channels. Using continuous 2-dimensional wavelet transform and time series analyses, we found that nuclear CREB-dependent transcriptional signaling is organized differentially in spatially and temporally separated microdomains of 4 distinct types. In rat neonatal cardiomyocytes, CREB-dependent transcription is mediated by the cAMP-initiated CaMKII-sensitive and Ca(v)1.2-initiated CaMKII-insensitive mechanisms. The latter microdomains show a tendency to exhibit periodic behavior correlated with spontaneous contraction of myocytes suggestive of frequency-dependent CREB-dependent transcriptional regulation in the heart.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Animais , Animais Recém-Nascidos , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Eletrofisiologia , Modelos Teóricos , Miócitos Cardíacos/metabolismo , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transcrição Gênica/genéticaRESUMO
Based on stable integration of recombinant DNA into a host genome, transgenic technology has become an important genetic engineering methodology. An organism whose genetic characteristics have been altered by the insertion of foreign DNA is supposed to exhibit a new phenotype associated with the function of the transgene. However, successful insertion may not be sufficient to achieve specific modification of function. In this study we describe a strain of transgenic mouse, G7-882, generated by incorporation into the mouse genome of human CaV 1.2 α(1C) cDNA deprived of 3'-UTR to exclude transcription. We found that, in response to chronic infusion of isoproterenol, G7-882 develops dilated cardiomyopathy, a misleading "transgenic artifact" compatible with the expected function of the incorporated "correct" transgene. Specifically, using magnetic resonance imaging (MRI), we found that chronic ß-adrenergic stimulation of G7-882 mice caused left ventricular hypertrophy and aggravated development of dilated cardiomyopathy, although no significant changes in the kinetics, density and voltage dependence of the calcium current were observed in G7-882 cardiomyocytes as compared to cells from wild type mice. This result illustrates the possibility that even when a functional transgene is expressed, an observed change in phenotype may be due to the artifact of "incidental incorporation" leading to misleading conclusions. To exclude this possibility and thus provide a robust tool for exploring biological function, the new transgenic phenotype must be replicated in several independently generated transgenic strains.
Assuntos
Canais de Cálcio Tipo L/genética , Técnicas Genéticas , Isoproterenol/farmacologia , Transgenes , Regiões 3' não Traduzidas , Animais , Canais de Cálcio Tipo L/metabolismo , Cardiomiopatia Dilatada/patologia , Humanos , Hipertrofia Ventricular Esquerda/patologia , Cinética , Imageamento por Ressonância Magnética/métodos , Imagem Cinética por Ressonância Magnética/métodos , Camundongos , Camundongos Transgênicos , Fenótipo , Receptores Adrenérgicos beta/metabolismo , Receptores de IgGRESUMO
Voltage-gated calcium channels conduct Ca(2+) ions in response to membrane depolarization. The resulting transient increase in cytoplasmic free calcium concentration is a critical trigger for the initiation of such vital responses as muscle contraction and transcription. L-type Ca(v)1.2 calcium channels are complexes of the pore-forming α(1C) subunit associated with cytosolic Ca(v)ß subunits. All major Ca(v)ßs share a highly homologous membrane associated guanylate kinase-like (MAGUK) domain that binds to α(1C) at the α-interaction domain (AID), a short motif in the linker between transmembrane repeats I and II. In this study we show that Ca(v)ß subunits form multimolecular homo- and heterooligomeric complexes in human vascular smooth muscle cells expressing native calcium channels and in Cos7 cells expressing recombinant Ca(v)1.2 channel subunits. Ca(v)ßs oligomerize at the α(1C) subunits residing in the plasma membrane and bind to the AID. However, Ca(v)ß oligomerization occurs independently on the association with α(1C). Molecular structures responsible for Ca(v)ß oligomerization reside in 3 regions of the guanylate kinase subdomain of MAGUK. An augmentation of Ca(v)ß homooligomerization significantly increases the calcium current density, while heterooligomerization may also change the voltage-dependence and inactivation kinetics of the channel. Thus, oligomerization of Ca(v)ß subunits represents a novel and essential aspect of calcium channel regulation.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Subunidades Proteicas/metabolismo , Western Blotting , Canais de Cálcio Tipo L/genética , Células Cultivadas , Eletrofisiologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Imunoprecipitação , Microscopia Confocal , Multimerização Proteica/genética , Multimerização Proteica/fisiologia , Subunidades Proteicas/genéticaRESUMO
Existing molecular knowledge base of cardiovascular diseases is rudimentary because of lack of specific attribution to cell type and function. The aim of this study was to investigate cell-specific molecular remodeling in human atrial and ventricular myocytes associated with ischemic cardiomyopathy. Our strategy combines two technological innovations, laser-capture microdissection of identified cardiac cells in selected anatomical regions of the heart and splice microarray of a narrow catalog of the functionally most important genes regulating ion homeostasis. We focused on expression of a principal family of genes coding for ion channels, exchangers and pumps (CE&P genes) that are involved in electrical, mechanical and signaling functions of the heart and constitute the most utilized drug targets. We found that (1) CE&P genes remodel in a cell-specific manner: ischemic cardiomyopathy affected 63 CE&P genes in ventricular myocytes and 12 essentially different genes in atrial myocytes. (2) Only few of the identified CE&P genes were previously linked to human cardiac disfunctions. (3) The ischemia-affected CE&P genes include nuclear chloride channels, adrenoceptors, cyclic nucleotide-gated channels, auxiliary subunits of Na(+), K(+) and Ca(2+) channels, and cell-surface CE&Ps. (4) In both atrial and ventricular myocytes ischemic cardiomyopathy reduced expression of CACNG7 and induced overexpression of FXYD1, the gene crucial for Na(+) and K(+) homeostasis. Thus, our cell-specific molecular profiling defined new landmarks for correct molecular modeling of ischemic cardiomyopathy and development of underlying targeted therapies.
Assuntos
Cardiomiopatias/genética , Canais Iônicos/genética , Bombas de Íon/genética , Isquemia Miocárdica/genética , Miócitos Cardíacos/química , Adulto , Idoso , Transporte Biológico , Cardiomiopatias/etiologia , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Átrios do Coração/química , Ventrículos do Coração/química , Humanos , Masculino , Microdissecção/métodos , Pessoa de Meia-Idade , Isquemia Miocárdica/complicações , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análiseRESUMO
BACKGROUND: Voltage-gated Ca(v)1.2 calcium channels play a crucial role in Ca(2+) signaling. The pore-forming alpha(1C) subunit is regulated by accessory Ca(v)beta subunits, cytoplasmic proteins of various size encoded by four different genes (Ca(v)beta(1)-beta(4)) and expressed in a tissue-specific manner. METHODS AND RESULTS: Here we investigated the effect of three major Ca(v)beta types, beta(1b), beta(2d) and beta(3), on the structure of Ca(v)1.2 in the plasma membrane of live cells. Total internal reflection fluorescence microscopy showed that the tendency of Ca(v)1.2 to form clusters depends on the type of the Ca(v)beta subunit present. The highest density of Ca(v)1.2 clusters in the plasma membrane and the smallest cluster size were observed with neuronal/cardiac beta(1b) present. Ca(v)1.2 channels containing beta(3), the predominant Ca(v)beta subunit of vascular smooth muscle cells, were organized in a significantly smaller number of larger clusters. The inter- and intramolecular distances between alpha(1C) and Ca(v)beta in the plasma membrane of live cells were measured by three-color FRET microscopy. The results confirm that the proximity of Ca(v)1.2 channels in the plasma membrane depends on the Ca(v)beta type. The presence of different Ca(v)beta subunits does not result in significant differences in the intramolecular distance between the termini of alpha(1C), but significantly affects the distance between the termini of neighbor alpha(1C) subunits, which varies from 67 A with beta(1b) to 79 A with beta(3). CONCLUSIONS: Thus, our results show that the structural organization of Ca(v)1.2 channels in the plasma membrane depends on the type of Ca(v)beta subunits present.
Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo N/metabolismo , Subunidades Proteicas/metabolismo , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo N/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Proteínas Luminescentes/metabolismo , Subunidades Proteicas/química , Proteína Vermelha FluorescenteRESUMO
Voltage-activated CaV1.2 calcium channels require association of the pore-forming alpha1C subunit with accessory CaVbeta and alpha2delta subunits. Binding of a single calmodulin (CaM) to alpha1C supports Ca2+-dependent inactivation (CDI). The human CaV1.2 channel is silent in the absence of CaVbeta and/or alpha2delta. Recently, we found that coexpression of exogenous CaM (CaMex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of CaVbeta. Here we discovered that CaMex and its Ca2+-insensitive mutant (CaM1234) rendered active alpha1C/CaVbeta channel in the absence of alpha2delta. Coexpression of CaMex with alpha1C and beta2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by approximately +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca2+) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaMex and CaM1234 accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal CaV1.2 channel. The data suggest a previously unknown action of CaM that in the presence of CaVbeta; translates into activation of the alpha2delta-deficient calcium channel and alteration of its properties.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Ativação do Canal Iônico , Animais , Células COS , Canais de Cálcio/genética , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Calmodulina/genética , Chlorocebus aethiops , Humanos , Potenciais da Membrana , Modelos Biológicos , Mutação , Fatores de Tempo , TransfecçãoRESUMO
It is generally accepted that to generate calcium currents in response to depolarization, Ca(v)1.2 calcium channels require association of the pore-forming alpha(1C) subunit with accessory Ca(v)beta and alpha(2)delta subunits. A single calmodulin (CaM) molecule is tethered to the C-terminal alpha(1C)-LA/IQ region and mediates Ca2+-dependent inactivation of the channel. Ca(v)beta subunits are stably associated with the alpha(1C)-interaction domain site of the cytoplasmic linker between internal repeats I and II and also interact dynamically, in a Ca2+-dependent manner, with the alpha(1C)-IQ region. Here, we describe a surprising discovery that coexpression of exogenous CaM (CaM(ex)) with alpha(1C)/alpha(2)delta in COS1 cells in the absence of Ca(v)beta subunits stimulates the plasma membrane targeting of alpha(1C), facilitates calcium channel gating, and supports Ca2+-dependent inactivation. Neither real-time PCR with primers complementary to monkey Ca(v)beta subunits nor coimmunoprecipitation analysis with exogenous alpha(1C) revealed an induction of endogenous Ca(v)beta subunits that could be linked to the effect of CaM(ex). Coexpression of a calcium-insensitive CaM mutant CaM(1234) also facilitated gating of Ca(v)beta-free Ca(v)1.2 channels but did not support Ca2+-dependent inactivation. Our results show there is a functional matchup between CaM(ex) and Ca(v)beta subunits that, in the absence of Ca(v)beta, renders Ca2+ channel gating facilitated by CaM molecules other than the one tethered to LA/IQ to support Ca2+-dependent inactivation. Thus, coexpression of CaM(ex) creates conditions when the channel gating, voltage- and Ca2+-dependent inactivation, and plasma-membrane targeting occur in the absence of Ca(v)beta. We suggest that CaM(ex) affects specific Ca(v)beta-free conformations of the channel that are not available to endogenous CaM.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Calmodulina/metabolismo , Ativação do Canal Iônico , Subunidades Proteicas/metabolismo , Animais , Células COS , Cálcio/farmacologia , Calmodulina/farmacologia , Chlorocebus aethiops , Eletrofisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/metabolismoRESUMO
Ca(v)beta subunits support voltage gating of Ca(v)1.2 calcium channels and play important role in excitation-contraction coupling. The common central membrane-associated guanylate kinase (MAGUK) region of Ca(v)beta binds to the alpha-interaction domain (AID) and the IQ motif of the pore-forming alpha(1C) subunit, but these two interactions do not explain why the cardiac Ca(v)beta(2) subunit splice variants differentially modulate inactivation of Ca(2+) currents (I(Ca)). Previously we described beta(2Deltag), a functionally active splice variant of human Ca(v)beta(2) lacking MAGUK. By deletion analysis of beta(2Deltag), we have now identified a 41-amino acid C-terminal essential determinant (beta(2)CED) that stimulates I(Ca) in the absence of Ca(v)beta subunits and conveys a +20-mV shift in the peak of the I(Ca)-voltage relationship. The beta(2)CED is targeted by alpha(1C) to the plasma membrane, forms a complex with alpha(1C) but does not bind to AID. Electrophysiology and binding studies point to the calmodulin-interacting LA/IQ region in the alpha(1C) subunit C terminus as a functionally relevant beta(2)CED binding site. The beta(2)CED interacts with LA/IQ in a Ca(2+)- and calmodulin-independent manner and need LA, but not IQ, to activate the channel. Deletion/mutation analyses indicated that each of the three Ca(v)beta(2)/alpha(1C) interactions is sufficient to support I(Ca). However, beta(2)CED does not support Ca(2+)-dependent inactivation, suggesting that interactions of MAGUK with AID and IQ are crucial for Ca(2+)-induced inactivation. The beta(2)CED is conserved only in Ca(v)beta(2) subunits. Thus, beta(2)CED constitutes a previously unknown integrative part of the multifactorial mechanism of Ca(v)beta(2)-subunit differential modulation of the Ca(v)1.2 calcium channel that in beta(2Deltag) occurs without MAGUK.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Subunidades Proteicas/metabolismo , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos/genética , Sítios de Ligação/fisiologia , Canais de Cálcio Tipo L/genética , Calmodulina/genética , Calmodulina/metabolismo , Linhagem Celular , Membrana Celular/genética , Humanos , Miocárdio/metabolismo , Estrutura Terciária de Proteína/fisiologia , Subunidades Proteicas/genética , Deleção de SequênciaRESUMO
A technique that utilizes the one-dimensional (1D) continuous wavelet transform (CWT) of linearized fluorescence resonance energy transfer (FRET) microscopic images has been extended to identify signaling macro- and microdomains in cell plasma membranes by incorporating the two-dimensional (2D) CWT of time-lapse fluorescence and/or FRET images. Signaling domains were identified from differences in wavelet coefficient matrices, and there was good agreement between the 1D and 2D methods on examining a), static fluorescent images of COS1 cells expressing calmodulin kinase II fused with enhanced yellow fluorescent protein, and b), time lapse FRET images of reporters of protein kinase C (PKC) (PKC activity reporter) and adenylyl cyclase dynamics (cAMP) activity within COS1 plasma membrane confines after stimulation by phorbol-12,13-dibutyrate or forskolin, respectively. The proposed 2D wavelet-based image analysis effectively detected phosphorylation/dephosphorylation signaling microdomains (PKC) as well as those reflective of cAMP without the limitation of requiring linearized signals imposed by the 1D approach. Illustrating successful application to the analysis of intracellular compartments, the 2D CWT was further used to identify signaling domains of cAMP response element-binding (CREB)-induced transcriptional activation in the nuclei of COS1 cells, which could not be achieved with the 1D approach. This technique may be eventually used to characterize complex cellular signaling and protein-protein interactions within localized cytoplasmic domains.
Assuntos
Algoritmos , Transferência Ressonante de Energia de Fluorescência/métodos , Interpretação de Imagem Assistida por Computador/métodos , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Transdução de Sinais/fisiologia , Animais , Células COS , Chlorocebus aethiops , Estrutura Terciária de ProteínaRESUMO
A three-day International Symposium entitled "Principles of Calcium Signaling" organized by James N. Weiss, Yale E. Goldman, Stéphane Hatem, Lars Cleemann and Nikolai M. Soldatov in honor of the research contributions of Professor Martin Morad was held at the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine. Support for this meeting was provided in part by GlaxoSmithKline, Leica Microsystems, Nikon Corp., St. Jude Medical, Inc., UCLA Cardiac Arrhythmia Center, Dr. Donald S. Orkand, Bob Hillis Family and OML, and Mount Desert Island Biological Laboratory. The symposium featured sessions on Cardiac physiology, Ion channels and Calcium signaling.
Assuntos
Sinalização do Cálcio , Animais , Arritmias Cardíacas/metabolismo , Canais de Cálcio/metabolismo , Células-Tronco Embrionárias/metabolismo , Humanos , Ativação do Canal Iônico , Potenciais da Membrana , Músculo Liso/metabolismo , Miócitos Cardíacos/metabolismo , Sinapses/metabolismoRESUMO
Atherosclerosis is an inflammatory process characterized by proliferation and dedifferentiation of vascular smooth muscle cells (VSMC). Ca(v)1.2 calcium channels may have a role in atherosclerosis because they are essential for Ca(2+)-signal transduction in VSMC. The pore-forming Ca(v)1.2alpha1 subunit of the channel is subject to alternative splicing. Here, we investigated whether the Ca(v)1.2alpha1 splice variants are affected by atherosclerosis. VSMC were isolated by laser-capture microdissection from frozen sections of adjacent regions of arteries affected and not affected by atherosclerosis. In VSMC from nonatherosclerotic regions, RT-PCR analysis revealed an extended repertoire of Ca(v)1.2alpha1 transcripts characterized by the presence of exons 21 and 41A. In VSMC affected by atherosclerosis, expression of the Ca(v)1.2alpha1 transcript was reduced and the Ca(v)1.2alpha1 splice variants were replaced with the unique exon-22 isoform lacking exon 41A. Molecular remodeling of the Ca(v)1.2alpha1 subunits associated with atherosclerosis caused changes in electrophysiological properties of the channels, including the kinetics and voltage-dependence of inactivation, recovery from inactivation, and rundown of the Ca(2+) current. Consistent with the pathophysiological state of VSMC in atherosclerosis, cell culture data pointed to a potentially important association of the exon-22 isoform of Ca(v)1.2alpha1 with proliferation of VSMC. Our findings are consistent with a hypothesis that localized changes in cytokine expression generated by inflammation in atherosclerosis affect alternative splicing of the Ca(v)1.2alpha1 gene in the human artery that causes molecular and electrophysiological remodeling of Ca(v)1.2 calcium channels and possibly affects VSMC proliferation.
Assuntos
Processamento Alternativo , Aterosclerose/genética , Canais de Cálcio Tipo L/genética , Idoso , Sequência de Aminoácidos , Aterosclerose/metabolismo , Aterosclerose/patologia , Sequência de Bases , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Proliferação de Células , Primers do DNA/genética , Éxons , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
T-type Ca2+ channels play essential roles in numerous cellular processes. Recently, we reported that phorbol-12-myristate-13-acetate (PMA) potently enhanced the current amplitude of Cav3.2 T-type channels reconstituted in Xenopus oocytes. Here, we have compared PMA modulation of the activities of Cav3.1, Cav3.2 and Cav3.3 channels, and have investigated the underlying mechanism. PMA augmented the current amplitudes of the three T-type channel isoforms, but the fold stimulations and time courses differed. The augmentation effects were not mimicked by 4alpha-PMA, an inactive stereoisomer of PMA, but were abolished by preincubation with protein kinase C (PKC) inhibitors, indicating that PMA augmented T-type channel currents via activation of oocyte PKC. The stimulation effect on Cav3.1 channel activity by PKC was mimicked by endothelin when endothelin receptor type A was coexpressed with Cav3.1 in the Xenopus oocyte system. Pharmacological studies combined with fluorescence imaging revealed that the surface density of Cav3.1 T-type channels was not significantly changed by activation of PKC. The PKC effect on Cav3.1 was localized to the cytoplasmic II-III loop using chimeric channels with individual cytoplasmic loops of Cav3.1 replaced by those of Cav2.1.
Assuntos
Canais de Cálcio Tipo T/efeitos dos fármacos , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo T/análise , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Relação Dose-Resposta a Droga , Endotelina-1/farmacologia , Ativação Enzimática/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Microinjeções , Mutação , Oócitos/química , Oócitos/metabolismo , Técnicas de Patch-Clamp , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptor de Endotelina A/efeitos dos fármacos , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Xenopus laevisRESUMO
The voltage-gated Kv2.1 channel is composed of four identical subunits folded around the central pore and does not inactivate appreciably during short depolarizing pulses. To study voltage-induced relative molecular rearrangements of the channel, Kv2.1 subunits were genetically fused with enhanced cyan fluorescent protein and/or enhanced yellow fluorescent protein, expressed in COS1 cells, and investigated using fluorescence resonance energy transfer (FRET) microscopy combined with patch clamp. Fusion of fluorophores to either or both termini of the Kv2.1 monomer did not significantly affect the gating properties of the channel. FRET between the N- and C-terminal tags fused to the same or different Kv2.1 monomers decreased upon activation of the channel by depolarization from -80 to +60 mV, suggesting voltage-gated relative rearrangement between the termini. Because FRET between the Kv2.1 N- or C-terminal tags and the membrane-trapped EYFP(N)-PH pleckstrin homology domains did not change on depolarization, voltage-gated relative movements between the Kv2.1 termini occurred in a plane parallel to the plasma membrane, within a distance of 1-10 nm. FRET between the N-terminal tags did not change upon depolarization, indicating that the N termini do not rearrange relative to each other, but they could either move cooperatively with the Kv2.1 tetramer or not move at all. No FRET was detected between the C-terminal tags. Assuming their randomized orientation in the symmetrically arranged Kv2.1 subunits, C termini may move outwards in order to produce relative rearrangements between N and C termini upon depolarization.