RESUMO
Expression of L-type calcium channels in cardiac myocytes and vascular smooth muscle cells (VSMC) critically regulates the contractile state of these cells. In order to discover the elements in the promoter region of the Ca(v)1.2 gene encoding the vascular/cardiac calcium channel alpha(1C) subunit that are important for the basal gene expression, approximately 2 kb of the 5'-flanking sequence of the Ca(v)1.2 gene has been cloned in our lab. In this study, using various lengths of the 5'-flanking DNA fused with a luciferase gene as a reporter, we have defined a 493-bp fragment of the cis-regulatory DNA which carries the majority of promoter activity in pulmonary artery smooth muscle (PAC1) cells. DNase I footprinting analysis of this 493-bp DNA using nuclear extracts from PAC1 cells revealed a 27-bp DNA sequence that contains a c-Ets like motif (CAGGATGC). Mutation of the Ets-like site and the respective flanking sequence within the DNase I footprinting protection region induced a marked change in the promoter activity in PAC1 cells. Electrophoretic mobility shift assays (EMSA) confirmed the presence of specific binding factor(s) in PAC1 cells' nuclear extracts for this 27-bp DNA. Competition studies with the wild-type and mutated DNA fragments established the importance of the 27 bp DNA sequence for high-affinity binding of the nuclear proteins to the promoter. We conclude that there is a 27 bp region in the promoter of the Ca(v)1.2 gene to which nuclear proteins from VSMC bind and strongly regulate the basal promoter activity.
Assuntos
Canais de Cálcio Tipo L/genética , Músculo Liso Vascular/metabolismo , Regiões Promotoras Genéticas/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Canais de Cálcio Tipo L/biossíntese , Linhagem Celular , Pegada de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Artéria Pulmonar , Ratos , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The alpha1C subunit is the pore-forming protein for the L-type calcium channel. Previous studies indicate that there is possible tissue-specific alternative splicing of this gene. In this study we cloned the entire open reading frame of the alpha1C subunit cDNA from adult rat cardiac myocytes in a single piece (6.64 kb). Using 75 positive clones that were identified by restriction enzyme mapping, we tested the alternative splicing patterns of the Ca(v) 1.2 gene that encodes the alpha1C subunit protein and focused on five loci: IS6, post-IS6, IIIS2, IVS3, and the c-terminus. The results indicate that: (1) alternative splicing occurs in most of the loci, giving rise to two or three different isoforms at those sites; (2) there is a predominant form for each splicing site, (3) there does not appear to be consistent coordination of splicing at multiple loci of this gene. Alternative splicing is not tissue-specific in most regions.
Assuntos
Processamento Alternativo , Canais de Cálcio Tipo L/genética , Sítios de Splice de RNA , Animais , Clonagem Molecular , Masculino , Miócitos Cardíacos/metabolismo , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Mapeamento por Restrição , Distribuição TecidualRESUMO
To test the hypothesis that mutated beta2-subunits of the L-type calcium channel could serve as a decoy and interdict calcium channel trafficking and function, we engineered a beta2 subunit that contained the beta interaction domain for alpha1c subunit interaction, but lacked N- and C-terminal domains that might be essential for sarcolemmal localization. An adenoviral vector was constructed containing the gene for the beta-interaction domain (BID) fused to green fluorescence protein (GFP), using a vector containing only GFP as control. Freshly plated, dissociated adult rat myocytes were infected and expression and function were assessed at 60 h. Fluorescence microscopy confirmed GFP expression; immunoblot analysis confirmed dose-dependent GFP-BID expression. Mechanical properties of adult rat ventricular myocytes were evaluated using a video edge-detection system. Contractility analysis (optical/video, field stimulation) demonstrated that contracting cells decreased from 60 to 2%. Contractile amplitude (percent shortening) decreases significantly from 5.6 vs. 2.4% with no change in time to peak twitch. Recombinant adenovirus overexpressing mutated beta2 subunits in adult mammalian myocytes can markedly alter excitation-contraction coupling. This paradigm may offer new approaches to understanding and modulating EC coupling.