Department of Molecular Biology and Histocompatibility, "Dr. Manuel Gea González" General Hospital, Mexico City, Mexico.
J Cell Physiol. 2014 May;229(5):551-60. doi: 10.1002/jcp.24432.
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 ∼1.5 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.
电压门控 T 型钙通道（CaV3）在发育和成熟细胞中介导许多生理事件，并与神经和心血管疾病有关。在哺乳动物中，有三个不同的 T 型通道基因（CACNA1G、CACNA1H 和 CACNA1I）编码的蛋白质（CaV3.1-CaV3.3）在其定位以及分子、生物物理和药理学特性上存在差异。CACNA1G 是一个包含 38 个外显子的大基因，位于染色体 17q22 上。仅对 CACNA1G 基因启动子区域的基本特征进行了研究，将其归类为不含 TATA 的序列，包含几个潜在的转录因子结合基序。在这里，我们克隆并鉴定了一个近端启动子区域，并开始使用小鼠 Cacna1g 基因作为模型分析控制 CaV3.1 通道表达的转录因子。我们分离了一个约 1.5kb 的 Cacna1g 5'上游区域，并验证了其在小鼠神经母细胞瘤 N1E-115 细胞系中的转录活性。计算机分析显示，该区域具有一个无 TATA 的最小启动子，包含两个潜在的转录起始位点和四个转录因子 Sp1 的结合位点。通过电泳迁移率变动分析证实了其中一个位点与转录因子相互作用的能力。与此一致的是，Sp1 的过表达增强了启动子活性，而 siRNA 介导的 Sp1 沉默显著降低了 N1E-115 细胞中 CaV3.1 蛋白的水平，并减少了全细胞 T 型 Ca(2+)电流的幅度。这些结果为控制 CaV3.1 通道表达的分子机制提供了新的见解。
