Citations

Marine teleost fish precipitate divalent cations as carbonate deposits in their intestine to minimize the potential for excessive calcium entry and to stimulate water absorption by removing osmolyte ions. This carbonate deposit formation therefore plays an important role in osmoregulation in the seawater environment and also requires controlled secretion of HCO 3 − to match the amount of calcium entering the intestinal lumen. Despite this physiological importance, the process of carbonate precipitate formation has not been characterized at the molecular level. We first isolated 24 candidate cDNA clones representing all possible membrane proteins capable of transporting HCO 3 − from the euryhaline pufferfish, mefugu Takifugu obscurus based on the DNA sequence information on the T. rubripes genome, then performed Northern blot analyses to select clones highly expressed in the intestine of seawater mefugu, and finally determined the cellular locations by immunohistochemistry. Based on these observations, we proposed a model for HCO 3 − excretion across the intestinal epithelium of marine teleost fish that includes a basolateral NBCe1 (Na + /bicarbonate cotransporter) and apical Slc26a6 (Cl − /HCO 3 − exchangers).

Mutations in the electrogenic Na + /HCO 3 − cotransporter (NBCe1) result in proximal renal tubular acidosis, glaucoma and cataracts in patients and are only recessive. Parents and siblings of these affected individuals are asymptomatic though their tissues should make some of the 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. For our studies we chose two severe NBCe1 mutations (<10% WT function individually): S427L (naturally occurring) and E91R (for N‐terminal structure studies). When we co‐expressed S427L and E91R, we measured ~55% wild‐type function which can only occur if the S427L‐E91R heterodimer is the functional unit. To further investigate this functional dimerization, we have engineered concatemeric hkNBCe1 constructs. When the hkNBCe1 tandem‐dimer (WT‐WT) cRNA was expressed in Xenopus oocytes, the electrophysiology data demonstrate the WT‐WT homodimer functions as a WT hkNBCe1. The electrophysiology data also showed that the WT‐S427L or WT‐E91R heterodimers functioned very similarly to WT hkNBCe1. Intriguingly, we observed ~50% WT function in both the S427L‐E91R and E91R‐S427L heterodimers constructs. These data illuminate why the "NBCe1 phenotype" only occurs recessively. An eYFP fragment (1‐158, YFP N ) was fused to the N‐ or C‐terminus of hkNBCe1 and another eYFP fragment (159‐238, YFP C ) was fused to N‐ or C‐terminus of hkNBCe1. The electrophysiology data demonstrate the eYFP‐hkNBCe1 fusion proteins have WT hkNBCe1 like function. The hkNBCe1 dimerization can also be visualized by biomolecular fluorescence complementation (BiFC) method. We plan to further use these molecular tools to probe the nature of the hkNBCe1 dimer interactions. Supported by EY017732.

Many diseases are caused by altered epithelial Cl − or HCO 3 − transport. Mutations in the SLC26A3 transporter are causative for congenital chloride‐losing diarrhea; however, no other SLC26 transporter plays such a direct genetic role in disorders of epithelial Cl − transport. SLC26A9 is expressed in the lung, stomach, intestine and kidney (lesser extent) but its physiological function in these epithelia is not clear. Thus far, no diseases result from SLC26A9 mutations. However, database analysis reveals three single nucleotide polymorphisms (SNPs). We reasoned that the human SLC26A9‐SNPs would provide insight into what a disease phenotype might be functionally manifest. Here we analyzed the ion transport characteristics of human SLC26A9 and its SNPs: V622L, V744M and H748R. Human SLC26A9 and its three SNPs were expressed in Xenopus laevis oocytes. Effects on ion transport were evaluated using ion selective electrodes and voltage clamping. Measuring Cl − and HCO 3 − flux, our results show that SLC26A9 is a Cl − /HCO 3 − exchanger. One SNP, V622L, is located in the C‐terminus just before the STAS domain of SLC26A9. V622L has ~50% function (current and Cl − transport). These results suggest that the possession of V622L allele in humans would decrease SLC26A9 function and modulate the risk for lung disease (cystic fibrosis), gut disease (gastric ulcer) or hypertension, due to reduced Cl − transport across epithelial cells.

The majority of patients with cystic fibrosis (CF) have a single codon deletion in the CFTR gene that impairs assembly of the multidomain glycoprotein, most of which succumbs to ER quality control and proteasomal degradation. Exit from the ER can be facilitated by specific manipulations i.e. growth of cells at reduced temperature, but the rescued ΔF508 is rapidly cleared from the distal secretory pathway and degraded in lysosomes. Recently, accumulation and redistribution of free cholesterol was observed in CF cells and attributed to the lack of CFTR function (White et al. (2004) Am J Respir Cell Mol Biol. 31:538‐43). Here we show that the presence of several misassembled mutant CFTR proteins including ΔF508 in the distal secretory pathway rather than the absence of a functional CFTR is responsible for lipid alterations. Unesterified cholesterol and BIODIPY‐lactosylceramide accumulate in punctate late endosomal structures and are unable to translocate to the Golgi and ER for esterification. This block can be overcome by Rab9 overexpression resulting in clearance of accumulating intracellular cholesterol. Similar alterations were observed on expression of a temperature‐rescued variant of the related ABC protein MRP1. Thus, on escape from ER quality control, misassembled mutants of CFTR and possibly other proteins may impair sterol and glycosphingolipid homeostasis which involves trafficking through the membranous compartments traversed by the aberrant proteins as they are cleared. Supported by the NIH (DK051870).

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.