The multi-subunit vacuolar-type H(+)-ATPase consists of a V(1) domain (A-H subunits) catalyzing ATP hydrolysis and a V(0) domain (a, c, c', c", d, e) responsible for H(+) translocation. The mammalian V(0) d subunit is one of the least-well characterized, and its function and position within the pump are still unclear. It has two different forms encoded by separate genes, d1 being ubiquitous while d2 is predominantly expressed at the cell surface in kidney and osteoclast. To determine whether it forms part of the pump's central stalk as suggested by bacterial A-ATPase studies, or is peripheral as hypothesized from a yeast model, we investigated both human d subunit isoforms. In silico structural modelling demonstrated that human d1 and d2 are structural orthologues of bacterial subunit C, despite poor sequence identity. Expression studies of d1 and d2 showed that each can pull down the central stalk's D and F subunits from human kidney membrane, and in vitro studies using D and F further showed that the interactions between these proteins and the d subunit is direct. These data indicate that the d subunit in man is centrally located within the pump and is thus important in its rotary mechanism.

Several of the 13 subunits comprising mammalian H+-ATPases have multiple alternative forms, encoded by separate genes and with differing tissue expression patterns. These may play an important role in the intracellular localization and activity of H+-ATPases. Here we report the cloning of a previously uncharacterized human gene, ATP6V0E2, encoding a novel H+-ATPase e-subunit designated e2. We demonstrate that in contrast to the ubiquitously expressed gene encoding the e1 subunit (previously called e), this novel gene is expressed in a more restricted tissue distribution, particularly kidney and brain. We show by complementation studies in a yeast strain deficient for the ortholog of this subunit, that either form of the e-subunit is essential for proper proton pump function. The identification of this novel form of the e-subunit lends further support to the hypothesis that subunit differences may play a key role in the structure, site and function of H+-ATPases within the cell.

The specialized H+-ATPases found in the inner ear and acid-handling cells in the renal collecting duct differ from those at other sites, as they contain tissue-specific subunits, such as a4 and B1, and in the kidney, C2, d2, and G3 as well. These subunits replace the ubiquitously expressed forms. Previously, we have shown that, in major organs of both mouse and man, G3 subunit expression is limited to the kidney. Here we have shown widespread transcription of murine G3 in specific segments of microdissected nephron, and demonstrated additional G3 expression in epithelial fragments from human inner ear. We raised a polyclonal G3-specific antibody, which specifically detects G3 from human, mouse, and rat kidney lysates, and displays no cross-reactivity with G1 or G2. However, immunolocalization using this antibody on human and mouse kidney sections was unachievable, suggesting epitope masking. Phage display analysis and subsequent enzyme-linked immunosorbent assay, using the G3 antibody epitope peptide as bait, identified a possible interaction between the G3 subunit and the a4 subunit of the H+-ATPase. This interaction was verified by successfully using purified, immobilized full-length G3 to pull down the a4 subunit from human kidney membrane preparations. This confirms that a4 and G3 are component subunits of the same proton pump and explains the observed epitope masking. This interaction was also found to be a more general feature of human H+-ATPases, as similar G1/a1, G3/a1, and G1/a4 interactions were also demonstrated. These interactions represent a novel link between the V-1 and V-0 domains in man, which is known to be required for H+-ATPase assembly and regulation.

The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pump's constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henle's loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target.

The endolymph in the endolymphatic sac (ES) is acidic (pH 6.6-7). Maintaining this acidic lumen is believed to be important for the normal function of the ES. The acid-base regulation mechanisms of the ES are unknown. Here we investigated the expression patterns of acid-base regulators, including vacuolar (v)H+-ATPase (proton pump), carbonic anhydrase (CA) II, and pendrin in the murine ES epithelium by immunohistochemistry (IHC) and compared their expression patterns by double immunostaining. We found that pendrin and vH+-ATPase were co-localized in the apical membrane of a specific type of ES epithelial cell. Pendrin- and vH+-ATPase-positive cells also expressed cytoplasmic CA II. Co-expression of pendrin, vH+-ATPase, and CA II in the same subgroup of ES cells suggests that this specific type of ES cell is responsible for the acid-base balance processes in the ES and pendrin, vH+-ATPase, and CA II are involved in these processes.

Genes and mechanisms involved in common complex diseases, such as the autoimmune disorders that affect approximately 5% of the population, remain obscure. Here we identify polymorphisms of the cytotoxic T lymphocyte antigen 4 gene (CTLA4)—which encodes a vital negative regulatory molecule of the immune system—as candidates for primary determinants of risk of the common autoimmune disorders Graves' disease, autoimmune hypothyroidism and type 1 diabetes. In humans, disease susceptibility was mapped to a non-coding 6.1?kb 3′ region of CTLA4, the common allelic variation of which was correlated with lower messenger RNA levels of the soluble alternative splice form of CTLA4. In the mouse model of type 1 diabetes, susceptibility was also associated with variation in CTLA-4 gene splicing with reduced production of a splice form encoding a molecule lacking the CD80/CD86 ligand-binding domain. Genetic mapping of variants conferring a small disease risk can identify pathways in complex disorders, as exemplified by our discovery of inherited, quantitative alterations of CTLA4 contributing to autoimmune tissue destruction.

Autosomal dominant distal renal tubular acidosis (ddRTA) is caused by mutations in SLC4A1 , which encodes the polytopic chloride–bicarbonate exchanger AE1 that is normally expressed at the basolateral surface of α-intercalated cells in the distal nephron. Here we report that, in contrast with many disorders in which mutant membrane proteins are retained intracellularly and degraded, ddRTA can result from aberrant targeting of AE1 to the apical surface.

Genome-wide linkage disequilibrium (LD) mapping of common disease genes could be more powerful than linkage analysis if the appropriate density of polymorphic markers were known and if the genotyping effort and cost of producing such an LD map could be reduced. Although different metrics that measure the extent of LD have been evaluated1,2,3, even the most recent studies2,4 have not placed significant emphasis on the most informative and cost-effective method of LD mapping—that based on haplotypes. We have scanned 135 kb of DNA from nine genes, genotyped 122 single-nucleotide polymorphisms (SNPs; approximately 184,000 genotypes) and determined the common haplotypes in a minimum of 384 European individuals for each gene. Here we show how knowledge of the common haplotypes and the SNPs that tag them can be used to (i) explain the often complex patterns of LD between adjacent markers, (ii) reduce genotyping significantly (in this case from 122 to 34 SNPs), (iii) scan the common variation of a gene sensitively and comprehensively and (iv) provide key fine-mapping data within regions of strong LD. Our results also indicate that, at least for the genes studied here, the current version of dbSNP would have been of limited utility for LD mapping because many common haplotypes could not be defined. A directed re-sequencing effort of the approximately 10% of the genome in or near genes in the major ethnic groups would aid the systematic evaluation of the common variant model of common disease.

The 116-kDa a-subunit of the vacuolar proton pump (H(+)-ATPase) exists as several isoforms encoded by different genes and with different patterns of tissue expression. Its function within the multisubunit pump complex has yet to be elucidated. To date, three isoforms have been identified in mouse (designated a1-a3). We now report the cloning and characterization of Atp6n1b, encoding a novel fourth murine isoform (a4). Murine a4 has 833 residues and shows 85% amino acid identity to the human kidney-specific ATP6N1B protein in which loss-of-function alterations cause autosomal recessive distal renal tubular acidosis. The human and murine genes have similar genomic organization; furthermore, Atp6n1b maps to a region of mouse chromosome 6 that is syntenic with the segment of human 7q33-34 containing ATP6N1B. Together these findings establish the two genes as orthologs. The mouse a4 protein is 61, 52, and 47% identical to a1, a2, and a3, respectively. Phylogenetic analysis confirms that among vertebrates there are four a-subunit families, with a4 most resembling a1. Northern blot analysis of Atp6n1b reveals a 3.7-kilobase a4 transcript in kidney but not other major organs, and a reverse transcription polymerase chain reaction in 12 mouse tissues detects expression in kidney alone. Immunofluorescence studies in murine kidney demonstrate high intensity a4 staining at the surface of intercalated cells, with additional expression in the proximal tubule (not previously reported in human kidney). Similar apical a4 immunostaining is also present in male genital tissue. Identification of this novel murine kidney-enriched 116-kDa a-subunit provides a molecular tool for investigation of the currently unknown role of this protein, which is essential for proper function of the apical renal vacuolar H(+)-ATPase in man.

The multi-subunit H+-ATPase pump is present at particularly high density on the apical (luminal) surface of α-intercalated cells of the cortical collecting duct of the distal nephron, where vectorial proton transport is required for urinary acidification1. The complete subunit composition of the apical ATPase, however, has not been fully agreed upon. Functional failure of α-intercalated cells results in a group of disorders, the distal renal tubular acidoses (dRTA), whose features include metabolic acidosis accompanied by disturbances of potassium balance, urinary calcium solubility, bone physiology and growth2. Mutations in the gene encoding the B-subunit of the apical pump (ATP6B1) cause dRTA accompanied by deafness3. We previously localized a gene for dRTA with preserved hearing to 7q33–34 (ref. 4). We report here the identification of this gene, ATP6N1B, which encodes an 840 amino acid novel kidney-specific isoform of ATP6N1A, the 116-kD non-catalytic accessory subunit of the proton pump. Northern-blot analysis demonstrated ATP6N1B expression in kidney but not other main organs. Immunofluorescence studies in human kidney cortex revealed that ATP6N1B localizes almost exclusively to the apical surface of α-intercalated cells. We screened nine dRTA kindreds with normal audiometry that linked to the ATP6N1B locus, and identified different homozygous mutations in ATP6N1B in eight. These include nonsense, deletion and splice-site changes, all of which will truncate the protein. Our findings identify a new kidney-specific proton pump 116-kD accessory subunit that is highly expressed in proton-secreting cells in the distal nephron, and illustrate its essential role in normal vectorial acid transport into the urine by the kidney.

Genetic analysis of a mouse model of major histocompatability complex (MHC)-associated autoimmune type 1 (insulin-dependent) diabetes mellitus (IDDM) has shown that the disease is caused by a combination of a major effect at the MHC and at least ten other susceptibility loci elsewhere in the genome 1 , 2 . A genome-wide scan of 93 affected sibpair families (ASP) from the UK (UK93) indicated a similar genetic basis for human type 1 diabetes, with the major genetic component at the MHC locus ( IDDM1 ) explaining 34% of the familial clustering of the disease (λ s = 2.5; Refs 3 , 4 ). In the present report, we have analysed a further 263 multiplex families from the same population (UK263) to provide a total UK data set of 356 ASP families (UK356). Only four regions of the genome outside IDDM1 /MHC, which was still the only major locus detected, were not excluded at λ s = 3 and lod = –2, of which two showed evidence of linkage: chromosome 10p13–p11 (maximum lod score (MLS) = 4.7, P = 3 × 10 –6 , λ s = 1.56) and chromosome 16q22–16q24 (MLS = 3.4, P = 6.5 × 10 –5 , λ s = 1.6). These and other novel regions, including chromosome 14q12–q21 and chromosome 19p13–19q13, could potentially harbour disease loci but confirmation and fine mapping cannot be pursued effectively using conventional linkage analysis. Instead, more powerful linkage disequilibrium-based 5 , 6 , 7 and haplotype mapping approaches 8 must be used; such data is already emerging for several type 1 diabetes loci detected initially by linkage 6 , 8 , 9 , 10 , 11 , 12 , 13 .

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, that encompasses 250 kb of genomic DNA, cause cystic fibrosis. More than 5-10% of CF patients in most populations studied carry undefined mutations and hence intragenic CA repeats are important tools in genetic counselling. To date, polymorphic intragenic repeats have been found in introns 6a, 8 and 17b. We have identified a novel CA repeat within intron 1 of the CFTR gene that lies about 70 kb 5' to intron 6a and so will be a useful additional diagnostic marker.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene exhibits a tightly regulated pattern of expression in human epithelial cells. The mechanism of this regulation is complex and is likely to involve a number of genetic elements that effect temporal and spatial expression. To date none of the elements that have been identified in the CFTR promoter regulate tissue-specific expression. We have identified a putative regulatory element within the first intron of the CFTR gene at 181+10kb. The region containing this element was first identified as a DNase I hypersensitive site that was present in cells that express the CFTR gene but absent from cells not transcribing CFTR. In vitro analysis of binding of proteins to this region of DNA sequence by gel mobility shift assays and DNase I footprinting revealed that some proteins that are only present in CFTR-expressing cells bound to specific elements, and other proteins that bound to adjacent elements were present in all epithelial cells irrespective of their CFTR expression status. When assayed in transient expression systems in a cell line expressing CFTR endogenously, this DNA sequence augmented reporter gene expression through activation of the CFTR promoter but had no effect in nonexpressing cells.

Pancreatic adenocarcinoma cell lines rarely express the CFTR gene, despite the high levels of CFTR protein that are present in primary pancreatic duct cells. We have attempted to generate a non-CF pancreatic adenocarcinoma cell line that stably produces high levels of CFTR mRNA and protein by transfecting a vector containing the CFTR cDNA, driven by a strong mammalian promoter, into the poorly differentiated pancreatic adenocarcinoma cell line, Panc-1. The pANS6 pancreatic duct cell line expresses substantial levels of CFTR mRNA, but little CFTR protein. Despite this we were able to detect low conductance chloride channels in 40% of patches, stimulated with cAMP, that have similar biophysical properties to CFTR.

The chromatin structure of 120kb of genomic DNA 5′ to the CFTR gene has been analysed in a number of CFTR expressing and non expressing cell types, including primary genital duct epithelial cells. Novel DNAse I hypersensitive sites have been observed at −79.5kb and −20.5kb 5′ to the ATG translation start codon of the CFTR coding sequence. Neither of these sites appears to show strong correlation with CFTR expression in the cell types investigated, hence they are unlikely to reflect the sites of binding of the major CFTR tissue specific regulator(s). However, they may still play an important part in the complex series of events involved in the regulation of CFTR transcription.