PAT-1 (Slc26a6) is the predominant apical membrane Cl-/HCO3- exchanger in the upper villous epithelium of the murine duodenum

Basal HCO(3)(-) secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl(-)/HCO(3)(-) exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl(-)/HCO(3)(-) exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl(-)/HCO(3)(-) exchange activity was reduced by 65-80% in the PAT-1(-) duodenum, 30-40% in the DRA(-) duodenum, and <5% in the AE4(-) duodenum compared with the WT duodenum. SO(4)(2-)/HCO(3)(-) exchange was eliminated in the PAT-1(-) duodenum but was not affected in the DRA(-) and AE4(-) duodenum relative to the WT duodenum. Intracellular pH (pH(i)) was reduced in the PAT-1(-) villous epithelium but increased to WT levels in the absence of CO(2)/HCO(3)(-) or during methazolamide treatment. Further experiments under physiological conditions indicated active pH(i) compensation in the PAT-1(-) villous epithelium by combined activities of Na(+)/H(+) exchanger 1 and Cl(-)-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl(-)/HCO(3)(-) and SO(4)(2-)/HCO(3)(-) exchange across the apical membrane and 2) PAT-1 plays a role in pH(i) regulation in the upper villous epithelium of the murine duodenum.     

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

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.     

Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl-/1HCO3- exchange

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl(-)/1HCO(3)(-) exchange suggests a required revision of classical concepts of electroneutral Cl(-) transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl(-)/HCO(3)(-) activity on apical membrane potential (V(a)) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl(-)/(OH(-))HCO(3)(-) exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo (36)Cl(-) fluxes and microfluorometry revealed that cecal Cl(-)/HCO(3)(-) exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline V(a) of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl(-) substitution. Subsequent studies indicated that Cl(-)-dependent V(a) depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl(-) for extracellular HCO(3)(-) is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl(-) removal or pharmacological blockade. Further, Dra-dependent (36)Cl(-) efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl(-)/1HCO(3)(-) exchange; and 2) acute induction of Dra Cl(-)/HCO(3)(-) exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO(3)(-) secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.     

Novel role for pendrin in orchestrating bicarbonate secretion in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing airway serous cells

In most HCO(3)(-)-secreting epithelial tissues, SLC26 Cl(-)/HCO(3)(-) transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO(3)(-) in the airways. Here we show that Cl(-)/HCO(3)(-) anion exchange makes a major contribution to transcellular HCO(3)(-) transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl(-)-dependent HCO(3)(-) transport across the luminal membrane via CFTR-dependent coupled Cl(-)/HCO(3)(-) anion exchange. The pharmacological and functional profile of the luminal anion exchanger was consistent with SLC26A4 (pendrin), which was shown to be expressed by quantitative RT-PCR, Western blot, and immunofluorescence. Pendrin-mediated anion exchange activity was confirmed by shRNA pendrin knockdown (KD), which markedly reduced cAMP-activated Cl(-)/HCO(3)(-) exchange. To establish the relative roles of CFTR and pendrin in net HCO(3)(-) secretion, transepithelial liquid secretion rate and liquid pH were measured in wild type, pendrin KD, and CFTR KD cells. cAMP/PKA increased the rate and pH of the secreted fluid. Inhibiting CFTR reduced the rate of liquid secretion but not the pH, whereas decreasing pendrin activity lowered pH with little effect on volume. These results establish that CFTR predominately controls the rate of liquid secretion, whereas pendrin regulates the composition of the secreted fluid and identifies a critical role for this anion exchanger in transcellular HCO(3)(-) secretion in airway serous cells.     

Slc26a6 regulates CFTR activity in vivo to determine pancreatic duct HCO3- secretion: relevance to cystic fibrosis

Fluid and HCO(3)(-) secretion are vital functions of the pancreatic duct and other secretory epithelia. CFTR and Cl(-)/HCO(3)(-) exchange activity at the luminal membrane are required for these functions. The molecular identity of the Cl(-)/HCO(3)(-) exchangers and their relationship with CFTR in determining fluid and HCO(3)(-) secretion are not known. We show here that the Cl(-)/HCO(3)(-) exchanger slc26a6 controls CFTR activity and ductal fluid and HCO(3)(-) secretion. Unexpectedly, deletion of slc26a6 in mice and measurement of fluid and HCO(3)(-) secretion into sealed intralobular pancreatic ducts revealed that deletion of slc26a6 enhanced spontaneous and decreased stimulated secretion. Remarkably, inhibition of CFTR activity with CFTR(inh)-172, knock-down of CFTR by siRNA and measurement of CFTR current in WT and slc26a6(-/-) duct cells revealed that deletion of slc26a6 resulted in dis-regulation of CFTR activity by removal of tonic inhibition of CFTR by slc26a6. These findings reveal the intricate regulation of CFTR activity by slc26a6 in both the resting and stimulated states and the essential role of slc26a6 in pancreatic HCO(3)(-) secretion in vivo.     

A molecular mechanism for aberrant CFTR-dependent HCO(3)(-) transport in cystic fibrosis

Aberrant HCO(3)(-) transport is a hallmark of cystic fibrosis (CF) and is associated with aberrant Cl(-)-dependent HCO(3)(-) transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO(3)(-) current by CFTR cannot account for CFTR-activated HCO(3)(-) transport and that CFTR does not activate AE1-AE4. In contrast, CFTR markedly activates Cl(-) and OH(-)/HCO(3)(-) transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporters with isoform-specific stoichiometries. DRA activity occurred at a Cl(-)/HCO(3)(-) ratio > or =2. SLC26A6 activity is voltage regulated and occurred at HCO(3)(-)/Cl(-) > or =2. The physiological significance of these findings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and G551D mutants of CFTR. These findings provide a molecular mechanism for epithelial HCO(3)(-) transport (one SLC26 transporter-electrogenic transport; two SLC26 transporters with opposite stoichiometry in the same membrane domain-electroneutral transport), the CF-associated aberrant HCO(3)(-) transport, and reveal a new function of CFTR with clinical implications for CF and congenital chloride diarrhea.     

Relationship of delta-aminolevulinic acid-induced protoporphyrin IX levels to mitochondrial content in neoplastic cells in vitro

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a small conductance cAMP-activated chloride ion channel. In the CF pancreatic duct, mutations in CFTR cause a reduction in bicarbonate secretion. This is thought to result from CFTR operating in parallel with a chloride-bicarbonate (Cl(-)/HCO(-)(3)) exchanger, located in the apical membrane of pancreatic duct cells. The molecular basis of this Cl(-)/HCO(-)(3) exchanger has not been identified. A combination of screening cDNA libraries, RNase protection, and 5' RACE analysis was used to identify Cl(-)/HCO(-)(3) exchangers in human fetal pancreas. An AE2 Cl(-)/HCO(-)(3) exchanger was shown to be expressed in human fetal pancreas from the midtrimester of gestation, at a time when CF-associated pathology commences. In addition, an AE1 Cl(-)/HCO(3) was identified in fetal pancreas but was absent from the adult pancreas and cultured ductal epithelial cells from fetal and adult pancreas.     

Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells

The mechanism of the pancreatic ductal HCO secretion defect in cystic fibrosis (CF) is not well defined. However, a lack of apical Cl(-)/HCO exchange may exist in CF. To test this hypothesis, we examined the expression of Cl(-)/HCO exchangers in cultured pancreatic duct epithelial cells with physiological features prototypical of CF [CFPAC-1 cells lacking a functional CF transmembrane conductance regulator (CFTR)] or normal duct cells (CFPAC-1 cells transfected with functional wild-type CFTR, CFPAC-WT). Cl(-)/HCO exchange activity, assayed with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in cells grown on coverslips, increased about twofold in cells transfected with functional CFTR. This correlated with increased apical (36)Cl influx in cells expressing functional CFTR and grown on permeable support. Northern hybridizations indicated the induction of downregulated in adenoma (DRA) in cells expressing functional CFTR. The expression of putative anion transporter PAT1 also increased significantly in cells expressing functional CFTR. DRA was detected at high levels in native mouse pancreas by Northern hybridization and localized to the apical domain of the duct cells by immunohistochemical studies. In conclusion, CFTR upregulates DRA and PAT1 expression in cultured pancreatic duct cells. We propose that the pancreatic HCO secretion defect in CF patients is partly due to the downregulation of apical Cl(-)/HCO exchange activity mediated by DRA (and possibly PAT1).     

Specificity of anion exchange mediated by mouse Slc26a6

Recently, CFEX, the mouse orthologue of human SLC26A6, was localized to the brush border membrane of proximal tubule cells and was demonstrated to mediate Cl(-)-formate exchange when expressed in Xenopus oocytes. The purpose of the present study was to examine whether mouse Slc26a6 can mediate one or more of the additional anion exchange processes observed to take place across the apical membrane of proximal tubule cells. Influx of [(14)C]formate into Slc26a6-expressing oocytes was inhibited by sulfate, oxalate, and p-aminohippurate (PAH), indicating affinity for these anions. Measurements of uptake of [(14)C]oxalate, [(14)C]PAH, and [(35)S]sulfate indicated that Slc26a6 can mediate transport of oxalate and sulfate but not PAH. Studies of the effect of external anions on [(14)C]oxalate efflux demonstrated Slc26a6-mediated Cl(-)-oxalate, oxalate-formate, oxalate-oxalate, and oxalate-sulfate exchange. Two-electrode voltage clamp measurements indicated that Slc26a6-mediated Cl(-)-oxalate exchange is electrogenic. Intracellular pH recordings demonstrated that Slc26a6 can mediate Cl(-)-HCO(3)(-) exchange, but Cl(-)-OH(-) exchange was not detected. The presence of 100 microm oxalate inhibited the rate of Cl(-)-HCO(3)(-) exchange by 60%. We conclude that mouse Slc26a6 has affinity for oxalate, sulfate, and HCO(3)(-) in addition to Cl(-) and formate and can function in multiple exchange modes involving pairs of these anions. In the presence of high oxalate concentrations as found in renal tubular fluid and urine, Slc26a6 may largely function as an electrogenic Cl(-)-oxalate exchanger.     

Ion and solute transport by Prestin in Drosophila and Anopheles

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.     

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO(3)(-) secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y(2) nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca(2+)-activated Cl(-) secretion. However, the value of this treatment in facilitating transepithelial HCO(3)(-) secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca(2+)-dependent anion conductance during activation of luminal P2Y(2) receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current (I(sc)) that declined to a stable plateau phase lasting 30-60 min. The plateau I(sc) after UTP was Cl(-) independent, HCO(3)(-) dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I(sc) and serosal-to-mucosal HCO(3)(-) flux (J(s-->m)) during a 30-min period. Substitution of Cl(-) with gluconate in the luminal bath to inhibit Cl(-)/HCO(3)(-) exchange did not prevent the increase in J(s-->m) and I(sc) during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J(s-->m) and I(sc). We conclude that P2Y(2) receptor activation results in a sustained (30-60 min) increase in electrogenic HCO(3)(-) secretion that is mediated via an intracellular Ca(2+)-dependent anion conductance in CF gallbladder.     

Determinants of coupled transport and uncoupled current by the electrogenic SLC26 transporters

Members of the SLC26 family of anion transporters mediate the transport of diverse molecules ranging from halides to carboxylic acids and can function as coupled transporters or as channels. A unique feature of the two members of the family, Slc26a3 and Slc26a6, is that they can function as both obligate coupled and mediate an uncoupled current, in a channel-like mode, depending on the transported anion. To identify potential features that control the two modes of transport, we performed in silico modeling of Slc26a6, which suggested that the closest potential fold similarity of the Slc26a6 transmembrane domains is to the CLC transporters, despite their minimal sequence identity. Examining the predicted Slc26a6 fold identified a highly conserved glutamate (Glu(-); Slc26a6(E357)) with the predicted spatial orientation similar to that of the CLC-ec1 E148, which determines coupled or uncoupled transport by CLC-ec1. This raised the question of whether the conserved Glu(-) in Slc26a6(E357) and Slc26a3(E367) have a role in the unique transport modes by these transporters. Reversing the Glu(-) charge in Slc26a3 and Slc26a6 resulted in the inhibition of all modes of transport. However, most notably, neutralizing the charge in Slc26a6(E357A) eliminated all forms of coupled transport without affecting the uncoupled current. The Slc26a3(E367A) mutation markedly reduced the coupled transport and converted the stoichiometry of the residual exchange from 2Cl(-)/1HCO(3)(-) to 1Cl(-)/1HCO(3)(-), while completely sparing the current. These findings suggest the possibility that similar structural motif may determine multiple functional modes of these transporters.     

Solute carrier family 26 member a2 (Slc26a2) protein functions as an electroneutral SOFormula/OH-/Cl- exchanger regulated by extracellular Cl-

Slc26a2 is a ubiquitously expressed SO(4)(2-) transporter with high expression levels in cartilage and several epithelia. Mutations in SLC26A2 are associated with diastrophic dysplasia. The mechanism by which Slc26a2 transports SO(4)(2-) and the ion gradients that mediate SO(4)(2-) uptake are poorly understood. We report here that Slc26a2 functions as an SO(4)(2-)/2OH(-), SO(4)(2-)/2Cl(-), and SO(4)(2-)/OH(-)/Cl(-) exchanger, depending on the Cl(-) and OH(-) gradients. At inward Cl(-) and outward pH gradients (high Cl(-)(o) and low pH(o)) Slc26a2 functions primarily as an SO(4)(2-)(o)/2OH(-)(i) exchanger. At low Cl(-)(o) and high pH(o) Slc26a2 functions increasingly as an SO(4)(2-)(o)/2Cl(-)(i) exchanger. The reverse is observed for SO(4)(2-)(i)/2OH(-)(o) and SO(4)(2-)(i)/2Cl(-)(o) exchange. Slc26a2 also exchanges Cl(-) for I(-), Br(-), and NO(3)(-) and Cl(-)(o) competes with SO(4)(2-) on the transport site. Interestingly, Slc26a2 is regulated by an extracellular anion site, required to activate SO(4)(2-)(i)/2OH(-)(o) exchange. Slc26a2 can transport oxalate in exchange for OH(-) and/or Cl(-) with properties similar to SO(4)(2-) transport. Modeling of the Slc26a2 transmembrane domain (TMD) structure identified a conserved extracellular sequence (367)GFXXP(371) between TMD7 and TMD8 close to the conserved Glu(417) in the permeation pathway. Mutation of Glu(417) eliminated transport by Slc26a2, whereas mutation of Phe(368) increased the affinity for SO(4)(2-)(o) 8-fold while reducing the affinity for Cl(-)(o) 2 fold, but without affecting regulation by Cl(-)(o). These findings clarify the mechanism of net SO(4)(2-) transport and describe a novel regulation of Slc26a2 by an extracellular anion binding site and should help in further understanding aberrant SLC26A2 function in diastrophic dysplasia.     

Identification of an apical Cl(-)/HCO3(-) exchanger in the small intestine

HCO3(-) secretion is the most important defense mechanism against acid injury in the duodenum. However, the identity of the transporter(s) mediating apical HCO3(-) secretion in the duodenum remains unknown. A family of anion exchangers, which include downregulated in adenoma (DRA or SLC26A3), pendrin (PDS or SLC26A4), and the putative anion transporter (PAT1 or SLC26A6) has recently been identified. DRA and pendrin mediate Cl(-)/base exchange; however, the functional identity and distribution of PAT1 (SLC26A6) is not known. In these studies, we investigated the functional identity, tissue distribution, and membrane localization of PAT1. Expression studies in Xenopus oocytes demonstrated that PAT1 functions in Cl(-)/HCO3(-) exchange mode. Tissue distribution studies indicated that the expression of PAT1 is highly abundant in the small intestine but is low in the colon, a pattern opposite that of DRA. PAT1 was also abundantly detected in stomach and heart. Immunoblot analysis studies identified PAT1 as a approximately 90 kDa protein in the duodenum. Immunohistochemical studies localized PAT1 to the brush border membranes of the villus cells of the duodenum. We propose that PAT1 is an apical Cl(-)/HCO3(-) exchanger in the small intestine.     

Ca2+ activates cystic fibrosis transmembrane conductance regulator- and Cl- -dependent HCO3 transport in pancreatic duct cells

Pancreatic duct cells secrete bicarbonate-rich fluids, which are important for maintaining the patency of pancreatic ductal trees as well as intestinal digestive function. The bulk of bicarbonate secretion in the luminal membrane of duct cells is mediated by a Cl(-)-dependent mechanism (Cl(-)/HCO(3)(-) exchange), and we previously reported that the mechanism is CFTR-dependent and cAMP-activated (Lee, M. G., Choi, J. Y., Luo, X., Strickland, E., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 14670-14677). In the present study, we provide comprehensive evidence that calcium signaling also activates the same CFTR- and Cl(-)-dependent HCO(3)(-) transport. ATP and trypsin evoked intracellular calcium signaling in pancreatic duct-derived cells through the activation of purinergic and protease-activated receptors, respectively. Cl(-)/HCO(3)(-) exchange activity was measured by recording pH(i) in response to [Cl(-)](o) changes of the perfusate. In perfusate containing high concentrations of K(+), which blocks Cl(-) movement through electrogenic or K(+)-coupled pathways, ATP and trypsin highly stimulated luminal Cl(-)/HCO(3)(-) exchange activity in CAPAN-1 cells expressing wild-type CFTR, but not in CFPAC-1 cells that have defective (DeltaF508) CFTR. Notably, adenoviral transfection of wild-type CFTR in CFPAC-1 cells completely restored the stimulatory effect of ATP on luminal Cl(-)/HCO(3)(-) exchange. In addition, the chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA) treatment abolished the effect of calcium agonists on luminal Cl(-)/HCO(3)(-) exchange. These results provide a molecular basis for calcium-induced bicarbonate secretion in pancreatic duct cells and highlight the importance of CFTR in epithelial bicarbonate secretion induced by various stimuli.     

Functional comparison of mouse slc26a6 anion exchanger with human SLC26A6 polypeptide variants: differences in anion selectivity, regulation, and electrogenicity

The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared. Transport was studied as unidirectional fluxes of (36)Cl(-), [(14)C]oxalate, and [(35)S]sulfate; as net fluxes of HCO(3)(-) by fluorescence ratio measurement of intracellular pH; as current by two-electrode voltage clamp; and as net Cl(-) flux by fluorescence intensity measurement of relative changes in extracellular and intracellular [Cl(-)]. Four human SLC26A6 polypeptide variants each exhibited rates of bidirectional [(14)C]oxalate flux, Cl(-)/HCO(3)(-) exchange, and Cl(-)/OH(-) exchange nearly equivalent to those of mouse slc26a6. Cl(-)/HCO(3)(-) exchange by both orthologs was cAMP-sensitive, further enhanced by coexpressed wild type cystic fibrosis transmembrane regulator but inhibited by cystic fibrosis transmembrane regulator DeltaF508. However, the very low rates of (36)Cl(-) and [(35)S]sulfate transport by all active human SLC26A6 isoforms contrasted with the high rates of the mouse ortholog. Human and mouse orthologs also differed in patterns of acute regulation. Studies of human-mouse chimeras revealed cosegregation of the high (36)Cl(-) transport phenotype with the transmembrane domain of mouse slc26a6. Mouse slc26a6 and human SLC26A6 each mediated electroneutral Cl(-)/HCO(3)(-) and Cl(-)/OH(-) exchange. In contrast, whereas Cl(-)/oxalate exchange by mouse slc26a6 was electrogenic, that mediated by human SLC26A6 appeared electroneutral. The increased currents observed in oocytes expressing either mouse or human ortholog were pharmacologically distinct from the accompanying monovalent anion exchange activities. The human SLC26A6 polypeptide variants SLC26A6c and SLC26A6d were inactive as transporters of oxalate, sulfate, and chloride. Thus, the orthologous mouse and human SLC26A6 proteins differ in anion selectivity, transport mechanism, and acute regulation, but both mediate electroneutral Cl(-)/HCO(3)(-) exchange.     

Dynamic control of cystic fibrosis transmembrane conductance regulator Cl(-)/HCO3(-) selectivity by external Cl(-)

HCO(3)(-) secretion is a vital activity in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing epithelia. However, the role of CFTR in this activity is not well understood. Simultaneous measurements of membrane potential and pH(i) and/or current in CFTRexpressing Xenopus oocytes revealed dynamic control of CFTR Cl(-)/HCO(3)(-) permeability ratio, which is regulated by external Cl(-) (Cl(-)(o)). Thus, reducing external Cl(-) from 110 to 0-10 mm resulted in the expected increase in membrane potential, but with no corresponding OH(-) or HCO(3)(-) influx. Approximately 3-4 min after reducing Cl(o)(-) to 0 mm, an abrupt switch in membrane potential occurs that coincided with an increased rates of OH(-) and HCO(3)(-) influx. The switch in membrane permeability to OH(-)/HCO(3)(-) can also be recorded as a leftward shift in the reversal potential. Furthermore, an increased rate of OH(-) influx in response to elevating pH(o) to 9.0 was observed only after the switch in membrane potential. The time to switch increased to 11 min at Cl(o)(-) of 5 mm. Conversely, re-addition of external Cl(-) after the switch in membrane potential did not stop HCO(3)(-) influx, which continued for about 3.9 min after Cl(-) addition. Importantly, addition of external Cl(-) to cells incubated in Cl(-)-free medium never resulted in HCO(3)(-) efflux. Voltage and current clamp experiments showed that the delayed HCO(3)(-) transport is electrogenic. These results indicate that CFTR exists in two conformations, a Cl(-) only and a Cl(-) and OH(-)/HCO(3)(-) permeable state. The switch between the states is controlled by external Cl(-). Accordingly, a different tryptic pattern of CFTR was found upon digestion in Cl(-)-containing and Cl(-)-free media. The physiological significance of these finding is discussed in the context of HCO(3)(-) secretion by tissues such as the pancreas and salivary glands.     

Renal and intestinal transport defects in Slc26a6-null mice

SLC26A6 (PAT1, CFEX) is an anion exchanger that is expressed on the apical membrane of the kidney proximal tubule and the small intestine. Modes of transport mediated by SLC26A6 include Cl-/formate exchange, Cl-/HCO3- exchange, and Cl-/oxalate exchange. To study its role in kidney and intestinal physiology, gene targeting was used to prepare mice lacking Slc26a6. Homozygous mutant Slc26a6-/- mice appeared healthy and exhibited a normal blood pressure, kidney function, and plasma electrolyte profile. In proximal tubules microperfused with a low-HCO3-/high-Cl- solution, the baseline rate of fluid absorption (Jv), an index of NaCl transport under these conditions, was the same in wild-type and null mice. However, the stimulation of Jv by oxalate observed in wild-type mice was completely abolished in Slc26a6-null mice (P<0.05). Formate stimulation of Jv was partially reduced in null mice, but the difference from the response in wild-type mice did not reach statistical significance. Apical membrane Cl-/base exchange activity, assayed with the pH-sensitive dye BCPCF in microperfused proximal tubules, was decreased by 58% in Slc26a6-/- animals (P<0.001 vs. wild types). In the duodenum, the baseline rate of HCO3- secretion measured in mucosal tissue mounted in Ussing chambers was decreased by approximately 30% (P<0.03), whereas the forskolin-stimulated component of HCO3- secretion was the same in wild-type and Slc26a6-/- mice. We conclude that Slc26a6 mediates oxalate-stimulated NaCl absorption, contributes to apical membrane Cl-/base exchange in the kidney proximal tubule, and also plays an important role in HCO3- secretion in the duodenum.     

胰管细胞HCO3-分泌:囊性纤维化跨膜电导调节体和SLC26转运体

胰管细胞以至少6倍浓度差逆向分泌HCO3^-（人体浓度约140mmol／L）。HCO3^-跨顶膜转运的可能机制包括SLC26阴离子转运体的Cl-HCO3^-交换和囊性纤维化跨膜电导调节体（cystic fibrosis transmembrane conductance regulator,cFrR）对HCO3^-的传导扩散。SLC26家族成员介导上皮顶膜Cl^--HCO3^-交换,胰管中检测到SLC26A6和SLC26A3。共表达研究揭示,鼠类slc26a6和slc26a3通过slc26的STAS结构域与CFTR的R结构域相互作用,导致活性互相增强。研究显示这些交换体是产电的：slc26a6介导1Cl^--2HCO3^-交换,slc26a3介导2Cl^--1HCO3^-交换。近期slc26a6^-／-小鼠离体胰管研究显示,slc26a6介导大部分Cl^-依赖的HCO3^-跨顶膜分泌,与slc26a6的产电性一致。然而,因为人体能分泌非常高浓度的HCO3^-,SLC26A6在胰管HCO3^-分泌中的作用并不十分清楚。SLC26A6的作用只能在与人类似能分泌约140mmol／LHCO3^-的物种,如豚鼠中研究。现有的豚鼠研究数据显示,像slc26a6介导的1Cl^--2HCO3^-交换不可能完成这种高浓度差的HCO3^-分泌。另一方面,CFTR的HCO3^-电导性可以在理论上支持HCO3^-逆向分泌。所以,在豚鼠和人胰腺HCO3^-的分泌中,CFTR可能比SLC26A6发挥更大作用。