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FIGURE 1. 5′-RACE and RT-PCR analysis of Slc4a10 expression in small intestine of rat. (A) Diagram of structure of rat Slc4a10 gene. Rat Slc4a10 contains 30 exons and three promoters: promoter P1 (upstream of exon 1), P2 (located in the intron between exons 1 and 2), and P3 (located in the intron between exons 3 and 4). Exon 1 contains the coding sequence for unique Nt of MEIK-NBCn2. Exon 4 contains the coding sequence for the unique Nt of MCDL-NBCn2. Exons 6-26 boxed in gray encode the transmembrane domain of NBCn2. (B) Agrose gel analysis of 5′-RACE of Slc4a10 from small intestine. (C) Agrose gel analysis of RT-PCR product of full-length Slc4a10 transcripts from small intestine. Arrows a, b, and c in panel (A) indicate the approximate positions of the primers used for 5′-RACE. Arrowheads in panel (B) indicate the target bands of PCR products of Slc4a10.
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FIGURE 2. Sequence alignment of three partial clones obtained by 5′-RACE. All three clones of C4, C5, and C6 include exon 1 (encoding “MEIKâ€) and exon 4 (encoding “MCDLâ€). Clone C4 contains additional 4 nucleotides (nt) “ACAG†at the 5′-end of exon 4, and thus is predicted to express MCDL-NBCn2. Both clones 5 and 6 contain exon 1 and exon 4 in frame, and thus are predicted to express MEIK-NBCn2. Stars indicate the sequences identical in all three clones. The red boxes indicate the coding region of exon 1 for “MEIKâ‹…â‹…â‹…â‹…â‹…â‹…â€, whereas the blue boxes indicate the coding region of exon 4 for “MCDLâ‹…â‹…â‹…â‹…â‹…â‹…â€.
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FIGURE 3. Diagrams of partial-exon structures of rat Slc4a10 transcripts and full-length variants of NBCn2. (A) Partial-exon structures of Slc4a10 transcripts. (B) Diagrams of predicted primary structures of full-length NBCn2 variants. Not shown here are two full-length variants identified in mouse NBCn2-I and -J with minor variation at the Ct end, as well as four Nt-truncated NBCn2 variants originally identified from rat kidney (Wang et al., 2015). NBCn2-K is predicted to start with “MEIKâ€. However, the NBCn2 in small intestine can be detected with anti-MCDL, but not with anti-MEIK (see text in “Discussionâ€).
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FIGURE 4. Functional characterization of NBCn2 in Xenopus oocytes. (A) Representative recordings of Vm and pHi of an oocyte expressing rat NBCn2-G. (B) Representative recordings of Vm and pHi of an oocyte expressing rat NBCn2-K. (C) Representative recordings of Vm and pHi of a control oocyte injected with H2O. (D) Summary of pHi recovery rate dpHi/dt. The dpHi/dt of both NBCn2-G and NBCn2-K are not significantly different from each other, but both are significantly higher than that of H2O-injected control oocytes by one-way ANOVA analysis followed by Fisher’s comparison. The numerals in the parentheses in panel D indicate the number of individual oocytes included in each bar.
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FIGURE 5. Distribution of NBCn2, NHE3, and Na+-K+ pump in different segments of rat small intestine. (A) Image showing the tissue collection sites for different fragments of the small intestine. S1, duodenum; S2, proximal jejunum; S3, middle jejunum; S4, distal jejunum; S5, ileum. Scale bar, 5 cm. (B) Western blotting of NBCn2 (with anti-MCDL), NHE3, and Na+-K+ pump in segments S1-S5 of the small intestine. β-actin is used as loading control. (C) Fractional distribution of NBCn2 in S1-S5. (D) Fractional distribution of NHE3 in S1-S5. (E) Fractional distribution of Na+-K+ pump in S1-S5. The density of NBCn2 was normalized to that of actin of the same lane from blots like those shown in panel (B). The fractional distribution of NBCn2 in each segment was computed by dividing this normalized density by the sum of the normalized densities of S1-S5. The fractional distribution of NHE3 and Na-K pump was computed by a similar approach. Ns in panels C–E indicate the number of rat individuals included in each panel. One-way ANOVA followed by Fisher’s comparison was used for statistical analysis. P < 0.05 is considered significantly different. Bars not marked by a same alphabet are significantly different from each other.
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FIGURE 6. NBCn2 and NHE3 are expressed at the apical membrane of epithelium in small intestine. (A) Overview of staining with anti-MCDL (NBCn2) in the small intestine. (B) Overview of staining with anti-NHE3 in the small intestine. (C) Negative staining of anti-NBCn1 in the small intestine. In these experiments, the final concentrations of anti-MCDL, anti-NHE3, and anti-NBCn1 for immunofluorescence staining were 1.5 μg/ml (1:400 dilution). NBCn2 and NHE3 are mainly expressed in the villi of the small intestine. In panel C, no significant staining was observed for anti-NBCn1 when visualized under microscopy with parameters the same as those used for panel A and C. Inset in panel C shows that the non-specific background staining by anti-NBCn1, when visualized by a much higher exposure, is distributed throughout the cytosol of the epithelia. (D–F) High magnification view shows that NBCn2 is exclusively expressed at the apical membrane of small intestine epithelium. (G–I) High magnification view shows that NHE3 is exclusively expressed at the apical membrane of small intestine epithelium. In these experiments, the basolateral membrane is stained by α1 of Na+-K+ pump.
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FIGURE 7. High NaCl intake decreases expression of NBCn2 (A) and NHE3 (B) in small intestine of rat. Membrane preparations of the small intestine were used for western blotting to examine the expression of NBCn2 (probed with anti-MCDL) or NHE3. The density of the target transporter in each lane was normalized to that of actin of the same lane. This ratio of each lane was then normalized to the average of the ratios of the control lanes in the same blot. Compared to the control, NaCl treatment decreases the abundance of NBCn2 by 55% and that of NHE3 by 40%. Numerals in the parenthesis indicate the numbers of rats included in each condition. For statistical comparison, two-tailed student’s t-test was performed. “−†indicates the controls, whereas “+†indicates the rats treated with NaCl.
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FIGURE 8. Model to show the hypothetical role of NBCn2 in small intestine epithelium. In the apical membrane, NHEs (e.g., NHE2 and NHE3) in concert with SLC26 transporters mediate NaCl absorption. The NaCl absorption via this pathway involves the action of carbonic anhydrase (CA) in both the luminal side and the cytosol. The presence of NBCn2 in the apical membrane likely provides an alternate pathway for the absorption of Na+ and the direct uptake of luminal HCO3–. In the basolateral membrane, NaCl extrusion is mediated by the coupled action of Na+-K+ pump and Cl– channel ClC-2 (Lipecka et al., 2002; Pena-Munzenmayer et al., 2005). Kir7.1 expressed in the basolateral membrane provides a pathway for the cycling of K+ (Partiseti et al., 1998; Nakamura et al., 1999).
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