抄録
Hyperpolarization activated, cyclic nucleotide sensitive cation channels (Ih) of cardiac myocytes are composed of HCN1, HCN2 and HCN4. These molecules are highly expressed in the pacemaker cells in the heart. Low level of HCN2 are expressed in ventricular myocytes, and is upregulated in cardiac hypertrophy. In the present study, we aimed to clarify the transcriptional mechanism of HCN2 gene. Fisrt, We carried out in-silico search for enhancer elements in mouse hcn2 gene. By comparing rat, mouse, chicken, dog and human genome sequences using VISTA browser, we identified only one conserved region (–200 bps) in 5'region between neighboring gene, and designated it CNS#1. Proximal promoter region of hcn2 was also conserved, although its homology was lower than that of CNS#1. Proximal promoter lacked consensus TATA motif, but contained multiple CpG sites. We next carried out luciferase reporter assay using proximal promoter and CNS#1 of mouse hcn2 gene. Genomic fragments were subcloned into pGL4.10 vector, and transfected into primary cultured, neonatal cardiac myocytes. Deletion study of demonstrated that CNS#1 contained both positive- and negative regulatory function on the activity of proximal promoter. The genomic region other than CNS#1 and proximal promoter did not possess significant regulatory role. Thus, in silico approach appeared very useful to determine cis-element of ion channel genes. [J Physiol Sci. 2007;57 Suppl:S227]
