Cloning and Characterization of CLCN5, the Human Kidney Chloride Channel Gene Implicated in Dent Disease (an X-Linked Hereditary Nephrolithiasis)

Abstract

Dent disease, an X-linked familial renal tubular disorder, is a form of Fanconi syndrome associated with proteinuria, hypercalciuria, nephrocalcinosis, kidney stones, and eventual renal failure. We have previously used positional cloning to identify the 3' part of a novel kidney-specific gene (initially termed hClC-K2, but now referred to as CLCN5), which is deleted in patients from one pedigree segregating Dent disease. Mutations that disrupt this gene have been identified in other patients with this disorder. Here we describe the isolation and characterization of the complete open reading frame of the human CLCN5 gene, which is predicted to encode a protein of 746 amino acids, with significant homology to all known members of the ClC family of voltage-gated chloride channels. CLCN5 belongs to a distinct branch of this family, which also includes the recently identified genes CLCN3 and CLCN4. We have shown that the coding region of CLCN5 is organized into 12 exons, spanning 25-30 kb of genomic DNA, and have determined the sequence of each exon-intron boundary. The elucidation of the coding sequence and exon-intron organization of CLCN5 will both expedite the evaluation of structure/function relationships of these ion channels and facilitate the screening of other patients with renal tubular dysfunction for mutations at this locus.

Molecular Cloning and Characterization of an Intracellular Chloride Channel in the Proximal Tubule Cell Line, LLC-PK1

“…3A). The protein sequence of pCLC5 is the same length as and highly homologous with rat CLC5 (rCLC5, 97% amino acid identity (28)) and is also very similar to human CLC5 (98% amino acid identity in overlapping region (27)) except for the absence of a 20-residue stretch at the N-terminal of human CLC5. We conclude that pCLC5 is the porcine ortholog of CLC5.…”

“…Northern blots under high stringency conditions (hybridization at 65°C in the presence of 50% formamide, final washes at 65°C in 0.1ϫ SSCP) were performed on poly(A) ϩ RNA isolated from these cells, using digoxigenin-labeled antisense riboprobes of rat CLC5, CLC4, and CLC3 (11,26) and detected by chemiluminescence with CDP-Star and the Dig Genius System (Roche Molecular Biochemicals). cDNA Cloning and Generation of Expression Constructs-To clone the pCLC5 cDNA, degenerate oligonucleotide primers (sense, ATHTCT-GCNCAYTGGATGAC; antisense, TAYTTRTTRAARCARTGRCA) were designed to the conserved amino acid sequences of human (27), rat (28), and Xenopus (29) CLC5. By homology-based reverse transcription-polymerase chain reaction (PCR) 1 amplification of LLC-PK1 cell mRNA, a 427-base pair cDNA was isolated that was highly homologous to CLC5 in other species.…”

Identification of a novel splice site mutation of CLCN5 gene and characterization of a new alternative 5’ UTR end of ClC-5 mRNA in human renal tissue and leukocytes

“…3) showed that the isoform A (Fig. 3C) was indeed the most abundant in renal biopsies, whereas the isoform B, described by Fisher et al (1995), was not present in renal tissue as not in leukocytes. The new 5Õ UTR isoform (B1) (Fig.…”

“…Two different 5Õ UTR ends of ClC-5 cDNA have been described in the kidney. One of them (type B) was first described by Fisher et al (1994Fisher et al ( , 1995 and contains the untranslated exon 1b. The other (type A) was recently identified by Hayama et al (2000) and consists in a newly untranslated exon 1a that was found 2.1 kb upstream of exon 2 containing the translation start site.…”

Ion channelopathies in endocrinology: recent genetic findings and pathophysiological insights

“…Sixty percent of Dent's disease cases are caused by mutations in the chloride channel gene, CLCN5, that encodes the chloride-proton antiporter, CLC-5 (46). CLC-5 is found primarily in the kidneys and is expressed with proton-ATPase in subapical endosomes of the tubular proximal cells where it facilitates acidification (47).…”