Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter

被引:25
作者
Uchida, S [1 ]
Rai, T [1 ]
Yatsushige, H [1 ]
Matsumura, Y [1 ]
Kawasaki, M [1 ]
Sasaki, S [1 ]
Marumo, F [1 ]
机构
[1] Tokyo Med & Dent Univ, Dept Internal Med 2, Bunkyo Ku, Tokyo 113, Japan
关键词
reporter gene assay; transfection; cis element; gel-retardation assay; rat kidney;
D O I
10.1152/ajprenal.1998.274.3.F602
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
The rat CIC-KI: chloride channel is a kidney-specific member of the ClC chloride channel family found exclusively in the thin ascending limb of Henle's loop in the kidney. To gain insight into the mechanism(s) of kidney-specific expression of ClC-K1, a genomic clone that contains the 5'-flanking region of the rat ClC-K1 gene was isolated. A single transcription start site was located 84 bp upstream of the start codon. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-3 site, a glucocorticoid-responsive element, several AP-2 sites, and several E-boxes, but it lacked a TATA box. To functionally express the promoter, the similar to 2.5-kb pair 5'-flanking region was ligated to a luciferase reporter gene and transfected,into inner medullary (IM) cells, a stable ClC-K1-expressing cell line derived from the inner medulla of simian virus 40 transgenic mouse, and ClC-K1-nonexpressing cell lines. Luciferase activity was 7- to 24-fold greater in IM cells than those in nonexpressing cell lines, suggesting that the similar to 2.5-kb fragment contained cia-acting regulatory elements for cell-specific expression of the ClC-K1 gene. Deletion analysis revealed that this cell-specific promoter activity in IM cells was still present in the construct containing 51 bp of the 5'-flanking region but was lost in the -29 construct, clearly demonstrating that the 22 bp from -51 to -30 have a major role in the cell-specific activity of the ClC-K1 promoter. These 22 bp consist of purine-rich sequence (GGGGAGGGGGAGGGGAG), and gel-retardation analysis demonstrated the existence of a specific protein(s) binding to this element in IM cells. These results suggest that the novel purine-rich element may play a key role in the activity of the ClC-K1 gene promoter.
引用
收藏
页码:F602 / F610
页数:9
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