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Second Department of Internal Medicine, Tokyo Medical and Dental University, School of Medicine, 1-5-45 Yushima Bunkyo-ku, Tokyo 113, Japan
The rat ClC-K1 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 ~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 ~2.5-kb
fragment contained cis-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.
reporter gene assay; transfection; cis elememt; gel-retardation assay; rat kidney
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