A Slow pH-dependent Conformational Transition Underlies a Novel Mode of Activation of the Epithelial Na+/H+ Exchanger-3 Isoform

Allosteric control of Na+/H+ exchange by intracellular protons ensures rapid and accurate regulation of the intracellular pH. Although this allosteric effect was heretofore thought to occur almost instantaneously, we report here the occurrence of a slower secondary activation of the epithelial Na+/H+ exchanger (NHE)-3 isoform. This slow activation mode developed over the course of minutes and was unique to NHE3 and the closely related isoform NHE5, but was not observed in NHE1 or NHE2. Activation of NHE3 was not due to increased density of exchangers at the cell surface, nor was it accompanied by detectable changes in phosphorylation. The association of NHE3 with the cytoskeleton, assessed by its retention in the detergent-insoluble fraction, was similarly unaffected by acidification. In contrast to the slow progressive activation elicited by acidification, deactivation occurred very rapidly upon restoration of the physiological pH. We propose that NHE3 undergoes a slow pH-dependent transition from a less active to a more active state, likely by changing its conformation or state of association. Allosteric control of Na+/H+ exchange by intracellular protons ensures rapid and accurate regulation of the intracellular pH. Although this allosteric effect was heretofore thought to occur almost instantaneously, we report here the occurrence of a slower secondary activation of the epithelial Na+/H+ exchanger (NHE)-3 isoform. This slow activation mode developed over the course of minutes and was unique to NHE3 and the closely related isoform NHE5, but was not observed in NHE1 or NHE2. Activation of NHE3 was not due to increased density of exchangers at the cell surface, nor was it accompanied by detectable changes in phosphorylation. The association of NHE3 with the cytoskeleton, assessed by its retention in the detergent-insoluble fraction, was similarly unaffected by acidification. In contrast to the slow progressive activation elicited by acidification, deactivation occurred very rapidly upon restoration of the physiological pH. We propose that NHE3 undergoes a slow pH-dependent transition from a less active to a more active state, likely by changing its conformation or state of association. Na+/H+ exchangers (NHEs) 1The abbreviations used are: NHEsN+/H+ exchangerspHiintracellular pHBCECF2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluoresceinHAhemagglutininPBSphosphate-buffered salineMES4-morpholineethanesulfonic acid catalyze the electroneutral counter-transport of Na+ for H+across biological membranes (for review, see Refs. 1.Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar, 2.Wakabayashi S. Shigekawa M. Pouyssegur J. Physiol. Rev. 1997; 77: 51-74Crossref PubMed Scopus (563) Google Scholar, 3.Counillon L. Pouyssegur J. J. Biol. Chem. 2000; 275: 1-4Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). To date, seven different NHE isoforms have been cloned, all of which are integral membrane proteins with multiple transmembrane domains and an extensive cytosolic carboxyl-terminal domain. NHE1 and NHE3 are by far the most widely studied isoforms. The former is expressed ubiquitously and, by mediating Na+/H+ exchange across the plasma membrane, contributes to the regulation of cytosolic pH and cell volume (2.Wakabayashi S. Shigekawa M. Pouyssegur J. Physiol. Rev. 1997; 77: 51-74Crossref PubMed Scopus (563) Google Scholar). NHE3 is found in the brush border of epithelial cells in the kidney and gastrointestinal tract and is involved in the transepithelial (re)absorption of NaCl and, indirectly, HCO3− and water (4.Schultheis P.J. Clarke L.L. Meneton P. Miller M.L. Soleimani M. Gawenis L.R. Riddle T.M. Duffy J.J. Doetschman T. Wang T. Giebisch G. Aronson P.S. Lorenz J.N. Shull G.E. Nat. Genet. 1998; 19: 282-285Crossref PubMed Scopus (703) Google Scholar). N+/H+ exchangers intracellular pH 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein hemagglutinin phosphate-buffered saline 4-morpholineethanesulfonic acid Because Na+/H+ exchange is electroneutral with a 1:1 stoichiometry, thermodynamic equilibrium is predicted to be attained when [H+]i/[H+]o = ([Na+]i/[Na+]o)·Ke, where Ke is the equilibrium constant and the subscripts i and o refer to intra- and extracellular, respectively (5.Grinstein S. Cohen S. Rothstein A. J. Gen. Physiol. 1984; 83: 341-369Crossref PubMed Scopus (312) Google Scholar). In almost all cells, a steep inwardly directed Na+ gradient is maintained across the plasma membrane due to extrusion of Na+ by the Na+/K+-ATPase. Based on the concentrations recorded in most cases ([Na+]o being at least 10 times higher than [Na+]i), it is apparent that NHE could drive the intracellular pH (pHi) at least 1 unit above the external pH. Nevertheless, pHi rarely exceeds and is usually lower than the extracellular pH because NHE becomes virtually quiescent at pHi ≥7.2. This behavior has been attributed to the existence of an allosteric pH-sensitive site on the cytosolic aspect of NHE. The set point at which quiescence is dictated by this allosteric or “modifier” site is presumably intended to stabilize pHi near the physiological optimum level and to preclude deleterious alkalinization (6.Aronson P.S. Nee J. Suhm M.A. Nature. 1982; 299: 161-163Crossref PubMed Scopus (466) Google Scholar, 7.Grinstein S. Goetz J.D. Rothstein A. J. Gen. Physiol. 1984; 84: 585-600Crossref PubMed Scopus (51) Google Scholar). Although little is known about the molecular identity of the modifier site, the pH dependence of activation of NHE suggests that the rate of transport is modulated by protonation of one or more side chains of the protein. It has been postulated that the resulting change in surface potential may alter the local concentration of H+equivalents and thus their availability for the exchange reaction. Because protonation/deprotonation of side chains is expected to occur almost immediately after pH is altered, the effect of the modifier site has been tacitly assumed to occur instantaneously, providing rapid feedback and thereby accurate regulation of pHi. In the course of experiments using isolated brush-border membranes at subphysiological temperatures, we detected significant hysteresis in the inactivation of NHE3 (preliminary results are described in Ref. 8.Kinsella J.L. Heller P. Froehlich J.P. Biochem. Cell Biol. 1998; 76: 743-749Crossref PubMed Scopus (29) Google Scholar). This slow responsiveness, which developed over the course of minutes, cannot be easily reconciled with the simple side chain protonation model described above. We therefore decided to investigate whether slow activation and/or inactivation transitions are observed in intact cell systems and to examine the underlying mechanism. To this end, we used porcine kidney LLC-PK1 cells, which are known to express endogenous NHE3 (9.Shugrue C.A. Obermuller N. Bachmann S. Slayman C.W. Reilly R.F. J. Am. Soc. Nephrol. 1999; 10: 1649-1657Crossref PubMed Google Scholar). In addition, we compared the behavior of several isoforms of NHE expressed heterologously in antiport-deficient Chinese hamster cells. Our results revealed a novel mode of regulation that is unique to NHE3 and the related isoform NHE5 and that likely involves large conformational changes and/or interaction with other molecules. Nigericin, the acetoxymethyl ester of 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and Alexa 488-conjugated donkey anti-mouse antibody were obtained from Molecular Probes, Inc. (Eugene, OR). Mouse anti-hemagglutinin (HA) antibodies were from BabCo (Berkeley, CA). 125I-Labeled goat IgG and [32P]orthophosphate were from ICN (Costa Mesa, CA). Isotonic Na+ medium contained mm 140 mm NaCl, 3 mm KCl, 1 mmMgCl2, 1 mm CaCl2, and 20 mm HEPES (pH adjusted to 7.4 with Tris at room temperature). Na+-free solution was prepared by equimolar substitution with N-methyl-d-glucamine. Isotonic K+-rich medium had the same composition as Na+-rich medium, except that NaCl was replaced by KCl. LLC-PK1 cells were obtained from the American Type Culture Collection. For surface detection and immunoprecipitation experiments, LLC-PK1 cells were transfected with wild-type NHE3 containing three tandem copies of the influenza virus HA epitope (YPYDVPDYAS) in the first extracellular loop (between Arg38 and Phe39) as described previously (13.D'Souza S. Garcia-Cabado A. Yu F. Teter K. Lukas G. Skorecki K. Moore H.P. Orlowski J. Grinstein S. J. Biol. Chem. 1998; 273: 2035-2043Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). LLC-PK1 cells were transfected using FuGENE 6 (Roche Molecular Biochemicals) following the manufacturer's instructions. For selection of stable lines, cells were cotransfected with the pCMV plasmid (which contains the aminoglycoside phosphotransferase gene that confers resistance to G418), selected by limiting dilution cloning in the presence of 500 μg/ml G418, and screened by immunofluorescence for expression of HA-tagged NHE3 (NHE3′38HA3). Cells were maintained in 1:1 Dulbecco's modified Eagle's medium/nutrient mixture F-12 with 10% fetal bovine serum in an atmosphere containing 5% CO2. AP-1 is a cell line devoid of endogenous Na+/H+exchange activity that was isolated from WT5 Chinese hamster ovary cells as previously described (10.Rotin D. Grinstein S. Am. J. Physiol. 1989; 257: C1158-C1165Crossref PubMed Google Scholar). NHE cDNA constructs were transfected into AP-1 cells by the calcium phosphate/cDNA coprecipitation technique, and stable clones were selected for survival by imposing acute NH4Cl-induced acid loads (11.Franchi A. Perucca-Lostanlen D. Pouyssegur J. Proc. Natl. Acad. S. A. 83: PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Cells were maintained in medium with 10% fetal bovine serum in an atmosphere containing 5% CO2. pHi was by of the of using The activity of NHE was as the rate of pHi after acid with LLC-PK1 cells to on were with μg/ml ester mm and after 10 the cells were with Na+-free Na+/H+ exchange was by of extracellular Na+ at the of the cells to the were selected for pHi cells that were in the pHi likely occurred NHE. To the of from the cells, the was at using a The of was and as described previously (13.D'Souza S. Garcia-Cabado A. Yu F. Teter K. Lukas G. Skorecki K. Moore H.P. Orlowski J. Grinstein S. J. Biol. Chem. 1998; 273: 2035-2043Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The was from the pHi changes observed in to at were by the rate of pHi by the at the pH were at room LLC-PK1 cells were and to We not cells because of potential of extracellular Na+ was by the cells in Na+-free Na+ was by using as an intracellular Na+ the cells were with medium containing mm and 10 mm (pH The cells were with a into solution the were and was and the was used for was by the of cells Cells were with a LLC-PK1 cells were and to The cells were with phosphate-buffered saline and, where for with as described above for 20 using in the cells were three times with and with in for 10 The cells were with 5% in for and with antibody for 1 three times to the cells were with Alexa 488-conjugated donkey anti-mouse antibody for 1 and the were with medium CA). The of was assessed in transfected LLC-PK1 cells in and to the cells were first to pH as described above. The cells were with medium and with 1 of medium containing mm NaCl, mm mm mm and mixture Molecular and or The pH of the medium was adjusted to or 7.4 as on for 6 on an the was and The was with and with a into 1 of containing and mixture concentration was by the of S. S. J.J. Biochem. PubMed Scopus Google and by of were to and were with 5% and to antibody or secondary antibody was and were using and by To surface NHE3 LLC-PK1 cells were and to The cells were with and, where for with as described above. The cells were with with 10% goat serum in for on and for on with antibody in medium with 10% goat times to the cells were with goat anti-mouse IgG in medium with 10% goat serum on cells were times with to The antibodies were with 1 of and was using a of the assessed by was to on were for by with of Dulbecco's modified Eagle's medium containing of the cells were three times with and, where with for as described above. To the in NHE3 to we prepared brush-border membrane from the by the of M. J. 1984; PubMed Scopus Google with The cells were with a into 1 of medium containing mm mm mm and 10 mm (pH and with a for was to a concentration of 10 by for on and for at The was and to at The resulting containing the brush-border was in 1 of mm NaCl, mm and 20 mm at pH 7.4 or by a and on a for at at for to the was to that were with and the was for at The were and times with proteins were by in of at for of of was by and to a membrane The of NHE3 in the were by and using the the from the same membranes were using a and using are as the of the of were by or as The dependence of the activation of NHE3 was studied in LLC-PK1 cells by the of an acid cytosolic was by the A. Grinstein S. S. Orlowski J. J. Biol. Chem. Full Text PDF PubMed Google and Na+/H+ exchange was when by of 140 mm Na+ to the It is that in the of pHi was pH and the acid was at the of of Na+ the rate of pH was by an to due to intracellular in the of the 1 after of was a This rate of was in experiments and was to the of NHE on the rate of were to that the activity of NHE3 was assessed from which is expressed in the membrane of LLC-PK1 cells R.F. F. D. Pouyssegur J. Aronson P.S. Slayman C.W. P. Am. J. Physiol. Google Scholar). the cells were to and Na+ was was to NHE1 J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the used = the was slow when the cells were for 1 the rate of alkalinization increased as the of was The in the results of the rate of Na+/H+ exchange increased to and The rate observed was than the rate at 1 were not because the acid was 1 of experiments were using kidney cells, with results not that the slow activation of Na+/H+ exchange is a and that the in LLC-PK1 cells were to NHE3 because kidney cells express but not NHE1 J. Physiol. PubMed Scopus Google Scholar, L. D. Pouyssegur J. J. Physiol. PubMed Scopus Google Scholar). The rate observed after is likely to increased activity of it is that the cytosolic over after acidification, resulting in a at constant NHE The was using an in the pHi by and solution was whether the was 1 or after was The and at 1 and respectively a in and other in the of an acid of in Na+-free medium for different of This may have in intracellular Na+ at the of NHE. The observed in activity may therefore have from an increased for Na+/H+ exchange or from of a as has been for the regulation of NHE1 A. S. Proc. Natl. Acad. S. A. 1999; PubMed Scopus Google Scholar). To this cells were in Na+-free medium for 10 and to acid and with extracellular in the rate of after 1 of was not different from that recorded in control cells in 1 = the of NHE3 activity in the the rate of pH was higher after than after 1 and To that of Na+ was not the underlying we the intracellular of this by all the of extracellular Na+ to the acid intracellular Na+ by to 20 The of the was not different = when Na+ was for that occurred the acid in results that intracellular not to the activation of To the effect of pHi on the of NHE3 activation concentrations of were used the The dependence of the activation was recorded at all the that we (between and and in all the 1 and was when rate the to when the was for = but the was after 1 = This suggests that a more the activation of NHE3 is at 1 activation is by the expected is In as as in expression NHE3 is in at least the plasma membrane and an that (13.D'Souza S. Garcia-Cabado A. Yu F. Teter K. Lukas G. Skorecki K. Moore H.P. Orlowski J. Grinstein S. J. Biol. Chem. 1998; 273: 2035-2043Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, D. T. Aronson P.S. Am. J. Physiol. 1997; 273: PubMed Google Scholar, P. A. S. M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In kidney cells, it was that to the activity of NHE3 and that an is accompanied by of NHE3 to the cell surface M. Am. J. Physiol. 2000; PubMed Google Scholar). It was therefore that the of surface NHE3 may change acute intracellular acidification. To this the of NHE3 was by we a stable of an of The epitope was in an extracellular loop to detection of NHE3 in intact cells Ref. K. G. Orlowski J. Grinstein S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google for and of intact cells and after revealed in the density or of NHE3 to more surface we used in the surface density of NHE3 not change following acidification. results that the and not the of was increased when acid was for It is that NHE3 on and that is accompanied by changes in transport activity M. J. PubMed Scopus Google Scholar, K. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, Am. J. Physiol. 1999; PubMed Google Scholar, L. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, may a in NHE3 regulation because of exchange by is with activation of M. A. Miller Proc. Natl. Acad. S. A. PubMed Scopus Google Scholar). To whether the more acute here was similarly to cells were with [32P]orthophosphate and to acidification. The cells were immediately in the and NHE3 was of the following by revealed that the of of NHE3 was and after for a other than for the observed activation of We previously a interaction NHE3 and the in AP-1 cells K. S. K. Orlowski J. Grinstein S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In that the activity of the was modulated by the of the that the may to the pH-dependent To this we the association of NHE3 with the detergent-insoluble of which contains the Cells were or were to pH as described for the of exchange the cells were in at pH or and the and of NHE3 were by For the of and were in the using a of NHE3 was of the pH of the solution pH 7.4 and This that the of the exchangers are with the the the may known as K. Nature. 1997; PubMed Scopus Google Scholar). It was therefore that the of as to its dictated the of the This was using with and from biological a of NHE3 than the that the exchanger is its interaction with the the of the exchangers that were by was not when the cells were and/or the and The of and in were and results were obtained with not results that the interaction of NHE3 with the is not by the acid It therefore that the a in the and pH-dependent activation of the This was by in cells transfected with a of NHE3 that was to be to K. S. K. Orlowski J. Grinstein S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). in the rate of Na+/H+ exchange in was when the was as above for the endogenous exchanger of epithelial cells. The rate of was after 3 than after 1 that the is not the of the activation of We assessed the rate at which NHE3 following slow activation by a acid The cells were first for to NHE3 Na+ from the medium to preclude pH this the cytosolic pH was increased as as by an of found to pH to near Na+ was to the medium to Na+/H+ of Na+ to pH the that NHE was not more active than it was to acid This that NHE3 rapidly 1 after restoration of inactivation to be than the slow activation which developed over It could be that the slow activation of NHE3 above is a of the LLC-PK1 cells in which its activity was than being of this isoform. This was by the behavior of NHE3 expressed heterologously in AP-1 cells. AP-1 cells, a of Chinese hamster ovary cells, are devoid of endogenous NHE activity (10.Rotin D. Grinstein S. Am. J. Physiol. 1989; 257: C1158-C1165Crossref PubMed Google and are therefore to the activity of heterologously expressed in a activation was in AP-1 cells. This that the slow pH-dependent activation of NHE3 is an of this isoform that is in epithelial and It was of to other NHE isoforms similarly a slow activation following acidification. To this end, we the behavior of AP-1 cells transfected with isoforms of the described AP-1 cells to the exchange activity to the heterologously transfected NHE isoform of results are in the by NHE1 following acid for 1 or 3 in the rate of Na+/H+ exchange was not by the of the acid results were obtained in AP-1 cells transfected with NHE2. NHE5, which the known NHE isoforms the with NHE3 M. Shull G.E. Orlowski J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google a activity after 3 than after 1 of acidification, the was less than in the of the and pH-dependent activation of NHE is in NHE3 and the closely related isoform NHE5, but not in NHE1 and NHE2. Our results revealed the existence of a activation of which to This slow transition is to the rate of protonation of of the side chains of NHE3 and may from conformational changes of the exchanger and/or of proteins that its changes could or stabilize an active conformation of the is widely in the of NHE is to in an and one or more active this transition by the rate of or by its rate of To the that other isoforms a pH-dependent but not the slow transition by NHE3 and NHE5, we that and for NHE3 and NHE5 but one for the other isoforms. 1 The slow activation from to be dictated by which is presumably lower than and therefore limiting to the activation that to be which with that the course of the slow transition was not different when the of acid was The molecular of the mediating the slow activation of NHE3 to be J.L. Heller P. Froehlich J.P. Biochem. Cell Biol. 1998; 76: 743-749Crossref PubMed Scopus (29) Google the that large of exchangers may upon acidification. experiments to that higher are when the is NHE3 may into detergent-insoluble as has been for proteins in the K. Nature. 1997; PubMed Scopus Google Scholar). This was by the that the of NHE3 in a that was in control and cells. NHE3 and the had been K. S. K. Orlowski J. Grinstein S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google on the of Our that most of NHE3 in LLC-PK1 cells was not by or are with association of the exchangers with the of the cells with which and M. G. J. G. J. PubMed Scopus Google Scholar, J. M. M. K. Nature. PubMed Scopus Google increased the of NHE3 that is by the interaction of the with the was not by cytosolic and a of NHE3 by the the and pH-dependent and We therefore that the activation of the exchanger is not by in its We the that the transition a change in the conformation of the The slow of the transition that the a large activation that the conformational change is be to this The that NHE3 undergoes a slow activation transition to the of regulation of this isoform by In to the rapid activation in all isoforms of acid the and the slower described to be the a slower of NHE3 by was described S. A. Proc. Natl. Acad. S. A. PubMed Scopus Google Scholar, P. Reilly R.F. Am. J. Physiol. Google Scholar, M. J. M. Full Text PDF PubMed Scopus Google Scholar). This of transport from the one described in that it several to involves of and proteins M. J. M. Full Text PDF PubMed Scopus Google and is accompanied by increased of intracellular NHE3 molecules. are of regulation of NHE3 by acid are for the acute and regulation of the intracellular pH. over minutes may be intended to for acute in pH and and likely and more in the rate of This is by at the of In this it is that the secondary activation described in this report to rapidly upon restoration of the cytosolic pH to the feedback In the into the brush border of the and to a in the rate of acid extrusion when the pH is the of regulation are not but and for or to the and pH and

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