Romero, Michael F.

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.

SMCTe (SLC5A8) transports several monocarboxylates, including short‐chain fatty acids, ketone‐bodies, pyruvate, lactate, and keto‐isocaproic acid (KIC). We found SMCTe mRNA in zebrafish pancreas ( in situ hybridization); other groups have demonstrated that SLC5A8 gene silencing is associated with pancreatic cancer. Extracellular KIC stimulates insulin secretion in pancreas, suggesting a potential role for SMCTe. However, the physiological role of SMCTe in pancreas is unknown. Using RT‐PCR with rat SMCTe‐specific primers, we found that SMCTe mRNA is expressed in rat pancreas. Using Western Blot analysis with specific SMCTe antibodies, we also detected SMCTe protein in rat pancreas (~75 kDa). We next tested the effect of insulin on SMCTe function using 22 Na + uptake in Xenopus oocytes injected with human SMCTe (hSMCTe) or co‐injected with hSGK1 cRNA. Specifically, we evaluated transport 22 Na + uptake elicited by 40 U insulin in presence or absence of KIC. Insulin reduced KIC‐mediated 22 Na + uptake transport by ~25% in hSMCTe‐oocytes. Human SGK1 also reduced KIC‐mediated 22 Na + uptake transport by ~25% in hSMCTe‐oocytes. Co‐expression of hSMCTe and hSGK1 in oocytes did not further reduce KIC‐mediated 22 Na + uptake. These results indicate that the signal transduction pathway by which insulin and SKG1 reduce SMCTe transport activity overlaps. CONACYT48709MJ(CP), EY017732 (MFR), DK070756 (DM), DK64635(GG)

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.

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 .

The electroneutral (zSMCTn/Slc5a12) and electrogenic (zSMCTe/Slc5a8) Na + /monocarboxylate (MC − ) transporters of zebrafish, move short chain fatty acids, ketone‐bodies, lactate, pyruvate and nicotinate but with clear differences in Na + : MC − stoichiometry and affinity. The affinity of zSMCTn for pyruvate, lactate and nicotinate is lower than zSMCTe (~6, ~10 and ~40 times respectively). It is known that valproate and nonsteroidal anti‐inflammatory drugs, such as ibuprofen, valproate reduce the activity of mammalian SMCTe, but this effect has not been characterized for SMCTn. Using 22 Na + uptake in Xenopus oocytes injected with either zSMCT's or hSMCT cRNA, we evaluated the kinetics of Na + propionate cotransport by SMCT's (hSMCTe, zSMCTe, zSMCTn). We also determined cotransport inhibition by ibuprofen, ketoprofen, naproxen and valproate of these oocytes in presence or absence of propionate. Our results indicate that zSMCTn (Km =830±110μM) has only 3 times less affinity for propionate than zSMCTe (Km =240±50μM) and hSMCTe (Km =200±20μM). Ibuprofen, ketoprofen, naproxen and valproate cause a significant reduction in 22 Na + uptake for zSMCTe, hSMCTe as well as zSMCTn in presence of propionate. Ibuprofen inhibition is more potent for zSMCTn (K i ~200μM) than zSMCTe (K i ~600μM) and hSMCTe (K i ~800μM). These functional and inhibitory differences may help define the binding site for MC − and Na + of SMCT proteins.

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).

The gas in the swimbladder of teleost fish is composed primarily of O 2 , and the swimbladder volume is regulated by O 2 transfer between the luminal space of the swimbladder and the blood. In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the “Root effect.” To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 16 members of the H + /monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute‐specific transcellular transport. Electrophysiological analyses demonstrated that MCT1b is a high‐affinity lactate transporter whereas MCT4b is a low‐affinity/high‐conductance lactate transporter. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b.

Background Kidney stones (nephrolithiasis) are an expensive and painful health problem with a complex etiology with ~70% of stones composed of calcium oxalate (CaOx). The Slc26 gene family contains several proteins that transport oxalate in the gut and kidney. Slc26a6 and its homologs exchange Cl − for oxalate (ox 2− ), as well as SO 4 2− , HCO 3 − and formate. Slc26a6 −/− mice have hyperoxaluria and CaOx stones illustrating a physiologic collaboration between the gut and kidney. We developed a Drosophila genetic model for CaOx stones (dPrestin, Slc26a5/6, manipulation) allowing rapid gut, renal and whole‐body physiological assessment (min/h v. mo/y). Methods (Tissue) First, Malpighian tubules (MT, renal tubule) were dissected from wild type and dPrestin knockdown mutants and soaked in ox 2− solutions. CaOx stones were visualized by DIC microscopy. Second, we generated Drosophila expressing eYFP in the MT, MT+gut or whole body. eYFP is a halide (Cl − ) sensor, allowing quantification of Cl − /ox 2− exchange by native dPrestin. (Whole body) Wild type and dPrestin knockdown flies were fed ox 2− enriched food, and flies imaged (CaOx quantified) by micro computed tomography (MicroCT). Results Exposing dPrestin‐knockdowns to bath ox 2− elicited almost no CaOx crystals. However, exposing wt‐MT to ox 2− elicited many CaOx crystals in 20 min. Increased CaOx crystal formation was also coupled with an eYFP intensity increase, matching dPrestin Cl − /ox 2− exchange characteristics. MicroCT showed more CaOx crystals in flies that fed on ox 2− longer. Conclusions Our observations indicate that Drosophila and dPrestin manipulation can be used for rapid, detailed studies of gut, renal and whole organism oxalate metabolism & CaOx stone formation. [DK83007]