Ion and solute transport by Prestin in Drosophila and Anopheles

Abstract

The gut and Malpighian tubules of insects are the primary sites of active solute and water transport for controlling hemolymph and urine composition, pH, and osmolarity. These processes depend on ATPase (pumps), channels and solute carriers (Slc proteins). Maturation of genomic databases enables us to identify the putative molecular players for these processes. Anion transporters of the Slc4 family, AE1 and NDAE1, have been reported as HCO(3)(-) transporters, but are only part of the story. Here we report Dipteran (Drosophila melanogaster (d) and Anopheles gambiae (Ag)) anion exchangers, belonging to the Slc26 family, which are multi-functional anion exchangers. One Drosophila and two Ag homologues of mammalian Slc26a5 (Prestin) and Slc26a6 (aka, PAT1, CFEX) were identified and designated dPrestin, AgPrestinA and AgPrestinB. dPrestin and AgPrestinB show electrogenic anion exchange (Cl(-)/nHCO(3)(-), Cl(-)/SO(4)(2-) and Cl(-)/oxalate(2-)) in an oocyte expression system. Since these transporters are the only Dipteran Slc26 proteins whose transport is similar to mammalian Slc26a6, we submit that Dipteran Prestin are functional and even molecular orthologues of mammalian Slc26a6. OSR1 kinase increases dPrestin ion transport, implying another set of physiological processes controlled by WNK/SPAK signaling in epithelia. All of these mRNAs are highly expressed in the gut and Malpighian tubules. Dipteran Prestin proteins appear suited for central roles in bicarbonate, sulfate and oxalate metabolism including generating the high pH conditions measured in the Dipteran midgut lumen. Finally, we present and discuss Drosophila genetic models that integrate these processes.

Fig. 1. Phylogenetic tree of HCO₃⁻ transporters (A: SLC4 and B: SLC26) in Drosophila

Results of the Drosophila melanogaster BLAST search revealed that there are two Slc4 clones and nine Slc26 clones. Neighbor-joining trees (Saitou 1987) were constructed based on the deduced amino acid sequences of Slc4 and Slc26 from Drosophila (black) and human (grey). Accession numbers are as follows: CG4675 (NDAE1), AF047468; CG8177, NM_140100; SLC4A1, NM_000342; SLC4A2, NM_003040; SLC4A3, NM_005070; SLC4A4, NM_003759; SLC4A5, NM_133478; SLC4A7, NM_003615; SLC4A8, NM_004858; SLC4A9, NM_031467; SLC4A10, NM_022058; SLC4A11, NM_032034; CG5485 (Drosophila Prestin), NM_140767; CG5002, AY240021; CG5404, NM_142225; CG6125, AY240022; CG6928, AY240023; CG7005, NM_079766; CG7912, NM_143504; CG9702, AY240025; CG9717, NM_143555; SLC26A1, AF297659; SLC26A2, BC059390; SLC26A3, BC025671; SLC26A4, AF030880; SLC26A5 (prestin), AF523354; SLC26A6, NM_134263; SLC26A7, AF331521; SLC26A8, BC025408; SLC26A9, BC136538; SLC26A10, NM_133489; SLC26A11, AF345195. Phylogenetic trees were constructed using the Clustal W computer program. The scale bar represents a genetic distance of 0.1 amino acid substitutions per site.

Fig. 2. Drosophila Prestin function in Xenopus oocytes

A. Representative trances of intracellular pH (pH_i) and membrane potential (V_m) of oocytes injected with Drosophila Prestin (dPrestin) cRNAs (a), mouse Slc26a6 (mSlc26a6) cRNAs (b), or water (control) (c) are shown. In the continuous presence of 33 mM HCO₃⁻/5%CO₂ (dot square), the Cl⁻/HCO₃⁻ exchange activities were monitored as changes in pH_i and V_m when extracellular Cl⁻ was removed (grey shading) and readded. Model illustrating the suggested transport activity (electrogenic Cl⁻/nHCO₃⁻ exchange activity) is shown in d. B. C. D. Representative traces of intracellular Cl⁻ ([Cl⁻]_i) and membrane potential (V_m) of oocytes injected with dPrestin cRNA (a), mSlc26a6 cRNAs (b) and water (control) (c) are shown in each tested substrates: oxalate (B), sulfate (C), and formate (D). Results for solution changes from 20 mM-Cl⁻ ND96 (20Cl-ND96) to 20Cl-ND96 containing 0.33 mM oxalate, 5 mM sulfate, or 5 mM formate are indicated by dot square and results for solution changes to Cl⁻-free solution are indicated by gray shading. Models illustrating the suggested transport activity (electrogenic Cl⁻/oxalate²⁻, Cl⁻/sulfate²⁻ and Cl⁻/formate⁻ exchange activity) are shown in Bd, Cd and Dd, respectively.

Fig. 3. Phylogeny of Anopheles gambiae Prestin genes and their mRNA tissue distribution

A. Phylogenetic tree of AgSls26a5/a6 including those of other several species. Results of the Anopheles gambiae BLAST search revealed that there are two Prestin related isoforms. Neighbor-joining trees (Saitou 1987) were constructed based on the deduced amino acid sequences of slc26a5 and slc26a6 from human (h), chicken (c), fugu (f), zebrafish (z), fly (d), and Anopheles gambiae (Ag). The bootstrap values from a 5,000-replicate analysis are given as % at the nodes. Accession numbers are as follows: hSLC26A5, AF523354; mSlc26a5, AF529192; cSlc26a5, EF028087; fSlc26a5; BAE75795, zSlc26a5, BC054604; AgPrestinA, GQ332421; AgPrestinB, AB671171; dPrestin, NM_140767; hSLC26A6, NM_134263; mSlc126a6, AY049076; fSlc26a6A, AB200328; fSlc26a6B, BAE75797; fSlc26a6C, BAE75798; zSlc26a6, BC155340. Phylogenetic trees were constructed using the Clustal W computer program. The scale bar represents a genetic distance of 0.05 amino acid substitutions per site. B. Tissue distribution of AgPrestinA and AgPrestinB. mRNA expression level (signal intensity in Y axis) of AgPrestinA and B was examined by microarray analysis using in each tissue: salivary gland (SG), gastric cacae (GC), anterior midgut (AMG), posterior midgut (PMG) and hindgut (HG). AgPrestinA is highly expressed in salivary glands and gastric cacae and AgPrestinB is expressed in posterior midgut and hindgut.

Fig. 4. AgPrestinB, but not AgPrestinA, has transport function

A, Representative pH_i/V_m experiments for oocytes injected with AgPrestinB cRNA (a) or water –control (b) are shown. In the continuous presence of 33 mM HCO₃⁻/5%CO₂ (dot square), the Cl⁻/HCO₃⁻ exchange activities were monitored as changes in pH_i and V_m when extracellular Cl⁻ is removed (grey shading) followed by readdition. Model illustrating the suggested transport activity (electrogenic Cl−/nHCO₃⁻ exchange activity) is shown in (c). (B) Current-voltage (IV) relationships of oocytes expressing AgPrestinB in the presence of 0.33 mM oxalate (20 mM Cl⁻) or (C) 5 mM sulfate (70 mM Cl⁻) are shown. Values are means ± SE, n = 3–5. Oxalate-elicited or sulfate-elicited currents are calculated as I _{(oxalate or sulfate)} − I _{(no oxalate or ulfate)}.

Fig. 5. Drosophila Prestin transport is activated by dOSR1

A, B; Drosophila Prestin (dPrestin) activity at co-expression with dOSR1. Current-voltave (I-V) relationships of oocytes expressing dPrestin solely (filled square), dPrestin with wild-type dOSR1 (open square) and dOSR1 alone (open circle) in the presence of 0.33 mM oxalate (20 mM Cl⁻) (B) or 5 mM sulfate (20 mM Cl⁻) (C) are shown. Values are means ± SE, n = 3–5. Oxalate-elicited or sulfate-elicited currents are calculated as in Fig. 4

Fig 6

A model of Slc26a5/a5 (prestin) function of HCO₃⁻, SO₄²⁻ and oxalate²⁻ transport in Dipteran midgut.