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Figure 1. Urinary boric acid excretion by pufferfish in seawater (SW). Boric acid or boron concentrations of serum (n = 4–6), urine (n = 6–7), and rectal fluid (n = 4) of pufferfish acclimated to brackish water (BW), fresh water (FW), and natural SW are shown. Dots represent individual data. Bar graphs represent means ± SD. ∗p < 0.0001, ∗∗p < 0.0002, and ∗p < 0.05.
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Figure 2. Renal expression of Slc4a11A. A, phylogenetic tree of boric acid transporters in relation to the other human SLC4 family members. The boric acid transport activity of Takifugu Slc4a11A is shown in this study. The scale bar represents 0.1 amino acid substitution per site. B, tissue distribution of Slc4a11A and Slc4a11B. Semiquantitative RT–PCR was performed on various tissues of river pufferfish. Numbers indicate PCR cycles. Results from 27 PCR cycles show tissues with relatively high expression of the indicated genes, and those of 32 cycles show all tissues expressing the indicated genes from low to high levels (111, 162, 65). C, real-time PCR quantification of mRNAs for Slc4a11A and Slc4a11B in the kidneys of river pufferfish acclimated to FW and SW. Values are expressed relative to GAPDH. Dots represent individual data. Bar graphs represent means ± SD, n = 5. ∗p < 0.05. D, in situ hybridization of Slc4a11A and Slc4a11B in the kidney of river pufferfish in SW. Sense probes did not show labeling (data not shown). AE, anion exchanger; c, chicken; F, FW; NBC, Na+-HCO3– cotransporter; NDCBE, Na+-driven Cl–/HCO3– exchanger; S, SW; SLC4, solute carrier family 4; z, zebrafish.
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Figure 3. Multiple alignment of amino acid sequences of Slc4a11 family. The amino acid residues that are conserved among Slc4a11 family members are shaded. Transmembrane (TM) regions are indicated by solid bars labeled as TM1–TM12. The accession numbers of mfSlc4a11A, mfSlc4a11B, and hSLC4A11 are AB534190, AB534191, and NM_032034, respectively. h, human; mf, mefugu. Red box of mfSlc4a11A indicates the peptide antigen used to generate the mfSlc4a11A antibody.
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Figure 4. Validation of the antibody against Slc4a11. A, Western blot analysis of HEK293 cells transfected with pcDNA3 (mock), pcDNA3-Slc4a11A, or pcDNA3-Slc4a11B. The membrane fractions of the cells were incubated with (+) or without (–) glycosidases and analyzed using anti-Slc4a11 antiserum (left) and antigen-absorbed antiserum (right). B, polarized distribution of Slc4a11A and Slc4a11B in MDCK cells. Anti-Slc4a11 antiserum (green), anti-ZO-1 antibody (red), and Hoechst 33342 (blue) were used to stain MDCK cells transiently transfected with pcDNA3-Slc4a11A or pcDNA3-Slc4a11B. Confocal XY maximum projection image and XZ (vertical) sections are shown. C-E, anti-Slc4a11 antiserum (left panel), preimmune serum (center panel), or antigen-absorbed antiserum (right panel) (green) were used with anti-ZO-1 antibody (red) and Hoechst 33342 (blue) to stain MDCK cells transiently transfected with pcDNA3-Slc4a11A, pcDNA3-Slc4a11B, or pcDNA3 (mock). Confocal XY maximum projection images are shown.
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Figure 5. Immunolocalization of Slc4a11 in renal tubules of SW-acclimated river pufferfish. Serial frozen sections of mefugu kidney were stained with anti-Slc4a11 antiserum (A and C) or antigen-absorbed anti-Slc4a11 antiserum (B and D) (green), anti-Na+-K+-ATPase (NKA) antibody (red), and Hoechst (blue). Bars represent 20 μm. c, collecting duct; p, proximal tubule; Slc4, solute carrier family 4; SW, seawater.
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Figure 6. Boric acid transport mediated by Slc4a11A. A, representative traces of boric acid–elicited currents and changes in intracellular pH of voltage-clamped oocytes (holding potential Vh: −60 mV) injected with Slc4a11A or water (control). B, current–voltage (I–V) relationships of oocytes expressing Slc4a11A and control oocytes in the presence or the absence of 20 mM boric acid. Values are means ± SD, n = 5 to 8. C, representative traces of boric acid–elicited changes of membrane potential (Vm) of oocytes injected with Slc4a11A and water (control). D, Michaelis–Menten curve fitted to boric acid–elicited currents of oocytes expressing Slc4a11A at +60 mV. Boric acid–elicited currents were measured by the addition of 1, 3, 5, 10, and 20 mM boric acid and were calculated as I(boric acid) – I(no boric acid). Maximum current (Imax) and Michaelis–Menten constant (Km) are shown. Values are means ± SEM, n = 3. E, boric acid uptake by voltage-clamped oocytes. Slc4a11A oocytes and control oocytes were voltage clamped (Vh: 0 mV) in ND96 containing 10 mM boric acid for 10 min, and the amount of boron in each oocyte was measured by ICP-MS. Dots represent individual data. Bar graphs represent means ± SD, n = 6. F, time course of boric acid uptake by unclamped oocytes. Oocytes were incubated in an ND96 medium containing 20 mM boric acid for 30, 60, and 90 min, and the amount of boron in each oocyte was measured by ICP-MS. Values are means ± SD, n = 4. G, boric acid uptake by unclamped oocytes. Oocytes were incubated in test solutions for 24 to 40 h, and the amount of boron in each oocyte was measured by ICP-MS. Dots represent individual data. Bar graphs represent means ± SD, n = 4. No VC, unclamped. H, rates of boric acid influx in voltage-clamped (Vh: 0 mV) and unclamped oocytes. The rates were calculated from data shown in (E and F). Dots represent individual data. Bar graphs represent means ± SD, n = 4 to 6. I, time course of boric acid efflux by unclamped oocyte. Oocytes were incubated in an ND96 medium containing 20 mM boric acid for 24 h until saturated, followed by incubation in ND96 for 5, 10, and 20 min, and the amount of boron in each oocyte was measured by ICP-MS. Values are means ± SD, n = 4. ICP-MS, inductively coupled plasma mass spectrometry; Slc4, solute carrier family 4.
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Figure 7. Voltage-clamp analyses of Na+-independent electrogenic boric acid transport activity of Slc4a11A. A, current–voltage (I–V) relationships of Slc4a11A or water-injected (control) oocytes in a solution containing 20 mM boric acid and various cations. Boric acid–elicited currents calculated by subtraction are shown (n = 3 to 6). B, dose-dependent inhibition of boric acid transport activity of Slc4a11A by NMDG. Boric acid–elicited currents of Slc4a11A oocytes (holding potential Vh: +60 mV) in the presence of various concentrations of NMDG are shown. Values are means ± SD (n = 4 to 10). NMDG, N-methyl-d-glucamine; Slc4, solute carrier family 4.
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Figure 8. Ion-selective microelectrode analysis during Vm clamping and pH dependence of Na+-independent electrogenic boric acid transport activity of Slc4a11A. Representative traces of boric acid–elicited currents (holding potential Vh: −60 or 0 mV) and changes in intracellular pH of Slc4a11A (A) and control (B) oocytes. Oocytes were analyzed in ND96 (indicated by Na+) or similar media in which Na+ was replaced with Li+, choline, or K+. C, representative traces of boric acid–elicited currents (holding potential, −20 mV) and intracellular [Na+] of Slc4a11A oocyte. D, current–voltage (I–V) relationship of 5 mM boric acid–elicited currents in various pH conditions. Values are means (n = 4 to 5). Slc4, solute carrier family 4.
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Figure 9. Activity of Slc4a11A in HEK293 and yeast cells. A, the whole-cell current was measured in untransfected HEK293 cells (control) or HEK293 cells expressing eGFP-Slc4a11A. The cells were incubated in a solution containing 20 mM boric acid and 145 mM Na+ or Na+-free media in which all Na+ was replaced with choline or NMDG. B, concentration of boron in the yeast cells expressing Slc4a11s or AtBOR1. Cells were incubated in a medium containing 20 mM boric acid; boron concentration in these cells was measured by ICP-MS. Dots represent individual data. Values are means ± SD. ∗p < 0.002. eGFP, enhanced GFP; HEK293, human embryonic kidney 293 cell line; ICP-MS, inductively coupled plasma mass spectrometry; NMDG, N-methyl-d-glucamine; Slc4, solute carrier family 4.
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Figure 10. Model of electrogenic boric acid transport activity of Slc4a11A. A, schematic representation of B(OH)4− uniporter (left) or B(OH)3-OH− cotransporter (right) activity of Slc4a11A in Xenopus oocytes. B(OH)3/H+ exchange activity is equivalent with B(OH)3-OH− cotransport activity. B, hypothetical model of the epithelial secretion system for boric acid in the collecting duct cell of SW fish. Apical Slc4a11A mediates the B(OH)4− uniport (left) or B(OH)3-OH− cotransport (right), and the negative membrane potential and acidic pH of urine may be the driving forces for the luminal boric acid secretion. The activity of Slc4a11A may be coupled with apical H+-efflux systems, such as the Na+/H+ exchanger 3 (NHE3) or V-type H+-ATPase. Basolateral entry of boric acid from the plasma to the cytoplasm may be mediated by AQPs. AQP, aquaporin; Slc4a, solute carrier family 4; SW, seawater.
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