The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells.
ABSTRACT: The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl- for oxalate and HCO3-, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl-/oxalate exchange. Of note, both Cl-/oxalate exchange and Cl-/HCO3- exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6-/- mice, but HCO3- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.
Project description:The secretin-stimulated human pancreatic duct secretes HCO(3)(-)-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO(3)(-) secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl(-)/HCO(3)(-) exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ?140 mM HCO(3)(-) or more, mouse and rat ducts secrete ?40-70 mM HCO(3)(-). Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO(3)(-) secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl(-)/Cl(-) exchange and electroneutral Cl(-)/HCO(3)(-) exchange. gpSlc26a6 in Xenopus oocytes mediated Cl(-)/Cl(-) exchange and bidirectional exchange of Cl(-) for oxalate and sulfate, but Cl(-)/HCO(3)(-) exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl(-), oxalate, and sulfate transport but no detectable Cl(-)/HCO(3)(-) exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of (36)Cl(-) influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO(3)(-) secretion in species that share a high HCO(3)(-) secretory output.
Project description:The phylogenetically ancient SLC26 gene family encodes multifunctional anion exchangers and anion channels transporting a broad range of substrates, including Cl(-), HCO3(-), sulfate, oxalate, I(-), and formate. SLC26 polypeptides are characterized by N-terminal cytoplasmic domains, 10-14 hydrophobic transmembrane spans, and C-terminal cytoplasmic STAS domains, and appear to be homo-oligomeric. SLC26-related SulP proteins of marine bacteria likely transport HCO3(-) as part of oceanic carbon fixation. SulP genes present in antibiotic operons may provide sulfate for antibiotic biosynthetic pathways. SLC26-related Sultr proteins transport sulfate in unicellular eukaryotes and in plants. Mutations in three human SLC26 genes are associated with congenital or early onset Mendelian diseases: chondrodysplasias for SLC26A2, chloride diarrhea for SLC26A3, and deafness with enlargement of the vestibular aqueduct for SLC26A4. Additional disease phenotypes evident only in mouse knockout models include oxalate urolithiasis for Slc26a6 and Slc26a1, non-syndromic deafness for Slc26a5, gastric hypochlorhydria for Slc26a7 and Slc26a9, distal renal tubular acidosis for Slc26a7, and male infertility for Slc26a8. STAS domains are required for cell surface expression of SLC26 proteins, and contribute to regulation of the cystic fibrosis transmembrane regulator in complex, cell- and tissue-specific ways. The protein interactomes of SLC26 polypeptides are under active investigation.
Project description:The nonselective anion exchanger Slc26a6, also known as putative anion transporter 1 and chloride/formate exchanger, is thought to play a major role in HCO3- transport in exocrine glands. In this study, <i>Slc26a6</i> null mice were used to explore the function of Slc26a6 in the exocrine pancreas. Slc26a6 primarily localized to the apical membrane of pancreatic exocrine acinar cells. The volume of stimulated juice secretion by the ex vivo pancreas was significantly reduced ~35% in <i>Slc26a6<sup>-/-</sup></i> mice, but no changes occurred in the gross structure or gland weights of Slc26a6 null mice. The secretion of pancreatic juice by <i>Slc26a6<sup>+/+</sup></i> mice was dependent on HCO3- while, in contrast, fluid secretion by <i>Slc26a6<i><sup>-/-</sup></i></i> mice was independent of HCO3-, suggesting that Slc26a6 mediates the HCO3--dependent component of fluid secretion. Consistent with these observations, disruption of <i>Slc26a6</i> also significantly reduced HCO3- secretion by the pancreas ~35%. Taken together, these results demonstrate that the apical Slc26a6 anion exchanger in acinar cells is involved in HCO3--dependent fluid secretion but that another major HCO3--independent pathway is the primary driver of the fluid secretion process in the mouse pancreas.
Project description:CFTR has been recognized to function as both an anion channel and a key regulator of Slc26 anion transporters in heterologous expression systems. Whether this regulatory relationship between CFTR and Slc26 transporters is seen in native intestine, and whether this effect is coupled to CFTR transport function or other features of this protein, has not been studied. The duodena of anesthetized CFTR-, NHE3-, Slc26a6-, and Scl26a3-deficient mice and wild-type (WT) littermates were perfused, and duodenal bicarbonate (HCO(3)(-)) secretion (DBS) and fluid absorptive or secretory rates were measured. The selective NHE3 inhibitor S1611 or genetic ablation of NHE3 significantly reduced fluid absorptive rates and increased DBS. Slc26a6 (PAT1) or Slc26a3 (DRA) ablation reduced the S1611-induced DBS increase and reduced fluid absorptive rates, suggesting that the effect of S1611 or NHE3 ablation on HCO(3)(-) secretion may be an unmasking of Slc26a6- and Slc26a3-mediated Cl(-)/HCO(3)(-) exchange activity. In the absence of CFTR expression or after application of the CFTR(inh)-172, fluid absorptive rates were similar to those of WT, but S1611 induced virtually no increase in DBS, demonstrating that CFTR transport activity, and not just its presence, is required for Slc26-mediated duodenal HCO(3)(-) secretion. A functionally active CFTR is an absolute requirement for Slc26-mediated duodenal HCO(3)(-) secretion, but not for Slc26-mediated fluid absorption, in which these transporters operate in conjunction with the Na(+)/H(+) exchanger NHE3. This suggests that Slc26a6 and Slc26a3 need proton recycling via NHE3 to operate in the Cl(-) absorptive mode and Cl(-) exit via CFTR to operate in the HCO(3)(-) secretory mode.
Project description:The gut and Malpighian tubules of insects are the primary sites of active solute and water transport for controlling hemolymph and urine composition, pH, and osmolarity. These processes depend on ATPase (pumps), channels and solute carriers (Slc proteins). Maturation of genomic databases enables us to identify the putative molecular players for these processes. Anion transporters of the Slc4 family, AE1 and NDAE1, have been reported as HCO(3)(-) transporters, but are only part of the story. Here we report Dipteran (Drosophila melanogaster (d) and Anopheles gambiae (Ag)) anion exchangers, belonging to the Slc26 family, which are multi-functional anion exchangers. One Drosophila and two Ag homologues of mammalian Slc26a5 (Prestin) and Slc26a6 (aka, PAT1, CFEX) were identified and designated dPrestin, AgPrestinA and AgPrestinB. dPrestin and AgPrestinB show electrogenic anion exchange (Cl(-)/nHCO(3)(-), Cl(-)/SO(4)(2-) and Cl(-)/oxalate(2-)) in an oocyte expression system. Since these transporters are the only Dipteran Slc26 proteins whose transport is similar to mammalian Slc26a6, we submit that Dipteran Prestin are functional and even molecular orthologues of mammalian Slc26a6. OSR1 kinase increases dPrestin ion transport, implying another set of physiological processes controlled by WNK/SPAK signaling in epithelia. All of these mRNAs are highly expressed in the gut and Malpighian tubules. Dipteran Prestin proteins appear suited for central roles in bicarbonate, sulfate and oxalate metabolism including generating the high pH conditions measured in the Dipteran midgut lumen. Finally, we present and discuss Drosophila genetic models that integrate these processes.
Project description:Activation of an apical Ca(2+)-dependent Cl(-) channel (CaCC) is the rate-limiting step for fluid secretion in many exocrine tissues. Here, we compared the properties of native CaCC in mouse submandibular salivary gland acinar cells to the Ca(2+)-gated Cl(-) currents generated by Tmem16A and Best2, members from two distinct families of Ca(2+)-activated Cl(-) channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca(2+)-activated Cl(-) currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca(2+)-activated Cl(-) current found in native salivary acinar cells. Best2(-/-) and Tmem16A(-/-) mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca(2+)-activated Cl(-) current in submandibular gland acinar cells from Best2-deficient mice were the same as in wild type cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable with that in wild type mice. In contrast, submandibular gland acinar cells from Tmem16A(-/-) mice lacked a Ca(2+)-activated Cl(-) current and a Ca(2+)-mobilizing agonist failed to stimulate Cl(-) efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wild type and Best2(-/-) mice. Our results demonstrate that both Tmem16A and Best2 generate Ca(2+)-activated Cl(-) current in vitro with similar properties to those expressed in native cells, yet only Tmem16A appears to be a critical component of the acinar Ca(2+)-activated Cl(-) channel complex that is essential for saliva production by the submandibular gland.
Project description:Carbonic anhydrases (CA) catalyze the reversible conversion of CO2 to HCO3-. Some bicarbonate transporters bind CA, forming a complex called a transport metabolon, to maximize the coupled catalytic/transport flux. SLC26A6, a plasma membrane Cl-/HCO3- exchanger with a suggested role in pancreatic HCO3- secretion, was found to bind the cytoplasmic enzyme CAII. Mutation of the identified CAII binding (CAB) site greatly reduced SLC26A6 activity, demonstrating the importance of the interaction. Regulation of SLC26A6 bicarbonate transport by protein kinase C (PKC) was investigated. Angiotensin II (AngII), which activates PKC, decreased Cl-/HCO3- exchange in cells coexpressing SLC26A6 and AT1a-AngII receptor. Activation of PKC reduced SLC26A6/CAII association in immunoprecipitates. Similarly, PKC activation displaced CAII from the plasma membrane, as monitored by immunofluorescence. Finally, mutation of a PKC site adjacent to the SLC26A6 CAB site rendered the transporter unresponsive to PKC. PKC therefore reduces CAII/SLC26A6 interaction, reducing bicarbonate transport rate. Taken together, our data support a mechanism for acute regulation of membrane transport: metabolon disruption.
Project description:Sulfate (SO(4)(2-)) is the second most abundant anion in seawater (SW), and excretion of excess SO(4)(2-) from ingested SW is essential for marine fish to survive. Marine teleosts excrete SO(4)(2-) via the urine produced in the kidney. The SO(4)(2-) transporter that secretes and concentrates SO(4)(2-) in the urine has not previously been identified. Here, we have identified and characterized candidates for the long-sought transporters. Using sequences from the fugu database, we have cloned cDNA fragments of all transporters belonging to the Slc13 and Slc26 families from mefugu (Takifugu obscurus). We compared Slc13 and Slc26 mRNA expression in the kidney between freshwater (FW) and SW mefugu. Among 14 clones examined, the expression of a Slc26a6 paralog (mfSlc26a6A) was the most upregulated (30-fold) in the kidney of SW mefugu. Electrophysiological analyses of Xenopus oocytes expressing mfSlc26a6A, mfSlc26a6B, and mouse Slc26a6 (mSlc26a6) demonstrated that all transporters mediate electrogenic Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-), and Cl(-)/nHCO(3)(-) exchanges and electroneutral Cl(-)/formate(-) exchange. Two-electrode voltage-clamp experiments demonstrated that the SO(4)(2-)-elicited currents of mfSlc26a6A is quite large (approximately 35 microA at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and HCO(3)(-) are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest SO(4)(2-) affinity as well as capacity. Immunohistochemical analyses demonstrated that mfSlc26a6A localizes to the apical (brush-border) region of the proximal tubules. Together, these findings suggest that mfSlc26a6A is the most likely candidate for the major apical SO(4)(2-) transporter that mediates SO(4)(2-) secretion in the kidney of marine teleosts.
Project description:Ae4 (Slc4a9) belongs to the Slc4a family of Cl(-)/HCO3 (-) exchangers and Na(+)-HCO3 (-) cotransporters, but its ion transport cycle is poorly understood. In this study, we find that native Ae4 activity in mouse salivary gland acinar cells supports Na(+)-dependent Cl(-)/HCO3 (-) exchange that is comparable with that obtained upon heterologous expression of mouse Ae4 and human AE4 in CHO-K1 cells. Additionally, whole cell recordings and ion concentration measurements demonstrate that Na(+) is transported by Ae4 in the same direction as HCO3 (-) (and opposite to that of Cl(-)) and that ion transport is not associated with changes in membrane potential. We also find that Ae4 can mediate Na(+)-HCO3 (-) cotransport-like activity under Cl(-)-free conditions. However, whole cell recordings show that this apparent Na(+)-HCO3 (-) cotransport activity is in fact electroneutral HCO3 (-)/Na(+)-HCO3 (-) exchange. Although the Ae4 anion exchanger is thought to regulate intracellular Cl(-) concentration in exocrine gland acinar cells, our thermodynamic calculations predict that the intracellular Na(+), Cl(-), and HCO3 (-) concentrations required for Ae4-mediated Cl(-) influx differ markedly from those reported for acinar secretory cells at rest or under sustained stimulation. Given that K(+) ions share many properties with Na(+) ions and reach intracellular concentrations of 140-150 mM (essentially the same as extracellular [Na(+)]), we hypothesize that Ae4 could mediate K(+)-dependent Cl(-)/HCO3 (-) exchange. Indeed, we find that Ae4 mediates Cl(-)/HCO3 (-) exchange activity in the presence of K(+) as well as Cs(+), Li(+), and Rb(+) In summary, our results strongly suggest that Ae4 is an electroneutral Cl(-)/nonselective cation-HCO3 (-) exchanger. We postulate that the physiological role of Ae4 in secretory cells is to promote Cl(-) influx in exchange for K(+)(Na(+)) and HCO3 (-) ions.
Project description:Patients with cystic fibrosis have an increased incidence of hyperoxaluria and calcium oxalate nephrolithiasis. Net intestinal absorption of dietary oxalate results from passive paracellular oxalate absorption as modified by oxalate back secretion mediated by the SLC26A6 oxalate transporter. We used mice deficient in the cystic fibrosis transmembrane conductance regulator gene (Cftr) to test the hypothesis that SLC26A6-mediated oxalate secretion is defective in cystic fibrosis. We mounted isolated intestinal tissue from C57BL/6 (wild-type) and Cftr<sup>-/-</sup> mice in Ussing chambers and measured transcellular secretion of [<sup>14</sup>C]oxalate. Intestinal tissue isolated from Cftr<sup>-/-</sup> mice exhibited significantly less transcellular oxalate secretion than intestinal tissue of wild-type mice. However, glucose absorption, another representative intestinal transport process, did not differ in Cftr<sup>-/-</sup> tissue. Compared with wild-type mice, Cftr<sup>-/-</sup> mice showed reduced expression of SLC26A6 in duodenum by immunofluorescence and Western blot analysis. Furthermore, coexpression of CFTR stimulated SLC26A6-mediated Cl<sup>-</sup>-oxalate exchange in Xenopus oocytes. In association with the profound defect in intestinal oxalate secretion, Cftr<sup>-/-</sup> mice had serum and urine oxalate levels 2.5-fold greater than those of wild-type mice. We conclude that defective intestinal oxalate secretion mediated by SLC26A6 may contribute to the hyperoxaluria observed in this mouse model of cystic fibrosis. Future studies are needed to address whether similar mechanisms contribute to the increased risk for calcium oxalate stone formation observed in patients with cystic fibrosis.