Chang, Min‐Hwang

We previously cloned and analyzed the electrogenic Na + ‐Bicarbonate Cotransporter from zebrafish (zNBCe1; 80% identity to mammalian NBCe1; Sussman 2009, PMC2770747 ). As other fish NBCe1 clones, zNBCe1 is the only NBCe1 isoform, verified by functional data, and is most similar to the pancreatic form of NBCe1 (Slc4a4‐B). IRBIT, an inositol 1,4,5‐trisphosphate (IP 3 ) receptor‐binding protein, specifically binds to and activates this N‐terminus (B & C isoforms) but not the shorter kidney‐type/A‐isoform. This binding depends on IRBIT phosphorylation at multiple serine residues. We tested the hypothesis that zNBCe1, as a NBCe1‐B, could functionally interact with zIRBIT rather than no regulatory interaction observed in NBCe1‐A isoforms. The zIRBIT cRNA was co‐expressed with zNBCe1 or an N‐terminal truncation (Δ91aa) of zNBCe1 in Xenopus oocytes and functionally tested using two‐electrode voltage clamp. We also investigated whether IRBIT binding would activate NBCe1‐B specific human cSNPs (identified NHLBI 6500 genome) in the autoinhibitory domain (AID). Our data demonstrate that zNBCe1 is a functional ortholog of mammalian NBCe1‐B, which both binds IRBIT via the N‐terminus and stimulates transport activity. Nevertheless, zNBCe1 transport activity was not augmented by AID removal at SPAAER motif (Δ91aa) as observed in mammalian NBCe1‐B or NBCe1‐C. Moreover, while human NBCe1‐B specific coding SNPs (G50A, N63S) show diminished transport function (vs. WT NBCe1‐B), IRBIT can still stimulate transport to NBCe1‐A levels. Together these data implicate that IRBIT regulation of NBCe1‐function is evolutionary preserved in teleost precedes NBCe1‐A appearance and that this regulation fine‐tunes NBCe1‐B transporting capacity in the presence of certain AID‐cSNPS. This IRBIT‐control might also play a physiological role when NBCe1‐A is deleted from particular cells or tissues. Support or Funding Information Mayo Foundation This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Euryhaline pufferfish ( Takifugu obscurus, mefugu) can live in freshwater (FW) with ion concentration less than 1/100 of its body fluid or acclimate to seawater (SW) which has more than 3 times the ion concentration of its body fluid. However, the body‐fluid ion concentrations are maintained stable by the function of epithelial cells in the gill, intestine, and kidney which transport ions directionally and selectively. To understand the mechanisms of the fish body‐fluid homeostasis at the molecular level, there are several essentials: identification of transporters expressed in the epithelia, determination of transporter localization, and characterization of transporter activities. Analyses of various transporter mRNA levels in mefugu tissues identified increases for Slc26a6A and Slc26a6B in the intestine and the kidney of SW‐acclimated mefugu. To study their functions for SW acclimation, we subcloned the cDNAs into a Xenopus expression vector and assayed their functions using microelectrodes in oocytes. The electrophysiological analyses of the oocytes revealed that (a) Slc26a6A and Slc26a6B are electrogenic Cl − / n HCO 3 − exchangers; (b) Slc26a6A and Slc26a6B are also exchangers of Cl − /formate, Cl − /oxalate, and Cl − /SO 4 2− ; and (c) the slope of I–V relationship of Slc26a6A in the presence of SO 4 2− is 70 times higher than that of Slc26a6B. These results suggest significant roles for Slc26a6A and Slc26a6B in intestinal HCO 3 − secretion and a role for Slc26a6A in renal SO 4 2− excretion during SW acclimation. This work was supported by MEXT Japan, NIH (EY017732) and CFF grant (Romero‐06G0).

Boron is a vital micronutrient and is toxic at high concentrations, however, little is known about whole‐body boric acid homeostasis in animals. Slc4a11 was reported to function as a Na + ‐coupled borate transporter in mammals, presumably for borate absorption. However, seawater (SW) contains 0.4 mM boric acid, and the bladder urine of a euryhaline pufferfish mefugu ( Takifugu obscures ) in SW contains ~20 mM boric acid. Therefore, mefugu kidney is a good model to study a borate efflux system. In the mefugu kidney, a paralog of Slc4a11 (Slc4a11A) was markedly induced after transfer to SW and localized to the apical membrane of renal tubules. When Xenopus oocytes expressing Slc4a11A were voltage‐clamped at a holding potential of −60 mV and exposed medium containing borate, intracellular pH was increased and an outward current (anion influx) was observed. The borate current was not altered when Na + was replaced with other cations such as choline and Li + , but was eliminated when Na + was replaced with borate chelating agent NMDG. The borate (boron) influx was confirmed by elemental analysis of the oocytes. These results indicate that Slc4a11A is a Na + ‐independent B(OH) 4 − channel which is suitable for borate secretion, and clarify at first the mechanism of the borate efflux system in animal that prevent marine fishes from the toxic effects of borate.

The ten‐member SLC26 gene family encodes anion transporters with a diversity of substrates and transport modes, functioning as electrogenic exchangers, anion sensors, and/or anion channels. Slc26a6 is perhaps the most versatile paralog, mediating electrogenic exchange of multiple divalent and monovalent anions. We have generated a cysteineless Slc26a6 for structure‐function studies, fully substituting 8 cysteines with threonine, serine, and alanine, as dictated by the functional data for single and compound mutants. Although absolute transport rates are reduced in the cysteineless Slc26a6 protein it exhibits equivalent anion specificity and electrophysiology (chloride‐bicarbonate exchange) to that of wild‐type. On Western blots, the oligomerization of wild‐type Slc26a6 is inhibited by dithiothreitol, suggesting a role for inter‐cysteine disulfide bonds; oligomerization is absent in the cysteineless protein. Co‐immunoprecipitation of FLAG‐ and MYC‐tagged Slc26a6 is easily detectable for wild‐type proteins but absent in the epitope‐tagged cysteineless proteins. The equivalent data for single and compound mutants implicate C‐terminal cytoplasmic cysteines in oligomerization of the transporter protein. In summary, the eight cysteines in Slc26a6 are dispensable for transport activity but appear to play a role in oligomerization of the transport protein.

The solute carrier 26 (Slc26) transporters are anion transporters with diverse substrate specificity. They are expressed mainly in epithelia and play a central role in secretion and absorption of anions such as chloride, oxalate (ox 2− ), sulfate (SO 4 2− ), bicarbonate (HCO 3 − ), iodide, formate and hydroxyl. In mammals the Slc26a6 protein is expressed in kidney and gut epithelia, and mediates electrogenic Cl − /ox 2− , Cl − / n HCO 3 − , Cl − /SO 4 2− exchange. Recently, chicken and zebrafish prestin (Slc26a5) were shown to mediate electrogenic Cl − /ox 2− and Cl − /SO 4 2− exchange rather than being an auditory sensor. We have cloned and characterized the Slc26a5 (fPrestin) transporter from the euryhaline pufferfish Takifugu obscurus (mefugu). This fPrestin mRNA is expressed ubiquitously unlike fSlc26a6A‐C which is found in kidney and gut. While the fSlc26a6A‐C mRNAs are increased in response to a freshwater to saltwater transition, fPrestin expression is unchanged. We next expressed fSlc26a5 in Xenopus oocytes and evaluated transport using microelectrodes. These experiments reveal that fPrestin mediates electrogenic Cl − /ox 2− , and Cl − /SO 4 2− as previously shown for chicken and zebrafish Slc26a5. Furthermore, fPrestin also can mediate electrogenic Cl − / n HCO 3 − exchange. These transport features are quite similar to mammalian Slc26a6 which has been implicated in renal Ca‐oxalate, stones.

The SLC26A9 protein mediates Cl‐ transport as a Cl‐ channel and a Cl‐/HCO3 exchanger in the several epithelial cells, e.g., gut, lung, kidney. Topology analysis from BicA, a member of the SulP/SLC26A family, suggests that Slc26 proteins consists of 12 predicted transmembrane regions. However, the topology model of human SLC26 transporters has not yet been directly investigated. In this study, 12 HA‐inserted SLC26A9 mutants were constructed and expressed in Xenopus oocytes. Chemiluminescence was used to determine which HA‐tags were accessible from the extracellular surface. 157‐160 and 352‐353 sites of human SLC26A9 were identified as extracellular sites. Interestingly, the SLC26A9 HA (159‐160) mutant has ~40% Cl‐ transport activity, but this decreased function could be rescued by coexpression with another low function cSNP (T127N‐SLC26A9). These results revealed that the topology of TM region of SLC26A9 may different from that of BicA and also indicate that human SLC26A9 functions as dimer. Grant Funding Source : Supported by NIH EY 017732 (MFR)

Zinc (Zn2+) is the second most abundant trace element, but is considered a micronutrient, as it is a cofactor for many enzymes and transcription factors. Whereas Zn2+ deficiency can cause cognitive immune or metabolic dysfunction and infertility, excess Zn2+ is nephrotoxic. As for other ions and solutes, Zn2+ is moved into and out of cells by specific membrane transporters: ZnT, Zip, and NRAMP/DMT proteins. ZIP10 is reported to be localized at the apical membrane of renal proximal tubules in rats, where it is believed to play a role in Zn2+ import. Renal regulation of Zn2+ is of particular interest in light of growing evidence that Zn2+ may play a role in kidney stone formation. The objective of this study was to show that ZIP10 homologs transport Zn2+, as well as ZIP10, kidney localization across species. We cloned ZIP10 from dog, human, and Drosophila ( CG10006), tested clones for Zn2+ uptake in Xenopus oocytes and localized the protein in renal structures. CG10006, rather than foi (fear-of-intimacy, CG6817) is the primary ZIP10 homolog found in Drosophila Malpighian tubules. The ZIP10 antibody recognizes recombinant dog, human, and Drosophila ZIP10 proteins. Immunohistochemistry reveals that ZIP10 in higher mammals is found not only in the proximal tubule, but also in the collecting duct system. These ZIP10 proteins show Zn2+ transport. Together, these studies reveal ZIP10 kidney localization, a role in renal Zn2+ transport, and indicates that CG10006 is a Drosophila homolog of ZIP10.

Mutations in the electrogenic Na+/HCO3−cotransporter (NBCe1) that cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts in patients are recessive. Parents and siblings of these affected individuals seem asymptomatic although their tissues should make some mutant NBCe1 protein. Biochemical studies with AE1 and NBCe1 indicate that both, and probably all, Slc4 members form dimers. However, the physiologic implications of dimerization have not yet been fully explored. Here, human NBCe1A dimerization is demonstrated by biomolecular fluorescence complementation (BiFC). An enhanced yellow fluorescent protein (EYFP) fragment (1–158, EYFPN) or (159–238, EYFPC) was fused to the NH2or COOH terminus of NBCe1A and mix-and-matched expressed in Xenopus oocyte. The EYFP fluorescent signal was observed only when both EYFP fragments are fused to the NH2terminus of NBCe1A (EYFPN-N-NBCe1A w/ EYFPC-N-NBCe1A), and the electrophysiology data demonstrated this EYFP-NBCe1A coexpressed pair have wild-type transport function. These data suggest NBCe1A forms dimers and that NH2termini from the two monomers are in close proximity, likely pair up, to form a functional unit. To explore the physiologic significance of NBCe1 dimerization, we chose two severe NBCe1 mutations (6.6 and 20% wild-type function individually): S427L (naturally occurring) and E91R (for NH2-terminal structure studies). When we coexpressed S427L and E91R, we measured 50% wild-type function, which can only occur if the S427L-E91R heterodimer is the functional unit. We hypothesize that the dominant negative effect of heterozygous NBCe1 carrier should be obvious if the mutated residues are structurally crucial to the dimer formation. The S427L-E91R heterodimer complex allows the monomers to structurally complement each other resulting in a dimer with wild-type like function.

Secretion of HCO3− at the apical side of the epithelial cells of the choroid plexus is an essential step in the formation of cerebrospinal fluid. Anion conductance with a high degree of HCO3− permeability has been observed and suggested to be the major pathway for HCO3− transport across the apical membrane. Recently, it was found that NBC (Na+/HCO3− co-transporter) 4, an electrogenic member of the NBC family, was expressed in the choroid plexus. We found that a novel variant of the NBC4 [NBC4g/Slc4a5 (solute carrier family 4, sodium bicarbonate co-transporter, member 5)] is almost exclusively expressed in the apical membrane of rat choroid plexus epithelium at exceptionally high levels. RNA interference-mediated knockdown allowed the functional demonstration that NBC4g is the major player in the HCO3− transport across the apical membrane of the choroid plexus epithelium. When combined with a recent observation that in choroid plexus epithelial cells electrogenic NBC operates with a stoichiometry of 3:1, the results of the present study suggest that NBC4g mediates the efflux of HCO3− and contributes to cerebrospinal fluid production.