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Characterization of the Drosophila melanogaster alkali-metal/proton exchanger (NHE) gene family

Maria E. Giannakou and Julian A. T. Dow*

Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, UK



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  		Fig. 1. Sensitivity of secretion by the Drosophila melanogaster Malpighian tubule to inhibition by amiloride and its derivatives. Dose/response curves for amiloride, 5-N,N-dimethyl amiloride (DMA), benzamil, 5-N-ethyl-N-isopropyl amiloride (EIPA) and 2',4'-dichlorobenzamil (DCB). The upper limits of each graph are determined by the solubility of the compounds. Values are means ± S.E.M. (N=10).

 


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  		Fig. 2. Malpighian tubules do not express epithelial Na+ channels (ENaCs). (A) Phylogenetic tree of all Drosophila ENaCs identified by BLASTP search using human amiloride-sensitive cation channel 2, neuronal hBNaC2 (GenBank accession number NP 064423) protein sequence as a probe. (B) RT-PCR for putative EnaCs (left-hand panels) and corresponding Southern blots with probes specific to each gene (right-hand panels). Labels refer to known genes or to Gadfly-predicted genes. Size markers denote expected sizes from genomic (black arrows) and cDNA (white arrows) templates. The ladder is a Gibco BRL 1 kb ladder. The templates are as follows: Genomic, genomic DNA; Whole fly, whole-fly cDNA; Head, head cDNA; Tubule, Malpighian tubule cDNA; No template, no template (negative control).

 


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  		Fig. 3. cDNA, predicted protein sequence (A) and genomic context (B,C) for DmNHE1. (A) The signal peptide and putative cleavage sites are marked in red. The polyadenylation signal is marked in blue. (B) Transcript structure. DmNHE1 is a simple gene, with five exons spanning 2.5 kb of genomic sequence. (C) Genomic context of DmNHE1 at 21B1 on chromosome 2, showing the surrounding gene-dense region, with seven putative genes within 45 kb (from Gadfly annotation). This sequence has been deposited in GenBank with the accession number AF142676.

 


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  		Fig. 4. cDNA, predicted protein sequence (A) and genomic context (B,C) for DmNHE3. (A) The signal peptide and putative cleavage sites are marked in red. The polyadenylation signal is marked in blue. (B) Transcript structure. (C) Genomic context of DmNHE2 at 27A1 on chromosome 2, showing gene-dense region containing nine putative surrounding genes within 50 kb (from Gadfly annotation). This sequence has been deposited in GenBank with the accession number AF199463.

 


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  		Fig. 5. cDNA, predicted protein sequence and genomic context for DmNHE2. (A) cDNA sequence and putative transcript for testes clone AT11019. The putative signal peptide and cleavage site are marked in red. The polyadenylation signal is marked in blue. (B) Transcript structure of DmNHE2. The long transcript is that of GenBank accession number AF235935 (X. Lin, D. C. Huang, W. Yan and D. L. Barber, unpublished). The short transcript is our sequence for testes cDNA clone AT11019. (C) Genomic context of DmNHE2 at 39B1 on chromosome 2, showing the surrounding genes within 53 kb, modified from Gadfly annotation, to reflect the larger size of DmNHE2. This sequence has been deposited in GenBank with the accession number AF239763.

 


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  		Fig. 6. Structure predictions for human NHE1, DmNHE1, DmNHE2 and DmNHE3. Transmembrane probability plots for human NHE1, DmNHE1, DmNHE2, Aedes aegypti NHE3 and DmNHE3 proteins according to the von Heijne (von Heijne, 1992) algorithm. Predictions were made using MacVector 7.0. The blue background indicates the N-terminal domain; white, the compact transmembrane domain; yellow, the C-terminal cytoplasmic domain. Putative signal peptide cleavage sites are marked with red arrowheads. All three gene models for DmNHE2 are shown (see text) with the Aedes aegypti sequence for comparison.

 


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  		Fig. 7. Alignments of conserved domains in Na+/H+ exchangers (NHEs). Alignment of Drosophila melanogaster NHEs and other representative NHE family members, produced using ClustalW and SeqVu software. (A) An alignment of the NHEs in the amiloride-binding region and (B) the alignment of NHEs in the highly conserved region. Blue denotes hydrophilic, and red hydrophobic, residues. Nomenclature is as for Fig. 8.

 


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  		Fig. 8. Phylogenetic tree for selected Na+/H+ exchangers (NHEs). Protein sequences are those aligned in Fig. 7, produced using TreeViewPPC and ClustalW software. The boxed names are the Drosophila NHEs, and the other abbreviations are as follows: Xenopus NHE, Xenopus laevis, CAA69925 (Busch, 1997); human NHE1, Homo sapiens, AAB59460 (Sardet et al., 1988); nNHE chr1, Homo sapiens, Hs1_5070 chromosome 1 working draft sequence; human NHE2, Homo sapiens, XP_010884 (NCBI Annotation Project; direct submission, 2001); human NHE4, Homo sapiens, P26434 (Orlowski et al., 1992); nNHE chr2, Homo sapiens, Hs2_5544 chromosome 2 working draft sequence; human NHE5, Homo sapiens, NP_004585 (Klanke et al., 1995); nNHE chr16, Homo sapiens, Hs16_10635 chromosome 16 working draft sequence; C. elegans NHE, Caenorhabditis elegans, P35449 (Marra et al., 1993); nNHE chr3, Homo sapiens, Hs3_19514 chromosome 3 working draft sequence; NHE Arabidopsis, Arabidopsis thaliana, AAF21755 (Quintero et al., 2000); DmNHE3, Drosophila melanogaster, AAF13702 (this work); nNHE chrX2, Homo sapiens, HsX_11991 chromosome X working draft sequence; human NHE6, Homo sapiens, AAC39643 (Numata et al., 1998); nNHE chr12, Homo sapiens, Hs12_9838 chromosome 12 working draft sequence; nNHE chrX, Homo sapiens, HsX_11725 chromosome X working draft sequence; S. cerevisiae NHE, Saccharomyces cerevisiae, Q04121 (Numata et al., 1998); S. pombe NHE, Schizosaccharomyces pombe, T37706 (L. Murphy, D. Harris, V. Wood, B. G. Barrell and M. A. Rajandream, unpublished); KIAA0939, Homo sapiens, CAB46030 (N. Corby, unpublished); DmNHE1, Drosophila melanogaster, AAD32689 (J. A. T. Dow, unpublished; and this work; nNHE chr13, Homo sapiens, Hs1_22216 chromosome 1 working draft sequence; nNHE chr20, Homo sapiens, Hs20_11518 chromosome 20 working draft sequence; nNHE chr10, Homo sapiens, Hs10_8739 chromosome 10 working draft sequence; nNHE chr12, Homo sapiens, Hs1_22246 chromosome 1 working draft sequence; nNHE chr22, Homo sapiens, Hs22_11677 chromosome 22 working draft sequence; human NHE3, Homo sapiens, P48764 (Brant et al., 1995); nNHE chr5, Homo sapiens, Hs5_7223 chromosome 5 working draft sequence; DmNHE2, Drosophila melanogaster, AAF53960 (this work); Aedes NHE3, Aedes aegypti, AF80554 (S. S. Gill, H. Wediak and L. S. Ross, unpublished); NHE crab, Carcinus maenas, AAC26968 (Towle et al., 1997). nNHE denotes a novel or undocumented gene in the human genome sequence.

 


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  		Fig. 9. Expression pattern of Drosophila Na+/H+ exchangers (NHEs). Left-hand panels: RT-PCR using primers specific for DmNHE1 (top), DmNHE2 (middle) and DmNHE3 (lower panel). Predicted sizes for genomic (black arrow) and cDNA (white arrow) templates are shown. Templates: Genomic, genomic DNA; Whole fly, whole-fly cDNA; Head, head cDNA; Body, body cDNA; Tubule, Malpighian tubule cDNA; Larva, mixed larval cDNA; Pupa, mixed pupal cDNA; No template, no DNA control. Ladder is a 1 kb marker (Promega). Right-hand panels: the identity of the major bands labelled was verified by Southern hybridisation using DIG-labelled cDNA probes derived from an EST clone for DmNHE1 and DmNHE3 and from the previously sequenced tubule cDNA RT-PCR product for DmNHE2.

 

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© The Company of Biologists Ltd 2001