Kumagai S et al. (2023), Boric acid transport activity of marine teleost...

XB-ART-59706

Physiol Rep 2023 Mar 01;116:e15655. doi: 10.14814/phy2.15655.

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

Kumagai S , Watanabe E , Hayashi N , Kimura Y , Kamiya T , Nagashima A , Ushio K , Imaizumi G , Kim J , Munakata K , Umezawa T , Hirose S , Kasai K , Fujiwara T , Romero MF , Kato A .

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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.

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Genes referenced: aqp12a aqp7

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	FIGURE 1. Tissue distribution of aqp family genes in the Japanese pufferfish Takifugu rubripes. Expression profiles of aqp in adult tissues were determined via semiquantitative RT‐PCR. Pseudo‐gel images of the PCR products were generated using a microchip electrophoresis system. actb (β‐Actin gene) was used as an internal control gene.
	FIGURE 2. Increase in the volume of TrAqp‐expressing and control oocytes at the indicated timepoints (sec). Relative cell volume to the initial cell volume (V/V 0 ) of oocytes in hypo‐osmotic solution (a), iso‐osmotic solution containing 180 mM glycerol (b), urea (c), or boric acid (d) was calculated from the swelling data. Values are shown as the mean ± SEM (n = 9–24).
	FIGURE 3. Water (a), glycerol (b), urea (c), and boric acid (d) permeability measurements of TrAqps by Xenopus oocyte swelling assay. Pwater, Pglycerol, Purea, and Pboric acid values are presented as interquartile range from the 25–75 percentiles (box), range (whiskers), outliers (>1.5× interquartile range above upper quartile), mean (square in the box), and median (line in the box). Statistical significance was evaluated by one‐way ANOVA followed by Dunnett's test (*p < 0.05).
	FIGURE 4. Boric acid uptake activity of TrAqp oocytes measured as whole‐cell boron content using inductively coupled plasma‐mass spectrometry. Values are presented as interquartile range from the 25–75 percentiles (box), range (whiskers), outliers (>1.5× interquartile range above upper quartile), mean (square in the box), and median (line in the box). Statistical significance was evaluated by one‐way ANOVA followed by Dunnett's test (*p < 0.05, n = 6–24).
	FIGURE 5. B(OH)3 permeability of oocytes expressing TrAqp3a, 7, 8bb, 9a, and 9b. A: Representative traces of changes in the intracellular pH (pHi) and membrane potential (Vm) of an oocyte expressing TrAqp0a, 3a, 7, 8bb, 9a, or 9b, or a control oocyte. 10BA, 10 mM boric acid. B: The summary of pH changes (dpHi/s) in oocytes expressing Aqps and control in solution containing 0, 1, 2, 5, or 10 mM boric acid. BA, boric acid. Values are presented as interquartile range from the 25–75 percentiles (box), range (whiskers), outliers (>1.5× interquartile range above upper quartile), mean (square in the box), and median (line in the box). Statistical significance was evaluated by one‐way ANOVA followed by Dunnett's test (*p < 0.05, n = 4–25).
	FIGURE 6. Scatter plots and Pearson's correlation coefficient (r) among water, glycerol, urea, and boric acid permeabilities of oocytes expressing pufferfish TrAqps. (A‐F) The average values of water and solute permeabilities (P water, P glycerol, P urea, and P boric acid, shown in Table 2) expressing pufferfish TrAqps are plotted in red, and those of water‐injected oocytes were plotted in gray. The average values of water and solute permeabilities of oocytes expressing human HsAqps were recalculated from our previous study (Ushio et al., 2022) (Table 2) were plotted as comparison in blue. The correlations (r) calculated from the average values of TrAqp and control (n = 11) were shown with p value in red, and those calculated from the average values of TrAqp, HsAqp, and each control (n = 21) were shown with p value in black. T0a, TrAqp0a; T1aa, TrAqp1aa; T1ab, TrAqp1ab; T3a, TrAqp3a; T4a, TrAqp4a; T7, TrAqp7; T8bb, TrAqp8bb; T9a, TrAqp9a; T9b, TrAqp9b; T10bb, TrAqp10bb; H1, HsAqp1; H2, HsAqp2; H3, HsAqp3; H4, HsAqp4; H5, HsAqp5; H7, HsAqp7; H8, HsAqp8; H9, HsAqp9; H10, HsAqp10; C, control.

References [+] :

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