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Cloning and characterization of 5'-flanking region of mouse non-selective cation channel 1
1Department of Pharmacology, Jichi Medical School, 3311-1 Minamikawachimachi, Tochigi 329-0498, Japan.
Abstract:
We have previously cloned mouse non-selective cation channel 1 (mNSC1) 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 mNSC1. 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.
Insights
Researchers identified regulatory elements in the mouse non-selective cation channel 1 (mNSC1) gene. These findings suggest insulin may activate calcium-activated non-selective (CAN) cation currents.
Area of Science:
- Molecular Biology
- Ion Channel Physiology
- Gene Regulation
Background:
- Mouse non-selective cation channel 1 (mNSC1) induces cation currents with characteristics of Ca2+-activated non-selective (CAN) channels.
- mNSC1 mRNA is found in various tissues, including the brain, heart, and lung, suggesting diverse physiological roles.
Purpose of the Study:
- To elucidate the molecular mechanisms governing the transcriptional regulation of the mNSC1 gene.
- To characterize the 5'-flanking region of mNSC1 and identify key regulatory elements.
Main Methods:
- Cloning and sequencing of the 987 bp 5'-flanking region of the mNSC1 gene.
- Bioinformatic analysis to identify consensus regulatory motifs (e.g., IRE, AP-2, PEA3, GC box).
- Reporter gene assays using deletion constructs in CHO and LA-4 cells to determine transcription start sites and active regions.
Main Results:
- The 5'-flanking region of mNSC1 lacks typical TATA and CAAT boxes but contains functional motifs like the insulin-responsive element (IRE).
- Transient transfection analyses identified specific regions of transcriptional activity.
- A significantly active region in CHO cells correlated with the IRE consensus, suggesting insulin's role in regulation.
Conclusions:
- The characterized 5'-flanking region contains regulatory elements crucial for mNSC1 gene transcription.
- The correlation between the IRE and transcriptional activity supports the hypothesis that insulin activates CAN currents via mNSC1.