Alper, Seth L

Shmukler, Boris E

Stewart, Andrew K

Vandorpe, David H

Tokushima University Institutional Repository

The majority of fluid and HCO3- in pancreatic juicearises from the pancreatic duct cells. Secretin stimu-lates HCO3- secretion via a mechanism that involvesactivation of the adenylate cyclase pathway, activa-tion of both basolateral K+ channels and the apicalCFTR Cl- channel, and stimulation of an apical Slc26-mediated Cl-/HCO3- exchanger (1). The Slc26 an-ion exchangers, Slc26a3 and Slc26a6, have bothbeen localized to the apical membrane of humanpancreatic duct and mouse pancreatic duct althoughtheir functional roles in pancreatic HCO3- secretionremain uncertain. The high rates of pancreatic bicar-bonate secretion shared by guinea pig (gp) and hu-man, in contrast to the low rates in mouse, promptedus to study the functional characteristics of gp Slc26anion transporters. Furthermore, recent data fromisolated guinea pig pancreatic duct experimentsdemonstrated that during stimulated HCO3- secre-tion an apical Slc26a6-like Cl-/HCO3- exchangeris functionally coupled with CFTR (2). This studyprompted us to clone cDNAs encoding Slc26a3 andSlc26a6 polypeptides from the gp pancreas and tocompare their functional characteristics in Xenopusoocytes with those of their mouse and humanorthologs (3-5), and with those of luminal Cl-/HCO3-exchange measured in isolated gp pancreatic inter-lobular ducts (2).Whereas gp Slc26a6 mediated Cl-/Cl- exchangeand bidirectional exchange of Cl- for oxalate andHCO3-, gp Slc26a6 mediated only sulfate influx butnot sulfate efflux in preliminary experiments. GpSlc26a3 mediated robust Cl-/Cl- exchange and mini-mal oxalate efflux in exchange for Cl-, but did notmediate detectable influx of oxalate or sulfate.Measurement of intracellular pH by BCECF ratiofluorimetry in HEK cells transfected with Slc26a6or in Slc26a3-expressing Xenopus oocytes duringremoval and restoration of Cl-o in the presence ofHCO3- indicated that both gp Slc26a6 and Slc26a3function as Cl-/HCO3- exchangers. These substratespecificity experiments reveal that whereas Slc26a3functions predominantly as a Cl-/HCO3- exchanger,Slc26a6 can exchange a wider range of substrates,functioning as a Cl-/HCO3- exchanger or a Cl-/anionexchanger. This finding allows for the possibilitythat Slc26a6 may have alternative roles in transepi-thelial solute transport in the pancreatic duct.The apparent affinity of gp Slc26a6 for extra-cellular Cl- (K1/2) was 2 mM during 36Cl-i/Cl-o ex-change and 30 mM during 14C-oxalatei/Cl-o ex-change. These experiments reveal that binding and/or translocation rates of extracellular Cl- with respectto gp slc26a6 is/are dependent on the internal trans-anion substrate and show species-specificity (5).The high affinity for extracellular Cl-o suggests thateven when duct luminal Cl- is low, such as might oc-cur with passage of pancreatic juice along the ductlumen, Slc26a6-mediated Cl-/HCO3- exchange wouldstill be active. Whereas gp Slc26a6-mediated iso-topic flux was moderately sensitive to DNDS (500μM), niflumic acid (100 μM) and DIDS (500 μM),gp Slc26a3 was minimally sensitive to inhibition byDIDS and DNDS but strongly sensitive to niflumicacid. These pharmacological data, combined withEXPANDED ABSTRACTMolecular characterization of Slc26a3 and Slc26a6 aniontransporters in guinea pig pancreatic ductAndrew K Stewart, Boris E Shmukler, David H Vandorpe, and Seth L AlperRenal Division and Molecular and Vascular Medicine Unit, Beth Israel Deaconess Medical Center,Harvard Medical School, Boston, MA, USAKeywords : Cl -/HCO3- exchange, CFTR, Slc26 anion transporter, Xenopus oocytesJ. Med. Invest. 56 Suppl. : 329-331, December, 2009Received for publication November 10, 2009 ; accepted Novem-ber 17, 2009.Address correspondence and reprint requests to Andrew Stewart,Renal Division and Molecular and Vascular Medicine Unit, BethIsrael Deaconess Medical Center, Harvard Medical School,Boston, MA, USA and Fax : +001-617-667-8040.The Journal of Medical Investigation Vol. 56 Supplement 2009329those from isolated guinea pig interlobular ductsidentifying a DIDS-sensitive Cl-/HCO3- exchangerthat mediates the majority of forskolin-stimulatedHCO3- secretion during recovery from an intracel-lular alkaline load, suggest a dominant role for Slc26a6 in guinea pig ductal HCO3- secretion. In con-trast, the role of Slc26a3-mediated Cl-/HCO3- ex-change in pancreatic ductal secretion remains inquestion.The ability of the pancreatic duct to secrete a highconcentration of HCO3- has been attributed, at leastin part, to electrogenic apical Cl-/HCO3- exchange.However, the stochiometry of Slc26a6-and Slc26a3-mediated Cl-/anion exchange in heterologous ex-pression systems (5, 6) and in intestinal mucosa (7,8) remains a point of controversy. Both gp Slc26a3and Slc26a6-mediated electroneutral Cl-/HCO3- ex-change in Xenopus oocytes, as judged by pH-sensi-tive microelectrode measurements and by two-elec-trode voltage clamp recording under the same ex-perimental conditions. In contrast, measurementsof membrane potential in isolated guinea pig inter-lobular ducts under conditions that activate apicalCl-/HCO3- exchange have suggested the presenceof an electrogenic, DIDS-sensitive apical Cl-/HCO3-exchanger consistent with electrogenic Slc26a6 (2).These discrepant results suggest either that Slc26a6requires an additional cofactor (such as CFTR) tomediate electrogenic anion exchange or, alterna-tively, that the DIDS-sensitive Cl-/HCO3- exchangemeasured in isolated ducts was not mediated bySlc26a6. Our current data reinforce the conclusionthat functional differences are present amongorthologous Slc26 anion exchangers of different spe-cies (3) and should be considered in modeling therole of Slc26 Cl-/HCO3- exchangers in pancreaticduct bicarbonate secretion.In conclusion, data obtained to date with clonedguinea pig Slc26 anion exchangers continue to high-light species-specific differences in Slc26 aniontransporter function. These differences may be im-portant in formulating a model for the secretion of140 mM HCO3- by guinea pig pancreatic duct, andmay suggest new insights into the molecular mecha-nism of human pancreatic bicarbonate secretion.ACKNOWLEDGEMENTSWe thank M Nakakuki for excellent technical as-sistance and Dr H Ishiguro for helpful discussionsand generous support.GRANTSThis work was supported by a Pilot and Feasibil-ity award from the Harvard Digestive Disease Cen-ter (Boston, MA ; National Institute of Health (NIH)Grant DK34854) and a Japan Society for the Pro-motion of Sciences Long Term Fellowship awardedto AK Stewart, SL Alper was supported by NIHGrants DK43495 and DK34854.REFERENCES1. Steward MC, Ishiguro H : Molecular and cel-lular regulation of pancreatic duct cell function.Curr Opin Gastroenterol 25 : 447-453, 20092. Stewart AK, Yamamoto A, Nakakuki M,Kondo T, Alper SL, Ishiguro H : Functionalcoupling of apical Cl-/HCO3- exchange withCFTR in stimulated HCO3- secretion by guineapig interlobular pancreatic duct. Am J PhysiolGastrointest Liver Physiol 296 : G1307-G1317,20093. Chernova MN, Jiang L, Friedman DJ, DarmanRB, Lohi H, Kere J, Vandorpe DH, Alper SL :Functional comparison of mouse slc26a6 anionexchanger with human SLC26A6 polypeptidevariants : differences in anion selectivity, regu-lation, and electrogenicity. J Biol Chem 280 :8564-8580, 20054. Chernova MN, Jiang L, Shmukler BE,Schweinfest CW, Blanco P, Freedman SD,Stewart AK, Alper SL : Acute regulation of theSLC26A3 congenital chloride diarrhoea anionexchanger (DRA) expressed in Xenopus oo-cytes. J Physiol 549 : 3-19, 20035. Clark JS, Vandorpe DH, Chernova MN,Heneghan JF, Stewart AK, Alper SL : Speciesdifferences in Cl- affinity and in electrogenicityof SLC26A6-mediated oxalate/Cl- exchangecorrelate with the distinct human and mousesusceptibilities to nephrolithiasis. J Physiol 586 :1291-306, 20086. Shcheynikov N, Wang Y, Park M, Ko SB,Dorwart M, Naruse S, Thomas PJ, MuallemS : Coupling modes and stochiometry of Cl-/HCO3- exchange by slc26a3 and slc26a6. J GenPhysiol 127 : 511-524, 20067. Walker NM, Simpson JE, Brazill JM, Gill RK,Dudeja PK, Schweinfest CW, Clarke LL : Roleof down-regulated in adenoma anion exchangerin HCO3- secretion across murine duodenum.A. K. Stewart, et al. Guinea Pig Slc26 anion transporters330Gastroenterology 136 : 893-901, 20098. Walker NM, Simpson JE, Yen PF, Gill RK,Rigsby EV, Brazill JM, Dudeja PK, SchweinfestCW, Clarke LL : Down-regulated in adenomaCl-/HCO3- exchanger couples with Na/H ex-changer 3 for NaCl absorption in murine smallintestine. Gastroenterology 135 : 1645 - 1653e1643, 2008The Journal of Medical Investigation Vol. 56 Supplement December 2009 331

Molecular characterization of Slc26a3 and Slc26a6 anion transporters in guinea pig pancreatic duct

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Molecular characterization of Slc26a3 and Slc26a6 anion transporters in guinea pig pancreatic duct

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