slc26a12-A novel member of the slc26 family, is located in tandem with slc26a2 in coelacanths, amphibians, reptiles, and birds.

Solute carrier family 26 (Slc26) is a family of anion exchangers with 11 members in mammals (named Slc26a1-a11). Here, we identified a novel member of the slc26 family, slc26a12, located in tandem with slc26a2 in the genomes of several vertebrate lineages. BLAST and synteny analyses of various jawed vertebrate genome databases revealed that slc26a12 is present in coelacanths, amphibians, reptiles, and birds but not in cartilaginous fishes, lungfish, mammals, or ray-finned fishes. In some avian and reptilian lineages such as owls, penguins, egrets, and ducks, and most turtles examined, slc26a12 was lost or pseudogenized. Phylogenetic analysis showed that Slc26a12 formed an independent branch with the other Slc26 members and Slc26a12, Slc26a1 and Slc26a2 formed a single branch, suggesting that these three members formed a subfamily in Slc26. In jawless fish, hagfish have two genes homologous to slc26a2 and slc26a12, whereas lamprey has a single gene homologous to slc26a2. African clawed frogs express slc26a12 in larval gills, skin, and fins. These results show that slc26a12 was present at least before the separation of lobe-finned fish and tetrapods; the name slc26a12 is appropriate because the gene duplication occurred in the distant past.

Electroneutral Na+/Cl- cotransport activity of zebrafish Slc12a10.1 expressed in Xenopus oocytes.

Na+/Cl- cotransporter 2 (Ncc2 or Slc12a10), is a membrane transport protein that belongs to the electroneutral cation-chloride cotransporter family. The Slc12a10 gene (slc12a10) is widely present in bony vertebrates but is deleted or pseudogenized in birds, some bony fishes, and most mammals. Slc12a10 is highly homologous to Ncc (Slc12a3 or Ncc1); however, there are only a few reports measuring the activity of Slc12a10. In this study, we focused on zebrafish Slc12a10.1 (zSlc12a10.1) and analyzed its activity using Xenopus oocyte electrophysiology. Analysis using Na+-selective microelectrodes showed that intracellular sodium activity (aNai) in zSlc12a10.1 oocytes was significantly decreased in Na+- or Cl--free medium and recovered when Na+ or Cl- was readded to the medium. Similar analysis using a Cl--selective microelectrode showed that intracellular chloride activity (aCli) in zSlc12a10.1 oocytes significantly decreased in Na+- or Cl--free medium and recovered when Na+ or Cl- was readded to the medium. When a similar experiment was performed with a voltage clamp, the membrane current did not change when aNai of zSlc12a10.1 oocytes were decreased in Na+-free medium. Molecular phylogenetic and synteny analyses suggest that gene duplication between slc12a10.2 and slc12a10.3 in zebrafish, is a relatively recent event, whereas gene duplication between slc12a10.1 and the ancestral gene of slc12a10.2/slc12a10.3 occurred at least about 2 million years ago. slc12a10 deficiency was observed in species belonging to Ictaluridae, Salmoniformes, Osmeriformes, Batrachoididae, Syngnathiformes, Gobiesociformes, Labriformes, and Tetraodontiformes. These results indicate that zebrafish Slc12a10.1 is an electroneutral Na+/Cl-cotransporter and establish its evolutionary position among various teleost slc12a10 paralogs.

Convergent gene losses and pseudogenizations in multiple lineages of stomachless fishes.

The regressive evolution of independent lineages often results in convergent phenotypes. Several teleost groups display secondary loss of the stomach, and four gastric genes, atp4a, atp4b, pgc, and pga2 have been co-deleted in agastric (stomachless) fish. Analyses of genotypic convergence among agastric fishes showed that four genes, slc26a9, kcne2, cldn18a, and vsig1, were co-deleted or pseudogenized in most agastric fishes of the four major groups. kcne2 and vsig1 were also deleted or pseudogenized in the agastric monotreme echidna and platypus, respectively. In the stomachs of sticklebacks, these genes are expressed in gastric gland cells or surface epithelial cells. An ohnolog of cldn18 was retained in some agastric teleosts but exhibited an increased non-synonymous substitution when compared with gastric species. These results revealed novel convergent gene losses at multiple loci among the four major groups of agastric fish, as well as a single gene loss in the echidna and platypus.

Functional Divergence in Solute Permeability between Ray-Finned Fish-Specific Paralogs of aqp10.

Aquaporin (Aqp) 10 is a member of the aquaglyceroporin subfamily of water channels, and human Aqp10 is permeable to solutes such as glycerol, urea, and boric acid. Tetrapods have a single aqp10 gene, whereas ray-finned fishes have paralogs of this gene through tandem duplication, whole-genome duplication, and subsequent deletion. A previous study on Aqps in the Japanese pufferfish Takifugu rubripes showed that one pufferfish paralog, Aqp10.2b, was permeable to water and glycerol, but not to urea and boric acid. To understand the functional differences of Aqp10s between humans and pufferfish from an evolutionary perspective, we analyzed Aqp10s from an amphibian (Xenopus laevis) and a lobe-finned fish (Protopterus annectens) and Aqp10.1 and Aqp10.2 from several ray-finned fishes (Polypterus senegalus, Lepisosteus oculatus, Danio rerio, and Clupea pallasii). The expression of tetrapod and lobe-finned fish Aqp10s and Aqp10.1-derived Aqps in ray-finned fishes in Xenopus oocytes increased the membrane permeabilities to water, glycerol, urea, and boric acid. In contrast, Aqp10.2-derived Aqps in ray-finned fishes increased water and glycerol permeabilities, whereas those of urea and boric acid were much weaker than those of Aqp10.1-derived Aqps. These results indicate that water, glycerol, urea, and boric acid permeabilities are plesiomorphic activities of Aqp10s and that the ray-finned fish-specific Aqp10.2 paralogs have secondarily reduced or lost urea and boric acid permeability.

Boric acid transport activity of marine teleost aquaporins expressed in Xenopus oocytes.

Marine teleosts ingest large amounts of seawater containing various ions, including 0.4 mM boric acid, which can accumulate at toxic levels in the body. However, the molecular mechanisms by which marine teleosts absorb and excrete boric acid are not well understood. Aquaporins (Aqps) are homologous to the nodulin-like intrinsic protein (NIP) family of plant boric acid channels. To investigate the potential roles of Aqps on boric acid transport across the plasma membrane in marine teleosts, we analyzed the function of Aqps of Japanese pufferfish (Takifugu rubripes) expressed in Xenopus laevis oocytes. Takifugu genome database contains 16 genes encoding the aquaporin family members (aqp0a, aqp0b, aqp1aa, aqp1ab, aqp3a, aqp4a, aqp7, aqp8bb, aqp9a, aqp9b, aqp10aa, aqp10bb, aqp11a, aqp11b, aqp12, and aqp14). When T. rubripes Aqps (TrAqps) were expressed in X. laevis oocytes, a swelling assay showed that boric acid permeability was significantly increased in oocytes expressing TrAqp3a, 7, 8bb, 9a, and 9b. The influx of boric acid into these oocytes was also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these TrAqps increase B(OH)3 permeability. These results indicate that TrAqp3a, 7, 8bb, 9a, and 9b act as boric acid transport systems, likely as channels, in marine teleosts.

Na+/Cl- cotransporter 2 is not fish-specific and is widely found in amphibians, non-avian reptiles, and select mammals.

Solute carrier 12 (Slc12) is a family of electroneutral cation-coupled chloride (Cl-) cotransporters. Na+/K+/2Cl- (Nkcc) and Na+/Cl- cotransporters (Ncc) belong to the Nkcc/Ncc subfamily. Human and mouse possess one gene for the Na+/Cl- cotransporter (ncc gene: slc12a3), whereas teleost fishes possess multiple ncc genes, slc12a3 (ncc1) and slc12a10 (ncc2), in addition to their species-specific paralogs. Amphibians and squamates have two ncc genes: slc12a3 (ncc1) and ncc3. However, the evolutionary relationship between slc12a10 and ncc3 remains unresolved, and the presence of slc12a10 (ncc2) in mammals has not been clarified. Synteny and phylogenetic analyses of vertebrate genome databases showed that ncc3 is the ortholog of slc12a10, and slc12a10 is present in most ray-finned fishes, coelacanths, amphibians, reptiles, and a few mammals (e.g., platypus and horse) but pseudogenized or deleted in birds, most mammals, and some ray-finned fishes (pufferfishes). This shows that slc12a10 is widely present among bony vertebrates and pseudogenized or deleted independently in multiple lineages. Notably, as compared with some fish that show varied slc12a10 tissue expression profile, spotted gar, African clawed frog, red-eared slider turtle, and horse express slc12a10 in the ovaries or premature gonads. In horse tissues, an unexpectedly large number of splicing variants for Slc12a10 have been cloned, many of which encode truncated forms of Slc12a10, suggesting that the functional constraints of horse slc12a10 are weakened, which may be in the process of becoming a pseudogene. Our results elaborate on the evolution of Nkcc/Ncc subfamily of Slc12 in vertebrates.NEW & NOTEWORTHY slc12a10 is not a fish-specific gene and is present in a few mammals (e.g., platypus and horse), non-avian reptiles, amphibians, but was pseudogenized or deleted in most mammals (e.g., human, mouse, cat, cow, and rhinoceros), birds, and some ray-finned fishes (pufferfishes).

Membrane Transport Proteins Expressed in the Renal Tubular Epithelial Cells of Seawater and Freshwater Teleost Fishes.

The kidney is an important organ that maintains body fluid homeostasis in seawater and freshwater teleost fishes. Seawater teleosts excrete sulfate and magnesium in small amounts of isotonic urine, and freshwater teleosts excrete water in large amounts of hypo-osmotic urine. The volume, osmolality, and ionic compositions of the urine are regulated mainly by membrane transport proteins expressed in the renal tubular epithelial cells. Gene expression, immunohistochemical, and functional analyses of the fish kidney identified membrane transport proteins involved in the secretion of sulfate and magnesium ions by the proximal tubules and reduction of urine volume by the collecting ducts in seawater teleosts, and excretion of water as hypotonic urine by the distal tubules and collecting ducts in freshwater teleosts. These studies promote an understanding of how the kidney contributes to the seawater and freshwater acclimation of teleosts at the molecular level.

Boric acid transport activity of human aquaporins expressed in Xenopus oocytes.

Boric acid is a vital micronutrient that is toxic at high concentrations in animals. However, the mechanisms underlying boric acid transport in animal cells remain unclear. To identify the plasma membrane boric acid channels in animals, we analyzed the function of human aquaporins (AQPs), which are homologous to the nodulin-like intrinsic protein family of plant boric acid channels. When human AQPs were expressed in Xenopus laevis oocytes, the results of the swelling assay showed that boric acid permeability significantly increased in oocytes expressing AQP3, 7, 8, 9, and 10, but not in those expressing AQP1, 2, 4, and 5. The boric acid influxes of these oocytes were also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these AQPs transported boric acid (B(OH)3 ) but not borate ions (B(OH)4- ). These results indicate that AQP3, 7, 8, 9, and 10 act as boric acid transport systems, likely as channels in humans.

Metabolism of Odorant Molecules in Human Nasal/Oral Cavity Affects the Odorant Perception.

In this study, we examined the mode of metabolism of food odorant molecules in the human nasal/oral cavity in vitro and in vivo. We selected 4 odorants, 2-furfurylthiol (2-FT), hexanal, benzyl acetate, and methyl raspberry ketone, which are potentially important for designing food flavors. In vitro metabolic assays of odorants with saliva/nasal mucus analyzed by gas chromatography mass spectrometry revealed that human saliva and nasal mucus exhibit the following 3 enzymatic activities: (i) methylation of 2-FT into furfuryl methylsulfide (FMS); (ii) reduction of hexanal into hexanol; and (iii) hydrolysis of benzyl acetate into benzyl alcohol. However, (iv) demethylation of methyl raspberry ketone was not observed. Real-time in vivo analysis using proton transfer reaction-mass spectrometry demonstrated that the application of 2-FT and hexanal through 3 different pathways via the nostril or through the mouth generated the metabolites FMS and hexanol within a few seconds. The concentration of FMS and hexanol in the exhaled air was above the perception threshold. A cross-adaptation study based on the activation pattern of human odorant receptors suggested that this metabolism affects odor perception. These results suggest that some odorants in food are metabolized in the human nasal mucus/saliva, and the resulting metabolites are perceived as part of the odor quality of the substrates. Our results help improve the understanding of the mechanism of food odor perception and may enable improved design and development of foods in relation to odor.

Transcriptional regulators involved in responses to volatile organic compounds in plants.

Field studies have shown that plants growing next to herbivore-infested plants acquire higher resistance to herbivore damage. This increased resistance is partly due to regulation of plant gene expression by volatile organic compounds (VOCs) released by plants that sense environmental challenges such as herbivores. The molecular basis for VOC sensing in plants, however, is poorly understood. Here, we report the identification of TOPLESS-like proteins (TPLs) that have VOC-binding activity and are involved in VOC sensing in tobacco. While screening for volatiles that induce stress-responsive gene expression in tobacco BY-2 cells and tobacco plants, we found that some sesquiterpenes induce the expression of stress-responsive genes. These results provided evidence that plants sense these VOCs and motivated us to analyze the mechanisms underlying volatile sensing using tobacco as a model system. Using a pulldown assay with caryophyllene derivative-linked beads, we identified TPLs as transcriptional co-repressors that bind volatile caryophyllene analogs. Overexpression of TPLs in cultured BY-2 cells or tobacco leaves reduced caryophyllene-induced gene expression, indicating that TPLs are involved in the responses to caryophyllene analogs in tobacco. We propose that unlike animals, which use membrane receptors for sensing odorants, a transcriptional co-repressor plays a role in sensing and mediating VOC signals in plant cells.

Enzymatic conversion of odorants in nasal mucus affects olfactory glomerular activation patterns and odor perception.

Odor information is decoded by a combination of odorant receptors, and thus transformed into discrete spatial patterns of olfactory glomerular activity. It has been found, however, that for some odorants, there are differences between the ligand specificity of an odorant receptor in vitro and its corresponding glomerulus in vivo. These observations led us to hypothesize that there exist prereceptor events that affect the local concentration of a given odorant in the nasal mucus, thus causing the apparent specificity differences. Here we show that odorants with functional groups such as aldehydes and esters are targets of metabolic enzymes secreted in the mouse mucus, resulting in their conversion to the corresponding acids and alcohols. The glomerular activation patterns elicited by an enzyme-targeted odorant in the olfactory bulb was different in the presence of an enzyme inhibitor in the mucosa, suggesting that the enzymatic conversion occurs fast enough to affect recognition of the odorant at the levels of olfactory sensory neurons. Importantly, olfactory discrimination tests revealed that mice behaviorally trained to associate an enzyme-targeted odorant to sugar rewards could not discriminate the odorant after treatment with the enzyme inhibitor. These results reveal that the enzymatic conversion of odorants in the nasal mucus appears be fast enough to affect olfactory perception, which sheds light on the previously unappreciated role of nasal mucosal enzymes in odor sensation.