|Budget Amount *help
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1996: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
To determine the intrarenal localization of three chloride channels isolated by us, we prepared antisera for rat ClC-K1, -K2, -3 using synthetic peptide as antigens. We obtained specific antisera to ClC-K1 and ClC-K2. Using these antisera, we performed immunohistochemical study to localize the intrarenal localizaton of ClC-K1 and ClC-K2. ClC-K1 is present in the both apical and basolateral plasma membranes of the thin ascending limb of Henle's loop, and ClC-K2 is present in the apical plasma membrane of the connecting tubules. These findings will shed light on the physiological roles if ClC-K1 and -K2 in the kidney.
Next, we studied structure- function relationship of ClC-K1 and ClC-3. The in vitro mutations of positively charged amino acids in the first extracellular loop of ClC-K1 into negatively charged amino acids greatly enhanced the chloride current expressed in Xenopus oocytes, suggesting the importance of this region. Further studies using chimera chloride channels of ClC-K1 and -K2 are in progress. As for ClC-3, a stably expressing cell line was established in CHO cells, and patch clamp analysis revealed the Ca-regulated nature of ClC-3. Finally, we isolated human ClC-K1 and -K2 cDNAs to study the involvements of these genes in certain human kidney diseases. Using these cDNA,we further isolated human ClC-K1 and -K2 genes and characterized the gene structure including exon-intron structures. We prepared PCR primer sets to cover all exons and exon-intron boundaries and could established the system to determine the sequences of ClC-K2 gene from patients by direct sequencing. Also, we determined the chromosomal localization of ClC-K1, -K2 and -3 by FISH method.