Cloning and characterization of 5′-flanking region of mouse non-selective cation channel 1

We have previously cloned mouse non-selective cation channel 1 (mNSCI) cDNA inducing cation current, from a mouse insulin secreting beta-cell line, MIN6. The current has characteristics of the Ca2+-activated non-selective (CAN) cation channel, and the mRNA is localized in the brain, heart, and lung. To understand the molecular mechanisms of the transcriptional regulation, we have cloned and characterized the 5'-flanking region of mNSCI. By the PCR method, we obtained 987 bp of mouse genomic fragment. The computer program-based analysis revealed that it contained several consensus motifs; insulin responsive element (IRE), AP-2, PEA3, and GC box-like region. But there were neither typical TATA box nor CAAT box. Primer extension analysis and RNase protection assay were performed to identify the transcription start site. Transient transfection analyses using a series of 5'-end deletion and reporter gene constructs with CHO and LA-4 cells demonstrated some relatively active regions. The significantly active border correlated with IRE consensus with CHO cells. This observation may support that CAN current is activated by insulin. (C) 1999 Elsevier Science B.V. All rights reserved.