Membrane Curvature Alters the Activation Kinetics of the Epithelial Na+/H+ Exchanger, NHE3

The epithelial Na+/H+ exchanger, NHE3, was found to activate slowly following an acute cytosolic acidification. The sigmoidal course of activation could not be explained by the conventional two-state model, which postulates that activation results from protonation of an allosteric modifier site. Instead, mathematical modeling predicted the existence of three distinct states of the exchanger: two different inactive states plus an active form. The interconversion of the inactive states is rapid and dependent on pH, whereas the conversion between the second inactive state and the active conformation is slow and pH-independent but subject to regulation by other stimuli. Accordingly, exposure of epithelial cells to hypoosmolar solutions activated NHE3 by accelerating this latter transition. The number of surface-exposed exchangers and their association with the cytoskeleton were not affected by hypoosmolarity. Instead, NHE3 is activated by the membrane deformation, a result of cell swelling. This was suggested by the stimulatory effects of amphiphiles that induce a comparable positive (convex) deformation of the membrane. We conclude that NHE3 exists in multiple states and that different physiological parameters control the transitions between them. The epithelial Na+/H+ exchanger, NHE3, was found to activate slowly following an acute cytosolic acidification. The sigmoidal course of activation could not be explained by the conventional two-state model, which postulates that activation results from protonation of an allosteric modifier site. Instead, mathematical modeling predicted the existence of three distinct states of the exchanger: two different inactive states plus an active form. The interconversion of the inactive states is rapid and dependent on pH, whereas the conversion between the second inactive state and the active conformation is slow and pH-independent but subject to regulation by other stimuli. Accordingly, exposure of epithelial cells to hypoosmolar solutions activated NHE3 by accelerating this latter transition. The number of surface-exposed exchangers and their association with the cytoskeleton were not affected by hypoosmolarity. Instead, NHE3 is activated by the membrane deformation, a result of cell swelling. This was suggested by the stimulatory effects of amphiphiles that induce a comparable positive (convex) deformation of the membrane. We conclude that NHE3 exists in multiple states and that different physiological parameters control the transitions between them. The activity of sodium-proton exchangers (NHEs) is fundamental to the maintenance of both intracellular and systemic [Na+] and pH. Multiple isoforms of NHE have been identified that differ in their tissue distribution and subcellular localization. Some isoforms function primarily in cytosolic cation homeostasis, whereas others are thought to regulate organellar cation transport, accounting for their differential subcellular localization. Similarly, differences in the pattern of expression likely underlie the functional roles of various NHEs; widely expressed isoforms, such as NHE1, have housekeeping activity, whereas those restricted to defined tissues have specialized functions. One such specialized isoform is NHE3, which is expressed almost exclusively on the apical pole of epithelial cells. In the gut and kidney, NHE3 mediates the (re)absorption of salt, bicarbonate, and water (1Lorenz J.N. Schultheis P.J. Traynor T. Shull G.E. Schnermann J. Am. J. Physiol. 1999; 277: F447-F453PubMed Google Scholar). Regardless of their precise function, the activity of all the isoforms studied to date is highly sensitive to the intracellular pH (pHi) (2Aronson P.S. Nee J. Suhm M.A. Nature. 1982; 299: 161-163Crossref PubMed Scopus (464) Google Scholar, 3Grinstein S. Cohen S. Rothstein A. J. Gen. Physiol. 1984; 83: 341-369Crossref PubMed Scopus (311) Google Scholar, 4Orlowski J. J. Biol. Chem. 1993; 268: 16369-16377Abstract Full Text PDF PubMed Google Scholar, 5Tse C.M. Levine S.A. Yun C.H. Brant S.R. Pouyssegur J. Montrose M.H. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9110-9114Crossref PubMed Scopus (117) Google Scholar, 6Vigne P. Frelin C. Lazdunski M. EMBO J. 1984; 3: 1865-1870Crossref PubMed Scopus (38) Google Scholar, 7Wakabayashi S. Fafournoux P. Sardet C. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2424-2428Crossref PubMed Scopus (238) Google Scholar). This exquisite pHi dependence has been attributed to the protonation of an allosteric site on the cytosolic face of the exchanger (2Aronson P.S. Nee J. Suhm M.A. Nature. 1982; 299: 161-163Crossref PubMed Scopus (464) Google Scholar, 8Wakabayashi S. Hisamitsu T. Pang T. Shigekawa M. J. Biol. Chem. 2003; 278: 43580-43585Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), which is distinct from the proton transport site (8Wakabayashi S. Hisamitsu T. Pang T. Shigekawa M. J. Biol. Chem. 2003; 278: 43580-43585Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). According to this two-state model, protonation of the allosteric site converts the exchanger from an inactive to an active form. In addition to pHi, some NHE isoforms are sensitive to alterations in osmolarity (9Ritter M. Fuerst J. Woll E. Chwatal S. Gschwentner M. Lang F. Deetjen P. Paulmichl M. Cell Physiol. Biochem. 2001; 11: 1-18Crossref PubMed Scopus (63) Google Scholar). The best studied example, NHE1, is activated by extracellular hyperosmolarity and inhibited by hypoosmolarity (10Garnovskaya M.N. Mukhin Y.V. Vlasova T.M. Raymond J.R. J. Biol. Chem. 2003; 278: 16908-16915Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Grinstein S. Cohen S. Goetz J.D. Rothstein A. Fed. Proc. 1985; 44: 2508-2512PubMed Google Scholar, 12Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar). The activation of NHE1 induced by hyperosmolarity is felt to be a compensatory response to cell shrinkage, because it causes net salt and water intake, leading to volume restoration. In contrast, hyperosmolarity inhibits epithelial NHE3 activity (12Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar, 13Good D.W. Di Mari J.F. Watts 3rd, B.A. Am. J. Physiol. 2000; 279: C1443-C1454Crossref PubMed Google Scholar, 14Watts 3rd, B.A. Good D.W. J. Clin. Investig. 1999; 104: 1593-1602Crossref PubMed Scopus (52) Google Scholar, 15Ambuhl P. Amemiya M. Preisig P.A. Moe O.W. Alpern R.J. J. Clin. Investig. 1998; 101: 170-177Crossref PubMed Scopus (48) Google Scholar, 16Nath S.K. Hang C.Y. Levine S.A. Yun C.H. Montrose M.H. Donowitz M. Tse C.M. Am. J. Physiol. 1996; 270: G431-G441PubMed Google Scholar, 17Soleimani M. Bookstein C. McAteer J.A. Hattabaugh Y.J. Bizal G.L. Musch M.W. Villereal M. Rao M.C. Howard R.L. Chang E.B. J. Biol. Chem. 1994; 269: 15613-15618Abstract Full Text PDF PubMed Google Scholar). The mechanisms underlying these divergent responses are not clear at present. The response of NHE3 to hypoosmolarity has been studied less extensively (13Good D.W. Di Mari J.F. Watts 3rd, B.A. Am. J. Physiol. 2000; 279: C1443-C1454Crossref PubMed Google Scholar, 14Watts 3rd, B.A. Good D.W. J. Clin. Investig. 1999; 104: 1593-1602Crossref PubMed Scopus (52) Google Scholar). Here we investigated the effect of reduced extracellular osmolarity on NHE3 activity. During the course of these studies, we found that the conventional two-state model of NHE activation was insufficient to account for the behavior of NHE3. We report that an additional inactive state of the exchanger is required to explain its kinetics of activation. The transition between the two inactive (or poorly active) states is pHi-independent and limits the rate of activation. Importantly, hypoosmotic stress was found to stimulate NHE3 by accelerating the rate-limiting transition between inactive states. This effect was not caused directly by the hypoosmolarity of the bathing solution but by the curvature imposed on the membrane upon cell swelling. Materials and Solutions—Nigericin, the acetoxymethyl ester of 2′,7′-bis(carboxyethyl)-5 (6)-carboxyfluorescein (BCECF), 3The abbreviations used are: BCECF, 2′,7′-bis(carboxyethyl)-5 (6)-carboxyfluorescein; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; HA, hemagglutinin; MDCK, Madin-Darby canine kidney; LPC, lysophosphatidylcholine; PC, phosphatidylcholine. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), Alexa 488-conjugated goat anti-mouse antibody and F(ab) fragment were obtained from Molecular Probes, Inc. Phosphatidylcholine, lysophosphatidylcholine, and O-phenylenediamine dihydrochloride were from Sigma. Anti-hemagglutinin (HA) mouse antibody and F(ab) fragment were from BabCo. Cy2- and Cy3-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. Monoclonal anti-phosphoserine-552 NHE3 antibody was generated as described (18Kocinsky H.S. Girardi A.C. Biemesderfer D. Nguyen T. Mentone S. Orlowski J. Aronson P.S. Am. J. Physiol. 2005; 289: F249-F258Crossref PubMed Scopus (98) Google Scholar). Isotonic Na+-rich medium contained 70 mm NaCl, 50 mm N-methylglucammonium chloride, 3 mm KCl, 1 mm MgCl2, and 20 mm HEPES-Tris (pH 7.4). Hypotonic Na+-rich medium contained 70 mm NaCl, 10 mm N-methylglucammonium chloride, 3 mm KCl, 1 mm MgCl2, and 20 mm HEPES-Tris (pH 7.4). Isotonic and hypotonic K+-rich medium had similar composition except that NaCl was replaced by KCl. Cells and Constructs—Madin-Darby canine kidney (MDCK)-II and opossum kidney cells were obtained from ATCC. The MDCK-II cells were with NHE3 three of the between the and second between and generated as described Orlowski J. Grinstein S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). a the cells were by in the of and by for expression of NHE3. and opossum kidney cells were in a of medium with in a The were at the had of Na+/H+ activity was in cells as the rate of pHi an of the of were used to pHi, as (12Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar). the cells were to on cell and on the of the were with acetoxymethyl ester and with 50 mm in at for 10 for and were with and Na+/H+ was by of in the hypoosmolar of NHE3 activity were in the of to the by pH (pHi) was by the cells with K+-rich medium to defined pH and 10 J.A. A. E. PubMed Scopus Google Scholar). was the of pHi for of cells were not to multiple of and of were as described Orlowski J. Grinstein S. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). In cells were with mouse F(ab) fragment in and with secondary Alexa 488-conjugated goat anti-mouse F(ab) fragment The in were on the of a and in hypoosmolar The apical was and two in were two of the was and the of both was The of the was to the of the two The was used to of the states of NHE3 activation were generated as in the were used to the rate of of pHi at the for of the for was used for this NHE3 of surface-exposed and NHE3 was by an as in J. P. Grinstein S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google to the of NHE3, the cells were with with for and with antibody for 1 at the cells with were with a anti-mouse antibody for 1 at similar was in to NHE3, except that the cells were with the secondary the cells were with 1 of for 10 at The was by of 3 The was and was at a In the with the of was in for by of the antibodies and was from of and NHE3 was as described in Orlowski J. Grinstein S. J. Gen. Physiol. PubMed Scopus Google The cells were a and and were with and were as described S. R. Orlowski J. Grinstein S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The are as the of the number of of MDCK-II cells an of NHE3, in Orlowski J. Grinstein S. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google and to as was used to the regulation of NHE3. The activity of NHE3 in of osmolarity was from of in the addition of to cells that had by an induced a the state pHi which was cells NHE1 on their these and all cells were in the of EIPA, is an at the used in the NHE1 activity, NHE3 J. J. Biol. Chem. 1993; 268: 16369-16377Abstract Full Text PDF PubMed Google Scholar, Lang Pouyssegur J. 1993; 44: Google Scholar). The effect of hypoosmolarity on NHE3 activity was was to a comparable and to the of to osmolarity the the osmolarity to the rate of pHi the the rate of was in hypoosmolar with of that the of transport was to NHE3 and not to inhibited we the effect of osmolarity on NHE activity in the in the of in 1 and hypoosmolar reduced the rate of pHi which is by NHE1 in this This was by the addition of EIPA, which the in these cells we the effect of reduced osmolarity in opossum kidney a to NHE3 but not NHE1 M. A. Moe O.W. Alpern R.J. Am. J. Physiol. 269: PubMed Google Scholar, A. D. Orlowski J. J. Biol. Chem. 270: Full Text Full Text PDF PubMed Scopus Google Scholar). NHE3 activity in opossum kidney cells The rate of proton is a function of both the of pHi and the was the activity of NHE3, hypoosmolarity reduced the of the accelerating the pHi at activity of the This was by of the of In three the of cells and and hypoosmolar for the of of some of the underlying the activation of the course of pH from an is sigmoidal whereas it is following hypoosmolar The effect of hypoosmolar is to the activation in cells. to be at with the conventional that NHE3 exists in two at physiological pH, the exchangers are thought to be in an inactive (or poorly active) of the to protonation of the the exchangers to an active that for the of these we used a mathematical model to the activity of NHE3 as a function of and pH. a two-state model the rate of of pHi following an is to the of the exchangers in the active and to the of the The of is dependent on pHi, and the rate at which it pHi is is by the rate of the to conversion these as explained in in the we the course of pHi caused by Na+/H+ following a rapid such as that by following a that of of membrane are the D. P. Nature. PubMed Scopus Google is from the model an rate of pH that is to the of acidification. the of the is upon acidification. The of the model was by these to the results obtained in NHE1 NHE3 were the the activity of NHE1 was in whereas NHE3 was in cells in the of in the by the model the behavior of NHE1 in which that for this isoform in a of other cell S.K. Hang C.Y. Levine S.A. Yun C.H. Montrose M.H. Donowitz M. Tse C.M. Am. J. Physiol. 1996; 270: G431-G441PubMed Google Scholar, S.A. Montrose M.H. Tse C.M. Donowitz M. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). The rate of transport is and with the of the and In contrast, because of the activation described the activity of NHE3 is not affected by pHi and Instead, the rate of transport a the pHi is the two-state model is to explain the behavior of NHE1, it not the sigmoidal response for NHE3. of NHE3 with a modeling that a two-state model is insufficient to the behavior of NHE3 and that additional states of this isoform the existence of an activation following that protonation of the allosteric site is not the the activation and that pH-independent We to the of the two-state The of is in This model postulates the existence of two distinct inactive and plus active The transition between inactive states is to be rapid and and the conversion from to the active conformation is slow and We to the model in a similar by and with results obtained from in cells. the predicted pHi following of generated the a slow transition between and the course of in this is to be the behavior of NHE3. because the slow transition between and was to be the rate of transport is of the of the as found for NHE3, but not for We conclude studied the kinetics of activation of NHE3 are best explained by a model of at three distinct activation states. the between a model that the behavior of NHE3 we to the by which hypoosmolarity this hypoosmolar to the cells are this is attributed in the model to the slow conversion between and the effect of reduced osmolarity be to an of this transition. be on the of this the conversion is the rate of from an in hypoosmolar medium and at the activity of NHE3 be affected by in were in the in In the activity of NHE3 was of of hypoosmolar hypoosmolarity a pHi dependence of the rate of which is not in This differential is a intracellular is imposed by of the activity of the exchanger is by the addition of extracellular The dependence of the response is in and at an pHi the of the activity of NHE3 is in hypoosmolar medium in medium the cells were at an pHi for a of the rate of pHi was rapid and not different in both and hypoosmolar that the in medium was in this because the the conversion of of to The in the of the be by the but not the two-state model the of the Importantly, of the model that hypoosmolar NHE3 by accelerating the transition between and This the conversion between and is it is sensitive to the osmolarity of the bathing solution to a such as in cell of the of the Molecular of NHE3 investigated the of the of NHE3. This isoform is not at the membrane but in intracellular and between has been suggested as a to the transport rate S. P. Musch M.W. Chang E.B. J.R. Proc. Natl. Acad. Sci. U. S. A. 101: PubMed Scopus Google Scholar, J.A. P. Am. J. Physiol. 2003; PubMed Google Scholar). We the osmolarity the expression of NHE3. We of the of the to the distribution of exchangers by Orlowski J. Grinstein S. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, Orlowski J. Grinstein S. J. Gen. Physiol. PubMed Scopus Google Scholar). of and cells of the of NHE3 that was surface-exposed following in hypoosmolar in was in the distribution of in the two precise was a In three similar we found the number of surface-exposed NHE3 to be between and hypoosmolar is of the activity of NHE In the of NHE3, of by is with an of activity R.L. Moe O.W. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We used a antibody to medium osmolarity the state of NHE3 cells to hypoosmolar medium were and the were to by with antibodies that the of (18Kocinsky H.S. Girardi A.C. Biemesderfer D. Nguyen T. Mentone S. Orlowski J. Aronson P.S. Am. J. Physiol. 2005; 289: F249-F258Crossref PubMed Scopus (98) Google Scholar). in NHE3 an in at to hypoosmolarity. at is to with a activity of NHE3 R.L. Moe O.W. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), the account for the of transport induced by hypoosmolar two of NHE3 are to on the of epithelial that is association with the cytoskeleton and that is in the of the membrane Orlowski J. Grinstein S. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). The transport activity of these be as found for the and of NHE3 D. Aronson P.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We the effects of hypoosmolarity on the association of NHE3 with the cytoskeleton and on its association was by the of the exchangers that is in the The of the in and hypoosmolar solutions was similar the of NHE3 the was The to between the apical and the association of the of NHE3 at the we its by We had that by is a of the of NHE3 with the cytoskeleton Orlowski J. Grinstein S. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). The in were a of of antibodies to HA, by Alexa 488-conjugated secondary we a of NHE3 is on the of cells not the in a in was that hypoosmolar not NHE3 from its and that the is of the of results in cell and a positive deformation of the membrane. of the apical membrane of cells the of be in the curvature of the D. Moe O.W. D.W. Proc. Natl. Acad. Sci. U. S. A. 101: PubMed Scopus Google Scholar, 2003; PubMed Scopus Google Scholar, E. A. Biol. PubMed Scopus Google Scholar), we NHE3 is to membrane this we a of deformation that not of the medium We to effect a deformation of the membrane by its D. Moe O.W. D.W. Proc. Natl. Acad. Sci. U. S. A. 101: PubMed Scopus Google Scholar, E. A. Biol. PubMed Scopus Google Scholar). is a that upon a positive curvature that the effects of cell a we used the We to NHE3 activity and found that the exchanger, whereas its PC, not and was from cells that were not with not The rate of pHi was not caused by a effect of the because was the addition of the activation following an the effect of hypoosmolar on NHE3. the are with membrane deformation the of the in activity, we the that NHE3 activity by NHE3 to be sensitive to membrane curvature in a This was an that deformation of the membrane J. Cell Biol. PubMed Scopus Google Scholar). LPC, which the exchanger, had effect on NHE3 activity NHE3 is activated by positive (convex) deformation of the membrane. The results of modeling that the conventional two-state model of NHE activation is insufficient to the results obtained with NHE3. the of activation of an acute is not with a model protonation of an allosteric site mediates the transition from the inactive to the active state of the found that protonation of those of membrane D. P. Nature. PubMed Scopus Google Scholar). a rapid protonation account for the required to the transition between the inactive and active states of NHE3. Instead, as suggested Orlowski J. Grinstein S. J. Biol. Chem. 277: Full Text Full Text PDF PubMed Scopus Google Scholar), a of the be modeling that the activation is best explained by the existence of at three functional with the of a slow following In two distinct inactive and are to in a a slow activation to the active A. could an model a slow conversion of to is by a that results in activation of could in account for We to the because the latter model is it the and rate are and the of NHE3 in the state is these are We the model in it is that three is the number of states that to the model the additional states could which not have been NHE3, the behavior of NHE1 be by the two-state this not the existence of three states for this The rapid of NHE1 to an acute for NHE1, the between inactive states at be to the because of the differential of this isoform to cell has that NHE1 in multiple states of activation J. M. C. EMBO PubMed Scopus Google Scholar). not with be required to the number of states of The model for NHE3 the existence of a of inactive exchangers at physiological pH. this it a to the the of a rate-limiting transition between and an additional that be for regulation by parameters other pH. Accordingly, we that the stimulatory effects of hypoosmolar medium are best explained by an of the conversion from to A. a different osmolarity in regulate NHE3 in a distinct from pH. hypoosmolar stress the conversion of to that of exchangers from to the apical membrane is not the activity result from the activity of a number of at the apical membrane. The association of NHE3 with the cytoskeleton not to be by to the that the the of NHE3 are in was for to hypoosmolar but it is not this to the in activity Similarly, of a at by is not likely to explain the of In contrast, membrane curvature to regulate the transition between and A. that positive (convex) deformation of the membrane the NHE3 activation the effects of cell a of hypoosmolar in membrane curvature the conformation state of NHE3, which could in its activity, as suggested J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). deformation of the apical membrane the state of association of NHE3 with other membrane such as F. R. J. C. Moe O.W. J. Biol. Chem. 279: Full Text Full Text PDF PubMed Scopus Google D. Moe O.W. D.W. Proc. Natl. Acad. Sci. U. S. A. 101: PubMed Scopus Google that are of the found for NHE3, curvature induced by were to the activity of NHE1 D. Moe O.W. D.W. Proc. Natl. Acad. Sci. U. S. A. 101: PubMed Scopus Google Scholar). and other that the effects of cell inhibited NHE1, an effect to that NHE1 and NHE3 are in to in to induced volume the is activated by cell shrinkage, whereas the latter is inhibited (12Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar). a the of NHE3 at membrane curvature and be by whereas is by deformation and the be for One in that such as and effects other deformation of the membrane. NHE activity by association with of the function of NHE in these in the activation of NHE3 by is not to the to those with hypoosmolar volume (13Good D.W. Di Mari J.F. Watts 3rd, B.A. Am. J. Physiol. 2000; 279: C1443-C1454Crossref PubMed Google Scholar, 14Watts 3rd, B.A. Good D.W. J. Clin. Investig. 1999; 104: 1593-1602Crossref PubMed Scopus (52) Google Scholar). that NHE3 S. P. Musch M.W. Chang E.B. J.R. Proc. Natl. Acad. Sci. U. S. A. 101: PubMed Scopus Google Scholar, J.R. Am. J. Physiol. 2001; PubMed Google Scholar). The of this activation is not the rapid of and water the to We that the for this activation of NHE3 is a in the membrane curvature induced by cell swelling. with

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