Hayashi, Naoko

AbstractMarine 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.

The kidney of marine teleosts is the major site of Mg2+excretion and produces urine with a high Mg2+concentration. However, the transporters involved in Mg2+excretion are poorly understood. The cyclin M (Cnnm; also known as ancient conserved domain protein) family comprises membrane proteins homologous to the bacterial Mg2+and Co2+efflux protein, CorC. To understand the molecular mechanism of Mg2+homeostasis in marine teleosts, we analyzed the expression of the C nnm family genes in the seawater (SW) pufferfish, torafugu ( Takifugu rubripes), and the closely related euryhaline species, mefugu ( Takifugu obscurus). Database mining and phylogenetic analysis indicated that the Takifugu genome contains six members of the Cnnm family: two orthologs of Cnnm1, one of Cnnm2, one of Cnnm3, and two of Cnnm4. RT-PCR analyses indicated that Cnnm2, Cnnm3, and Cnnm4a are expressed in the kidney, whereas other members are mainly expressed in the brain. Renal expression of Cnnm3 was upregulated in SW mefugu, whereas renal expression of Cnnm2 was upregulated in freshwater (FW) mefugu. No significant difference was observed in renal expression of Cnnm4a between SW and FW mefugu. In situ hybridization and immunohistochemical analyses of the SW mefugu kidney revealed that Cnnm3 is expressed in the proximal tubule, and its product localizes to the lateral membrane. When Cnnm3 was expressed in Xenopus laevis oocytes, whole cellular Mg2+content and free intracellular Mg2+activity significantly decreased. These results suggest that Cnnm3 is involved in body fluid Mg2+homeostasis in marine teleosts.

The second most abundant cation in seawater (SW), Mg2+, is present at concentrations of ∼53 mM. Marine teleosts maintain plasma Mg2+concentration at 1–2 mM by excreting Mg2+into the urine. Urine Mg2+concentrations of SW teleosts exceed 70 mM, most of which is secreted by the renal tubular epithelial cells. However, molecular mechanisms of the Mg2+secretion have yet to be clarified. To identify transporters involved in Mg2+secretion, we analyzed the expression of fish homologs of the Slc41 Mg2+transporter family in various tissues of SW pufferfish torafugu ( Takifugu rubripes) and its closely related euryhaline species mefugu ( Takifugu obscurus). Takifugu genome contained five members of Slc41 genes, and only Slc41a1 was highly expressed in the kidney. Renal expression of Slc41a1 was markedly elevated when mefugu were transferred from fresh water (FW) to SW. In situ hybridization analysis and immunohistochemistry at the light and electron microscopic levels revealed that Slc41a1 is localized to vacuoles in the apical cytoplasm of the proximal tubules. These results suggest that pufferfish Slc41a1 is a Mg2+transporter involved in renal tubular transepithelial Mg2+secretion by mediating Mg2+transport from the cytosol to the vacuolar lumen, and support the hypothesis that Mg2+secretion is mediated by exocytosis of Mg2+-rich vacuoles to the lumen.