Cloning and functional expression of a ClC Cl− channel from the renal cell line A6

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Abstract
Cl− channels are important for ion transport and cell volume regulation in A6 renal cells. In the present study, we used reverse transcriptase (RT)-polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) to identify proteins homologous to ClC Cl− channel proteins in A6 cells. Using degenerate primers designed on consensus sequences for members of the ClC family, we amplified an RT-PCR product that had significant homology to the ClC sequences. RACE-PCR was then used to isolate several full-length clones that had total lengths from 2,764 to 3,016 base pairs. Although the coding regions were identical, sequence differences occurred in the 5′ noncoding regions. The amino acid sequences of the clones had high homologies to rat and human ClC-5 (85 and 84%, respectively, if the 5th methionine of the open reading frame represents the start codon). Three parts of the protein (53, 80, and 63 amino acids in length) were 97–100% homologous to the mammalian sequences. Ribonuclease protection assay analysis revealed mRNA for this protein in oocytes, kidney, intestine, liver, brain, and blood, with lower amounts in stomach, muscle, and skin. Expression of the clones in Xenopus laevis oocytes resulted in an outwardly rectifying Cl−current that was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and possessed an anion selectivity of I− > Cl− >> gluconate.