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.     

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.     

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.     

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。     

Metabolon disruption: a mechanism that regulates bicarbonate transport

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.     

slc26a3 (dra)-deficient mice display chloride-losing diarrhea, enhanced colonic proliferation, and distinct up-regulation of ion transporters in the colon

Mutations in the SLC26A3 (DRA (down-regulated in adenoma)) gene constitute the molecular etiology of congenital chloride-losing diarrhea in humans. To ascertain its role in intestinal physiology, gene targeting was used to prepare mice lacking slc26a3. slc26a3-deficient animals displayed postpartum lethality at low penetrance. Surviving dra-deficient mice exhibited high chloride content diarrhea, volume depletion, and growth retardation. In addition, the large intestinal loops were distended, with colonic mucosa exhibiting an aberrant growth pattern and the colonic crypt proliferative zone being greatly expanded in slc26a3-null mice. Apical membrane chloride/base exchange activity was sharply reduced, and luminal content was more acidic in slc26a3-null mouse colon. The epithelial cells in the colon displayed unique adaptive regulation of ion transporters; NHE3 expression was enhanced in the proximal and distal colon, whereas colonic H,K-ATPase and the epithelial sodium channel showed massive up-regulation in the distal colon. Plasma aldosterone was increased in slc26a3-null mice. We conclude that slc26a3 is the major apical chloride/base exchanger and is essential for the absorption of chloride in the colon. In addition, slc26a3 regulates colonic crypt proliferation. Deletion of slc26a3 results in chloride-rich diarrhea and is associated with compensatory adaptive up-regulation of ion-absorbing transporters.     

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.     

SLC26A7 is a Cl- channel regulated by intracellular pH

Members of the SLC26 transporter family play an essential role in several epithelial functions, as revealed by diseases associated with mutations in members of the family. Several members were shown to function as Cl(-) and HCO(3)(-) transporters that likely play an important role in epithelial Cl(-) absorption and HCO(3)(-) secretion. However, the mechanism of most transporters is not well understood. SLC26A7 is a member of the SLC26 transporter family reported to be expressed in the basolateral membrane of the cortical collecting duct and parietal cells and functions as a coupled Cl(-)/HCO(3)(-) exchanger. In the present work we examined the transport properties of SLC26A7 to determine its transport characteristics and electrogenicity. We found that when expressed in Xenopus oocytes or HEK293 cells SLC26A7 functions as a pH(i)-regulated Cl(-) channel with minimal OH(-)/HCO(3)(-) permeability. Expression of SLC26A7 in oocytes or HEK293 cells generated a Cl(-) current with linear I/V and an instantaneous current that was voltage- and time-independent. Based on measurement of reversal potential the selectivity of SLC26A7 is NO(3)(-)>Cl(-)=Br(-)=I(-)>SO(4)(2-)=Glu(-), although I(-) partially inhibited the current. Incubating the cells with HCO(3)(-) or butyrate acidified the cytosol and increased the selectivity of SLC26A7 for Cl(-). Measurement of membrane potential and pH(i) showed minimal OH(-) and HCO(3)(-) transport by SLC26A7 when the cells were incubated in Cl(-)-containing or Cl(-)-free media. The activity of SLC26A7 was inhibited by all inhibitors of anion transporters tested, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylic acid, and glybenclamide. These findings reveal that SLC26A7 functions as a unique Cl(-) channel that is regulated by intracellular H(+).     

The branchial chloride pump in the goldfish Carassius auratus: relationship between Cl-/HCO3- and Cl-/Cl-- exchanges and the effect of thiocyanate.

1. The effect of thiocyanate on chloride and sodium fluxes across the gill was studied in the goldfish Carassius auratus. At low external chloride concentrations, addition of SCN- to the bathing solution markedly inhibited chloride influx and efflux, the net flux being reversed, SCN- injection was without effect. SCN- had no effect on sodium fluxes when injected or added to the external medium. 2. The inhibition of chloride influx by SCN- was of a mixed type involving simultaneous modifications of the affinity constant of the carrier for Cl- and of the maximal Cl- influx. The affinity constant of the carrier for SCN- was 10 times lower than that for Cl-. 3. The gill of the goldfish was found to be practically impermeable to SCN-. 4. In the presence of external SCN-, the Cl-/HCO3- exchange was reversed: Cl- was lost against HCO3- which is absorbed. This suggests an obligatory exchange. 5. Exchange diffusion for chloride was also demonstrated. 6. A kinetic model is proposed to explain chloride and bicarbonate transport across the gill of Carassius auratus.     

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.     

Na+, K+, and Cl- transport in resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

Sulfate-chloride exchange transport in a glioma cell line

Transport of SO4(2-) was studied in the glioma cell line LRM55 to determine whether it is mediated by the Cl-/HCO3- exchanger or the K+/Cl- cotransporter previously described in these cells (Wolpaw, E.W. and Martin, D.L. (1984) Brain Res. 297, 317-327). 35SO4(2-) influx was saturable with SO4(2-). External SO4(2-) stimulated 35SO4(2-) efflux, indicating an exchange mechanism. External Cl- was a competitive inhibitor of 35SO4(2-) influx. Internal Cl- stimulated 35SO4(2-) influx and external Cl- stimulated 35SO4(2-) efflux, indicating that Cl- is an exchange substrate for the SO4(2-) carrier. Also, SO4(2-) flux was sensitive to SITS, DIDS and furosemide. However, saturating external SO4(2-) did not inhibit 36Cl- influx and did not inhibit 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 35SO4(2-) influx as it does Cl- influx. These findings indicate that SO4(2-) transport into these cells is mediated by an exchange carrier distinct from both the Cl-/HCO3- exchanger and the K+/Cl- cotransporter. While Cl- is an alternative substrate for the SO4(2-) porter, this carrier is responsible for only a minor fraction of total Cl- flux in these cells.     

Urinary Metabolic Phenotyping the slc26a6 (Chloride-Oxalate Exchanger) Null Mouse Model

The prevalence of renal stone disease is increasing, although it remains higher in men than in women when matched for age. While still somewhat controversial, several studies have reported an association between renal stone disease and hypertension, but this may be confounded by a shared link with obesity. However, independent of obesity, hyperoxaluria has been shown to be associated with hypertension in stone-formers, and the most common type of renal stone is composed of calcium oxalate. The chloride-oxalate exchanger slc26a6 (also known as CFEX or PAT-1), located in the renal proximal tubule, was originally thought to have an important role in sodium homeostasis and thereby blood pressure control, but it has recently been shown to have a key function in oxalate balance by mediating oxalate secretion in the gut. We have applied two orthogonal analytical platforms (NMR spectroscopy and capillary electrophoresis with UV detection) in parallel to characterize the urinary metabolic signatures related to the loss of the renal chloride-oxalate exchanger in slc26a6 null mice. Clear metabolic differentiation between the urinary profiles of the slc26a6 null and the wild type mice were observed using both methods, with the combination of NMR and CE-UV providing extensive coverage of the urinary metabolome. Key discriminating metabolites included oxalate, m-hydroxyphenylpropionylsulfate (m-HPPS), trimethylamine-N-oxide, glycolate and scyllo-inositol (higher in slc26a6 null mice) and hippurate, taurine, trimethylamine, and citrate (lower in slc26a6 null mice). In addition to the reduced efficiency of anion transport, several of these metabolites (hippurate, m-HPPS, methylamines) reflect alteration in gut microbial cometabolic activities. Gender-related metabotypes were also observed in both wild type and slc26a6 null groups. Urinary metabolites that showed a sex-specific pattern included trimethylamine, trimethylamine-N-oxide, citrate, spermidine, guanidinoacetate, and 2-oxoisocaproate. The gender-dependent metabolic expression of the consequences of slc26a6 deletion might have relevance to the difference in prevalence of renal stone formation in men and women. The different composition of microbial metabolites in the slc26a6 null mice is consistent with the fact that the slc26a6 transporter is found in a range of tissues, including the kidney and intestine, and provides further evidence for the "long reach" of the microbiota in physiological and pathological processes.     

Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II. Regulation of H+ and HCO3- transporters by Ca2(+)-mobilizing agonists

Pancreatic acini loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to examine the effect of Ca2(+)-mobilizing agonists on the activity of acid-base transporters in these cells. In the accompanying article (Muallen, S., and Loessberg, P. A. (1990) J. Biol. Chem. 265, 12813-12819) we showed that in 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-buffered medium the main pHi regulatory mechanism is the Na+/H+ exchanger, a while in HCO3(-)-buffered medium pHi is determined by the combined activities of a Na+/H+ exchanger, a Na(+)-HCO3- cotransporter and a Cl-/HCO3- exchanger. In this study we found that stimulation of acini with Ca2(+)-mobilizing agonists in HEPES or HCO3(-)-buffered media is followed by an initial acidification which is independent of any identified plasma membrane-located acid-base transporting mechanism, and thus may represent intracellularly produced acid. In HEPES-buffered medium there was a subsequent large alkalinization to pHi above that in resting cells, which could be attributed to the Na+/H+ exchanger. Measurements of the rate of recovery from acid load indicated that the Na+/H+ exchanger was stimulated by the agonists. In HCO3(-)-buffered medium the alkalinization observed after the initial acidification was greatly attenuated. Examination of the activity of each acid-base transporting mechanism in stimulated acini showed that in HCO3(-)-buffered medium: (a) recovery from acid load in the presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS) (Na+/H+ exchange) was stimulated similar to that found in HEPES-buffered medium; (b) recovery from acid load in the presence of amiloride and acidification due to removal of external Na+ in the presence of amiloride (HCO3- influx and efflux, respectively, by Na(+)-HCO3- cotransport) were inhibited; and (c) HCO3- influx and efflux due to Cl-/HCO3- exchange, which was measured by changing the Cl- or HCO3- gradients across the plasma membrane, were stimulated. Furthermore, the rate of Cl-/HCO3- exchange in stimulated acini was higher than the sum of H+ efflux due to Na+/H+ exchange and HCO3- influx due to Na(+)-HCO3- cotransport. Use of H2DIDS showed that the latter accounted for the attenuated changes in pHi in HCO3(-)-buffered medium, as much as treating the acini with H2DIDS resulted in similar agonist-mediated pHi changes in HEPES- and HCO3(-)-buffered media. The effect of agonists on the various acid-base transporting mechanisms is discussed in terms of their possible role in transcellular NaCl transport, cell volume regulation, and cell proliferation in pancreatic acini.     

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.     

Identification of uterine ion transporters for mineralisation precursors of the avian eggshell

ABSTRACT: BACKGROUND: In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. RESULTS: A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1).We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane's Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters. CONCLUSIONS: Our Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.     

Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl-/HCO3- exchanger, AE1

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO2 to HCO3- + H+. Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl-/HCO3- exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl-/NO3- exchange assays, which were independent of CA activity, and in Cl-/HCO3- exchange assays. Transport was measured by following changes of intracellular [Cl-] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2',7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl-/NO3- exchange activity with EC50 values in the range 0.22-2.8 microM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 microM of each compound was only 22-53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl-/HCO3- exchange assays, which depend on functional CA to produce transport substrate, 40 microM celecoxib inhibited AE1 by 62+/-4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations.     

SLC26A9 is expressed in gastric surface epithelial cells, mediates Cl-/HCO3- exchange, and is inhibited by NH4+

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylori) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9.