RESUMO
It is well known that the CaVα2δ auxiliary subunit regulates the density of high voltage-activated Ca2+ channels in the plasma membrane and that alterations in their functional expression might have implications in the pathophysiology of diverse human diseases such as neuropathic pain. However, little is known concerning the transcriptional regulation of this protein. We previously characterized the promoter of CaVα2δ, and here we report its regulation by the transcription factor Egr-1. Using the neuroblastoma N1E-115 cells, we found that Egr-1 interacts specifically with its binding site in the promoter, affecting the transcriptional regulation of CaVα2δ. Overexpression and knockdown analysis of Egr-1 showed significant changes in the transcriptional activity of the CaVα2δ promoter. Egr-1 also regulated the expression of CaVα2δ at the level of protein. Also, functional studies showed that Egr-1 knockdown significantly decreases Ca2+ currents in dorsal root ganglion (DRG) neurons, while overexpression of the transcription factor increased Ca2+ currents in the F11 cell line, a hybrid of DRG and N18TG2 neuroblastoma cells. Studying the effects of Egr-1 on the transcriptional expression of CaVα2δ could help to understand the regulatory mechanisms of this protein in both health and disease.
Assuntos
Canais de Cálcio/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Regulação da Expressão Gênica , Animais , Canais de Cálcio/genética , Linhagem Celular Tumoral , Gânglios Espinais/metabolismo , Camundongos , Neurônios/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Subunidades Proteicas/metabolismoRESUMO
Voltage-gated T-type Ca(2+) (CaV 3) channels mediate a number of physiological events in developing and mature cells, and are implicated in neurological and cardiovascular diseases. In mammals, there are three distinct T-channel genes (CACNA1G, CACNA1H, and CACNA1I) encoding proteins (CaV 3.1-CaV 3.3) that differ in their localization as well as in molecular, biophysical, and pharmacological properties. The CACNA1G is a large gene that contains 38 exons and is localized in chromosome 17q22. Only basic characteristics of the CACNA1G gene promoter region have been investigated classifying it as a TATA-less sequence containing several potential transcription factor-binding motifs. Here, we cloned and characterized a proximal promoter region and initiated the analysis of transcription factors that control CaV 3.1 channel expression using the murine Cacna1g gene as a model. We isolated a â¼1.5 kb 5'-upstream region of Cacna1g and verified its transcriptional activity in the mouse neuroblastoma N1E-115 cell line. In silico analysis revealed that this region possesses a TATA-less minimal promoter that includes two potential transcription start sites and four binding sites for the transcription factor Sp1. The ability of one of these sites to interact with the transcription factor was confirmed by electrophoretic mobility shift assays. Consistent with this, Sp1 over-expression enhanced promoter activity while siRNA-mediated Sp1 silencing significantly decreased the level of CaV 3.1 protein and reduced the amplitude of whole-cell T-type Ca(2+) currents expressed in the N1E-115 cells. These results provide new insights into the molecular mechanisms that control CaV 3.1 channel expression.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Fator de Transcrição Sp1/metabolismo , Animais , Sequência de Bases , Canais de Cálcio Tipo T/genética , Linhagem Celular , Clonagem Molecular , Regulação da Expressão Gênica , Inativação Gênica , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/genética , Canais de Ânion Dependentes de VoltagemRESUMO
The α2δ proteins are auxiliary subunits of high-voltage-activated Ca(2+) channels associated with alterations of surface expression, kinetics, and voltage-dependent properties of the channel complex. Four mammalian genes and several splice α2δ subunit variants have been cloned and described, though very little information concerning the transcriptional mechanisms that regulate their expression is available. Here, we report the identification and characterization of the human α2δ-1 subunit gene promoter and its regulation by specific transcription factor 1 (Sp1). Transient transfection of human neuroblastoma SH-SY5Y cells with a promoter/luciferase reporter construct revealed a ~1.5 kb 5´-UTR fragment of the CACNA2D1 gene that produced high levels of luciferase activity. Deletional analysis of this sequence showed that the minimal promoter was located within a 413-bp region (nt -326 to +98) with respect to the transcription start site. In this region, no canonical TATA box was present, but a high GC content and five potential Sp1 binding sites were found. The ability of two of these sites to interact with the transcription factor was confirmed by electrophoretic mobility shift assays. Likewise, Sp1 overexpression enhanced promoter activity while siRNA-mediated Sp1 silencing significantly decreased the level of α2δ protein expressed in the SH-SY5Y cells, as well as reduced the amplitude of whole-cell patch clamp Ca(2+) currents in dorsal root ganglion neurons. This study thus represents the first identification of the transcriptional control region in the gene encoding the Ca(2+) channel α2δ-1 auxiliary subunit.
Assuntos
Regiões 5' não Traduzidas , Canais de Cálcio/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Potenciais de Ação , Animais , Composição de Bases , Sequência de Bases , Sítios de Ligação , Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Gânglios Espinais/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neurônios/metabolismo , Neurônios/fisiologia , Análise de Sequência de DNA , Fator de Transcrição Sp1/genética , TATA Box , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
High-voltage activated Ca(2+) (Ca(V)) channels play a key role in the regulation of numerous physiological events by causing transient changes in the intracellular Ca(2+) concentration. These channels consist of a pore-forming Ca(V)α(1) protein and three auxiliary subunits (Ca(V)ß, Ca(V)α(2)δ and Ca(V)γ). Ca(V)α(2)δ is an important component of Ca(V) channels in many tissues and of great interest as a drug target. It is well known that anticonvulsant agent gabapentin (GBP) binds to Ca(V)α(2)δ and reduces Ca(2+) currents by modulating the expression and/or function of the Ca(V)α(1) subunit. Recently, we showed that an adamantane derivative of GABA, AdGABA, has also inhibitory effects on Ca(V) channels. However, the importance of the interaction of AdGABA with the Ca(V)α(2)δ subunit has not been conclusively demonstrated and the mechanism of action of the drug has yet to be elucidated. Here, we describe studies on the mechanism of action of AdGABA. Using a combined approach of patch-clamp recordings and molecular biology we show that AdGABA inhibits Ca(2+) currents acting on Ca(V)α(2)δ only when applied chronically, both in a heterologous expression system and in dorsal root-ganglion neurons. AdGABA seems to require uptake and be acting intracellularly given that its effects are prevented by an inhibitor of the L-amino acid transport system. Interestingly, a mutation in the Ca(V)α(2)δ that abolishes GBP binding did not affect AdGABA actions, revealing that its mechanism of action is similar but not identical to that of GBP. These results indicate that AdGABA is an important Ca(V)α(2)δ ligand that regulates Ca(V) channels.