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The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers. The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. Here, the authors resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize it by PELDOR/DEER distance measurements, biochemical studies with MD simulations and spin-label ensemble refinement.

Bicarbonate uptake is one of the early steps of capacitation, but the identification of proteins regulating anion fluxes remains elusive. The aim of this study is to investigate the role of sperm solute carrier 4 (SLC4) A1 (spAE1) in the capacitation process. The expression, location, and tyrosine-phosphorylation (Tyr-P) level of spAE1 were assessed. Thereby, it was found that 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), an SLC4 family channel blocker, inhibited capacitation in a dose-dependent manner by decreasing acrosome reaction (ARC% 24.5 ± 3.3 vs. 64.9 ± 4.3, p < 0.05) and increasing the percentage of not viable cells (NVC%), comparable to the inhibition by I-172, a cystic fibrosis transmembrane conductance regulator (CFTR) blocker (AR% 30.5 ± 4.4 and NVC% 18.6 ± 2.2). When used in combination, a synergistic inhibitory effect was observed with a remarkable increase of the percentage of NVC (45.3 ± 4.1, p < 0.001). spAE1 was identified in sperm membrane as a substrate for Tyr-protein kinases Lyn and Syk, which were identified as both soluble and membrane-bound pools. spAE1-Tyr-P level increased in the apical region of sperm under capacitating conditions and was negatively affected by I-172 or DIDS, and, to a far greater extent, by a combination of both. In conclusion, we demonstrated that spAE1 is expressed in sperm membranes and it is phosphorylated by Syk, but above all by Lyn on Tyr359, which are involved in sperm viability and capacitation.

The oceanic carbonate system is changing rapidly due to rising atmospheric CO 2 , with current levels expected to rise to between 750 and 1,000 μatm by 2100, and over 1,900 μatm by year 2300. The effects of elevated CO 2 on marine calcifying organisms have been extensively studied; however, effects of imminent CO 2 levels on teleost acid–base and respiratory physiology have yet to be examined. Examination of these physiological processes, using a paired experimental design, showed that 24 h exposure to 1,000 and 1,900 μatm CO 2 resulted in a characteristic compensated respiratory acidosis response in the gulf toadfish ( Opsanus beta ). Time course experiments showed the onset of acidosis occurred after 15 min of exposure to 1,900 and 1,000 μatm CO 2 , with full compensation by 2 and 4 h, respectively. 1,900-μatm exposure also resulted in significantly increased intracellular white muscle pH after 24 h. No effect of 1,900 μatm was observed on branchial acid flux; however, exposure to hypercapnia and HCO 3 − free seawater compromised compensation. This suggests branchial HCO 3 − uptake rather than acid extrusion is part of the compensatory response to low-level hypercapnia. Exposure to 1,900 μatm resulted in downregulation in branchial carbonic anhydrase and slc4a2 expression, as well as decreased Na + /K + ATPase activity after 24 h of exposure. Infusion of bovine carbonic anhydrase had no effect on blood acid–base status during 1,900 μatm exposures, but eliminated the respiratory impacts of 1,000 μatm CO 2 . The results of the current study clearly show that predicted near-future CO 2 levels impact respiratory gas transport and acid–base balance. While the full physiological impacts of increased blood HCO 3 − are not known, it seems likely that chronically elevated blood HCO 3 − levels could compromise several physiological systems and furthermore may explain recent reports of increased otolith growth during exposure to elevated CO 2 .

The Na/HCO3 cotransporter NBCe1 is a member of SLC4A transporters that move HCO3− across cell membranes and regulate intracellular pH or transepithelial HCO3 transport. NBCe1 is highly selective to HCO3− and does not transport other anions; the molecular mechanism of anion selectivity is presently unclear. We previously reported that replacing Asp555 with a Glu (D555E) in NBCe1 induces increased selectivity to other anions, including Cl−. This finding is unexpected because all SLC4A transporters contain either Asp or Glu at the corresponding position and maintain a high selectivity to HCO3−. In this study, we tested whether the Cl− transport in D555E is mediated by an interaction between residues in the ion binding site. Human NBCe1 and mutant transporters were expressed in Xenopus oocytes, and their ability to transport Cl− was assessed by two-electrode voltage clamp. The results show that the Cl− transport is induced by a charge interaction between Glu555 and Lys558. The bond length between the two residues is within the distance for a salt bridge, and the ionic strength experiments confirm an interaction. This finding indicates that the HCO3− selectivity in NBCe1 is established by avoiding a specific charge interaction in the ion binding site, rather than maintaining such an interaction.

Bone remodeling requires osteoclasts to generate and maintain an acidified resorption compartment between the apical membrane and the bone surface to solubilize hydroxyapatite crystals within the bone matrix. This acidification process requires (i) apical proton secretion by a vacuolar H ⁺-ATPase, (ii) actin cytoskeleton reorganization into a podosome belt that forms a gasket to restrict lacunar acid leakage, and (iii) basolateral chloride uptake and bicarbonate extrusion by an anion exchanger to provide Cl ⁻ permissive for apical acid secretion while preventing cytoplasmic alkalinization. Here we show that osteoclast-targeted deletion in mice of solute carrier family 4 anion exchanger member 2 (Slc4a2) results in osteopetrosis. We further demonstrate a previously unrecognized consequence of SLC4A2 loss of function in the osteoclast: dysregulation of calpain-dependent podosome disassembly, leading to abnormal actin belt formation, cell spreading, and migration. Rescue of SLC4A2-deficient osteoclasts with functionally defined mutants of SLC4A2 indicates regulation of actin cytoskeletal reorganization by anion-exchange activity and intracellular pH, independent of SLC4A2’s long N-terminal cytoplasmic domain. These data suggest that maintenance of intracellular pH in osteoclasts through anion exchange regulates the actin superstructures required for bone resorption.

Primary biliary cirrhosis (PBC) is a cholestatic disease associated with autoimmune phenomena and alterations in both biliary bicarbonate excretion and expression of the bicarbonate carrier AE2. The bile acid ursodeoxycholic acid (UCDA) is currently used in treatment of cholestatic liver diseases and is the treatment of choice in PBC; however, a subset of PBC patients respond poorly to UDCA monotherapy. In these patients, a combination of UDCA and glucocorticoid therapy appears to be beneficial. To address the mechanism of this benefit, we analyzed the effects of UDCA and dexamethasone on AE2 gene expression in human liver cells from hepatocyte and cholangiocyte lineages. The combination of UDCA and dexamethasone, but not UDCA or dexamethasone alone, increased the expression of liver-enriched alternative mRNA isoforms AE2b1 and AE2b2 and enhanced AE2 activity. Similar effects were obtained after replacing UDCA with UDCA conjugates. In in vitro and in vivo reporter assays, we found that a UDCA/dexamethasone combination upregulated human AE2 alternate overlapping promoter sequences from which AE2b1 and AE2b2 are expressed. In chromatin immunoprecipitation assays, we demonstrated that combination UCDA/dexamethasone treatment induced p300-related interactions between HNF1 and glucocorticoid receptor on the AE2 alternate promoter. Our data provide a potential molecular explanation for the beneficial effects of the combination of UDCA and glucocorticoids in PBC patients with inadequate response to UDCA monotherapy.

Slc26a9 is a recently identified anion transporter that is abundantly expressed in gastric epithelial cells. To study its role in stomach physiology, gene targeting was used to prepare mice lacking Slc26a9. Homozygous mutant (Slc26a9⁻/⁻) mice appeared healthy and displayed normal growth. Slc26a9 deletion resulted in the loss of gastric acid secretion and a moderate reduction in the number of parietal cells in mutant mice at 5 weeks of age. Immunofluorescence labeling detected the H-K-ATPase exclusively on the apical pole of gastric parietal cells in Slc26a9⁻/⁻ mice, in contrast to the predominant cytoplasmic localization in Slc26a9⁺/⁺ mice. Light microscopy indicated that gastric glands were dilated, and electron micrographs displayed a distinct and striking absence of tubulovesicles in parietal cells and reductions in the numbers of parietal and zymogen cells in Slc26a9⁻/⁻ stomach. Expression studies indicated that Slc26a9 can function as a chloride conductive pathway in oocytes as well as a Cl⁻/HCO₃⁻ exchanger in cultured cells, and localization studies in parietal cells detected its presence in tubulovesicles. We propose that Slc26a9 plays an essential role in gastric acid secretion via effects on the viability of tubulovesicles/secretory canaliculi and by regulating chloride secretion in parietal cells.

Secretin induces bicarbonate-rich hydrocholeresis in healthy individuals, but not in untreated patients with primary biliary cirrhosis (PBC). Ursodeoxycholic acid (UDCA)--the first choice treatment for PBC--restores the secretin response. Compared with humans, secretin has poor effect in experimental normal-rat models with biliary drainage, although it may elicit hydrocholeresis when the bile-acid pool is maintained. In view of the benefits of UDCA in PBC, we used normal-rat models to unravel the acute contribution of UDCA (and/or taurine-conjugated TUDCA) for eliciting the biliary secretin response. Intravascular and/or intrabiliary administration of agonists and inhibitors was performed in normal rats with biliary monitoring. Secretin/bile-acid interplay was analyzed in 3D cultured rat cholangiocytes that formed expansive cystic structures with intralumenal hydroionic secretion. In vivo, secretin stimulates hydrocholeresis upon UDCA/TUDCA infusion, but does not modify the intrinsic hypercholeretic effect of dehydrocholic acid (DHCA). The former effect is dependent on microtubule polymerization, and involves PKCα, PI3K and MEK pathways, as shown by colchicine (i.p.) and retrograde biliary inhibitors. In vitro, while secretin alone accelerates the spontaneous expansion of 3D-cystic structures, this effect is enhanced in the presence of TUDCA, but not UDCA or DHCA. Experiments with inhibitors and Ca(2+)-chelator confirmed that the synergistic effect of secretin plus TUDCA involves microtubules, intracellular Ca(2+), PKCα, PI3K, PKA and MEK pathways. Gene silencing also demonstrated the involvement of the bicarbonate extruder Ae2. UDCA is conjugated in order to promote secretin-stimulated hydrocholeresis in rats through Ae2, microtubules, intracellular Ca(2+), PKCα, PI3K, PKA, and MEK.

As the only cell capable of bone resorption, the osteoclast is a central mediator of skeletal homeostasis and disease. To efficiently degrade mineralized tissue, these multinucleated giant cells secrete acid into a resorption lacuna formed between their apical membrane and the bone surface. For each proton pumped into this extracellular compartment, one bicarbonate ion remains in the cytoplasm. To prevent alkalinization of the cytoplasm, a basolateral bicarbonate/chloride exchanger provides egress for intracellular bicarbonate. However, the identity of this exchanger is unknown. Here, we report that the bicarbonate/chloride exchanger, solute carrier family 4, anion exchanger, member 2 (SLC4A2), is up-regulated during osteoclast differentiation. Suppression of Slc4a2 expression by RNA interference inhibits the ability of RAW cells, a mouse macrophage cell line, to differentiate into osteoclasts and resorb mineralized matrix in vitro. Accordingly, Slc4a2-deficient mice fail to remodel the primary, cartilaginous skeletal anlagen. Abnormal multinucleated giant cells are present in the bone marrow of Slc4a2-deficient mice. Though these cells express the osteoclast markers CD68, cathepsin K, and NFATc1, compared with their wild-type (WT) counterparts they are larger, fail to express tartrate-resistant acid phosphatase (TRAP) activity, and display a propensity to undergo apoptosis. In vitro Slc4a2-deficient osteoclasts are unable to resorb mineralized tissue and cannot form an acidified, extracellular resorption compartment. These data highlight SLC4A2 as a critical mediator of osteoclast differentiation and function in vitro and in vivo.

Human anion exchanger 2 (AE2) is a plasma membrane protein that regulates intracellular pH and cell volume. AE2 contributes to transepithelial transport of chloride and bicarbonate in normal colon and other epithelial tissues. We now report that AE2 overexpression in colon cancer cells is correlated with expression of the nuclear proliferation marker, Ki67. Survival analysis of 24 patients with colon cancer in early stage or 33 patients with tubular adenocarcinoma demonstrated that expression of AE2 is correlated with poor prognosis. Cellular and molecular experiments indicated that AE2 expression promoted proliferation of colon cancer cells. In addition, we found that transcription factor EGR1 underlies AE2 upregulation and the AE2 sequester p16INK4a (P16) in the cytoplasm of colon cancer cells. Cytoplasmic P16 enhanced ERK phosphorylation and promoted proliferation of colon cancer cells. Gastrin inhibited proliferation of colon cancer cells by suppressing expression of EGR1 and AE2 and by blocking ERK phosphorylation. Taken together, our data describe a novel EGR1/AE2/P16/P-ERK signaling pathway in colon carcinogenesis, with implications for pathologic prognosis and for novel therapeutic approaches.