A pH-dependent Conformational Change of NhaA Na+/H+ Antiporter of Escherichia coli Involves Loop VIII–IX, Plays a Role in the pH Response of the Protein, and Is Maintained by the Pure Protein in Dodecyl Maltoside

Abstract

Digestion with trypsin of purified His-tagged NhaA in a solution of dodecyl maltoside yields two fragments at alkaline pH but only one fragment at acidic pH. Determination of the amino acid sequence of the N terminus of the cleavage products show that the pH-sensitive cleavage site of NhaA, both in isolated everted membrane vesicles as well as in the pure protein in detergent, is Lys-249 in loop VIII–IX, which connects transmembrane segment VIII to IX. Interestingly, the two polypeptide products of the split antiporter remain complexed and co-purify on Ni2+-NTA column. Loop VIII–IX has also been found to play a role in the pH regulation of NhaA; three mutations introduced into the loop shift the pH profile of the Na+/H+ antiporter activity as measured in everted membrane vesicles. An insertion mutation introducing Ile-Glu-Gly between residues Lys-249 and Arg-250 (K249-IEG-R250) and Cys replacement of either Val-254 (V254C) or Glu-241 (E241C) cause acidic shift of the pH profile of the antiporter by 0.5, 1, and 0.3 pH units, respectively. Interestingly, the double mutant E241C/V254C introduces a basic shift of more than 1 pH unit with respect to the single mutation V254C. Taken together these results imply the involvement of loop VIII–IX in the pH-induced conformational change, which leads to activation of NhaA at alkaline pH. Digestion with trypsin of purified His-tagged NhaA in a solution of dodecyl maltoside yields two fragments at alkaline pH but only one fragment at acidic pH. Determination of the amino acid sequence of the N terminus of the cleavage products show that the pH-sensitive cleavage site of NhaA, both in isolated everted membrane vesicles as well as in the pure protein in detergent, is Lys-249 in loop VIII–IX, which connects transmembrane segment VIII to IX. Interestingly, the two polypeptide products of the split antiporter remain complexed and co-purify on Ni2+-NTA column. Loop VIII–IX has also been found to play a role in the pH regulation of NhaA; three mutations introduced into the loop shift the pH profile of the Na+/H+ antiporter activity as measured in everted membrane vesicles. An insertion mutation introducing Ile-Glu-Gly between residues Lys-249 and Arg-250 (K249-IEG-R250) and Cys replacement of either Val-254 (V254C) or Glu-241 (E241C) cause acidic shift of the pH profile of the antiporter by 0.5, 1, and 0.3 pH units, respectively. Interestingly, the double mutant E241C/V254C introduces a basic shift of more than 1 pH unit with respect to the single mutation V254C. Taken together these results imply the involvement of loop VIII–IX in the pH-induced conformational change, which leads to activation of NhaA at alkaline pH. transmembrane domain n-dodecyl β-d-maltoside polyacrylamide gel electrophoresis N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 1,3-bis[tris(hydroxymethyl)methylamino]propane Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals and microorganisms. They are involved in cell energetics and play primary roles in the regulation of intracellular pH, cellular Na+ content, and cell volume (reviewed in Refs. 1Padan E. Schuldiner S. Bakker E. Alkali Cation Transport Systems in Procaryotes. CRC Press, Boca Raton, FL1992: 3-24Google Scholar, 2Padan E. Schuldiner S. Biochim. Biophys. Acta. 1994; 1185: 129-151Crossref PubMed Scopus (143) Google Scholar, 3Schuldiner S. Padan E. Bakker E. Alkali Cation Transport Systems in Procaryotes. CRC Press, Boca Raton, FL1992: 25-51Google Scholar, 4Padan E. Oren A. Microbiology and Biochemistry of Hypersaline Environments. CRC Press, Boca Raton, FL1998: 163-175Google Scholar). Escherichia coli has two antiporters, NhaA (5Goldberg B.G. Arbel T. Chen J. Karpel R. Mackie G.A. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2615-2619Crossref PubMed Scopus (183) Google Scholar) and NhaB (6Pinner E. Padan E. Schuldiner S. J. Biol. Chem. 1992; 267: 11064-11068Abstract Full Text PDF PubMed Google Scholar), which specifically exchange Na+ or Li+ for H+ (3Schuldiner S. Padan E. Bakker E. Alkali Cation Transport Systems in Procaryotes. CRC Press, Boca Raton, FL1992: 25-51Google Scholar). nha A is indispensable for adaptation to high salinity, for challenging Li+ toxicity, and for growth at alkaline pH (in the presence of Na+) (7Padan E. Maisler N. Taglicht D. Karpel R. Schuldiner S. J. Biol. Chem. 1989; 264: 20297-20302Abstract Full Text PDF PubMed Google Scholar). Accordingly, expression of nha A, which is dependent on NhaR, a positive regulator, is induced by Na+, in a pH-dependent manner (8Karpel R. Alon T. Glaser G. Schuldiner S. Padan E. J. Biol. Chem. 1991; 266: 21753-21759Abstract Full Text PDF PubMed Google Scholar, 9Rahav-Manor O. Carmel O. Karpel R. Taglicht D. Glaser G. Schuldiner S. Padan E. J. Biol. Chem. 1992; 267: 10433-10438Abstract Full Text PDF PubMed Google Scholar, 10Carmel O. Rahav-Manor O. Dover N. Shaanan B. Padan E. EMBO J. 1997; 16: 5922-5929Crossref PubMed Scopus (28) Google Scholar). nha B by itself confers a limited sodium tolerance to the cells, but becomes essential when the lack of NhaA activity limits growth (11Pinner E. Kotler Y. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 1729-1734Abstract Full Text PDF PubMed Google Scholar). Both the NhaA and NhaB are electrogenic antiporters that have been purified to homogeneity and reconstituted in a functional form in proteoliposomes (12Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar, 13Pinner E. Padan E. Schuldiner S. J. Biol. Chem. 1994; 269: 26274-26279Abstract Full Text PDF PubMed Google Scholar, 14Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 5382-5387Abstract Full Text PDF PubMed Google Scholar). The H+/Na+stoichiometry of NhaA is 2H+/Na+ and that of NhaB 3H+/2Na+. NhaB but not NhaA is sensitive to amiloride derivatives, and the rate of activity of NhaA but not of NhaB is drastically dependent on pH, changing its Vmax over 3 orders of magnitude from pH 7 to pH 8 (12Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar). Interestingly, a strong pH sensitivity is characteristic of antiporters as well as other transporters that are involved in pH regulation (reviewed in Ref. 4Padan E. Oren A. Microbiology and Biochemistry of Hypersaline Environments. CRC Press, Boca Raton, FL1998: 163-175Google Scholar). Identifying the amino acid residues involved in the pH sensitivity of these proteins is important for understanding the mechanism of pH regulation. NhaA contains eight histidines, none of which were found essential for the Na+/H+ antiporter activity of NhaA (15Gerchman Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar). However, replacement of histidine 225 by Arg (H225R) suggested that His-225 has an important role in the pH sensitivity of the antiporter. Whereas the activation of the wild-type NhaA occurs between pH 7.5 and pH 8, that of H225R antiporter occurs between pH 6.5 and pH 7.5. In addition, while the wild-type antiporter remains almost fully active, at least up to pH 8.5, H225R is reversibly inactivated above pH 7.5, retaining only 10–20% of the maximal activity at pH 8.5 (15Gerchman Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar). Furthermore, replacement of His-225 with either cysteine (H225C) or serine (H225S) but not alanine (H225A) yielded an antiporter with a wild-type pH-sensitive phenotype, implying that polarity and/or hydrogen bonding, the common properties shared by His, Cys, and Ser, are essential at position 225 for pH regulation of NhaA (16Rimon A. Gerchman Y. Olami Y. Schuldiner S. Padan E. J. Biol. Chem. 1995; 270: 26813-26817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Glycine 338 affects the pH response of NhaA; its replacement with serine (G338S in TMS1 XI) produced a transporter, which in contrast to the wild-type protein lacks pH control; it is active between pH 6.5 and 8.5 A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have found that NhaA a its activation by pH which by trypsin A. Gerchman Y. Padan E. Schuldiner S. 1997; PubMed Scopus Google Scholar). acidic pH the protein in everted membrane vesicles is to while at alkaline pH it is in a the pH profile of the antiporter Furthermore, two with a pH profile are to trypsin in isolated membrane vesicles only at the pH are active and the of activity A. Gerchman Y. Padan E. Schuldiner S. 1997; PubMed Scopus Google Scholar). the mutant with a pH profile acidic pH, is at the pH it is which pH is active and to trypsin the pH of NhaA has many only two fragments were of isolated membrane vesicles at alkaline pH. that only one cleavage site is by pH while the other are that the trypsin cleavage site is and as a that of the protein which a conformational in response to pH. and of site in The results show that loop VIII–IX is important for the pH regulation of NhaA and the trypsin cleavage which is involved in the pH-dependent conformational of is by the pure protein in is an E. coli which is (11Pinner E. Kotler Y. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 1729-1734Abstract Full Text PDF PubMed Google Scholar). is nha nha to (12Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar). S. and were as for of were in in which by E. Maisler N. Taglicht D. Karpel R. Schuldiner S. J. Biol. Chem. 1989; 264: 20297-20302Abstract Full Text PDF PubMed Google pH the by and pH with were also in A sodium J. PubMed Google Scholar) with either or as a to were and/or and/or and/or to Li+ and Na+ as (15Gerchman Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar). and are A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google R. Olami Y. Taglicht D. Schuldiner S. Padan E. J. Biol. Chem. Full Text PDF PubMed Google the nha A and of the nha The nha A and nha R. is a which an to that of A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is a which by E. of is a Y. Rimon A. Gerchman Y. A. Padan E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and contains NhaA at its N terminus to the for over expression and at its terminus to a sequence in two cleavage and by of by and by with and of the The nha A NhaA, of which terminus is by a 1989; PubMed Scopus Google Scholar). of as a The and the are in for of NhaA at are in The introducing site are and from the are to the The sequence in the were and to amino and for amino between and and to at are in The introducing site are and from the are to the The sequence in the were in a In the of and the with and a fragment of which either to the fragment of to or or to the fragment of to or In the of the fragment as above to an fragment and as above to In the of the between Lys-249 and Arg-250 the fragment as above to an fragment and as above into both and to or respectively. of activity were on everted membrane vesicles PubMed Scopus Google Scholar). The of activity the of Na+ in the as (5Goldberg B.G. Arbel T. Chen J. Karpel R. Mackie G.A. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2615-2619Crossref PubMed Scopus (183) Google Scholar, S. PubMed Scopus Google Scholar). membrane vesicles were as everted membrane vesicles but the cell of the NhaA and its in membranes by as (16Rimon A. Gerchman Y. Olami Y. Schuldiner S. Padan E. J. Biol. Chem. 1995; 270: 26813-26817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). the wild-type and the antiporters, the His-tagged proteins in cells were The cells were in to induced with for to (12Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar), and for of high membranes either at or at NhaA or His-tagged NhaA were on as A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). antiporter to trypsin in a of antiporter of trypsin from 8, not 1 for at The by the of of trypsin from of of protein were on A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Digestion of membrane vesicles of of in of and and for 1 at which is also for the Na+/H+ antiporter activity and also with pure proteins as The protein in and on the were for to membranes in The were in for in in for in acid in and for in The were and to in a to Ref. T. PubMed Scopus Google of by an of NhaA, have NhaA at its terminus to two cleavage and residues NhaA of Ref. Y. Rimon A. Gerchman Y. A. Padan E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of both NhaA A. Gerchman Y. Padan E. Schuldiner S. 1997; PubMed Scopus Google Scholar) and NhaA A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) in everted membrane vesicles pH which is to that of the activity of the and has been suggested to a active conformational of the antiporter. Both are at acidic pH and above pH 7 to the at pH The products of protein are two fragments in that the trypsin a of the it to trypsin to of NhaA is by the pure protein in NhaA in to by trypsin at pH and the products on 1 two protein fragments in a pH-dependent in to that by trypsin of isolated membrane vesicles A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the acidic pH a single fragment that the cleavage which the pure protein at alkaline pH, is However, when the on the single at acidic pH found to than the implying the of an cleavage the at the terminus of NhaA, to Ni2+-NTA to the terminus is NhaA with trypsin either at acidic or alkaline pH and the of the products to on Ni2+-NTA the acidic the basic pH products to the implying that at both pH the terminus together with the results were when the trypsin on everted membrane vesicles NhaA not In contrast to NhaA, NhaA in isolated membrane vesicles not to by trypsin at its terminus A. Gerchman Y. Padan E. Schuldiner S. 1997; PubMed Scopus Google the of the protein acidic not with a the terminus the protein at acidic pH and the fragment at alkaline pH. not the to of the that the trypsin cleavage site at the terminus of NhaA introduced by the as the yielded His-tagged NhaA, which is not by trypsin at the The N terminus trypsin of pure as the fragment at alkaline pH with the sequence of the protein the trypsin cleavage site that is by pH and NhaA, of the two fragments and from the trypsin of at alkaline pH were isolated from the and to The N terminus of the fragment found than to that of the The fragment a fragment with a sequence that a sequence between Arg-250 and of loop In addition, it of two with in loop VIII–IX and in loop respectively. the cleavage site of trypsin that is at alkaline pH is in Lys-249 of loop The in the pH profile of the trypsin and the of the products have suggested that the cleavage site in in everted membrane vesicles is to that of the pure protein in However, it is a to the products that the the in by from the a from which His-tagged NhaA with cleavage membrane vesicles isolated from cells protein a Na+/H+ antiporter activity and a pH profile to that of the wild-type protein not everted membrane vesicles were to trypsin both at acidic not and alkaline pH the membranes in the on Ni2+-NTA column. and the on at acidic pH, the protein purified on the in in a to that of the one of a to the results that the remains in protein and the of the The results also show that the trypsin cleavage site at the terminus of the NhaA in the cleavage alkaline pH of His-tagged NhaA to the two fragments were on the Ni2+-NTA and a fragment in to that of NhaA, and a fragment as on the of the between the two of and His-tagged In none of the from NhaA to the were purified by the is that the trypsin split in His-tagged NhaA two fragments co-purify the the fragment with and the fragment that both and fragments by the trypsin split are to other and not in that the trypsin cleavage site of His-tagged NhaA in (in the is to that of the pure the fragments from the trypsin of His-tagged NhaA membranes were isolated by and to The results show that the trypsin cleavage site that is in at alkaline pH is to that in the pure protein in Lys-249 of loop to the pure protein is a split in at loop VIII–IX its with pH, the as to loop role in the activity of NhaA or its regulation by pH. three of mutations have been introduced to loop The is a mutation amino from to mutant to a of the expression of the and not in the presence of either at pH 7 or at pH measured in isolated membrane the mutant not show Na+/H+ antiporter activity at pH 7 but at pH 8.5 a but activity with the wild-type of of the of activity by the mutant only activity to the the membranes from cells with the not show of loop NhaA B cells with the were to in The membrane by (16Rimon A. Gerchman Y. Olami Y. Schuldiner S. Padan E. J. Biol. Chem. 1995; 270: 26813-26817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), with and for the of which wild-type nha A, and other nha A are in a B cells with the were to in The membrane by (16Rimon A. Gerchman Y. Olami Y. Schuldiner S. Padan E. J. Biol. Chem. 1995; 270: 26813-26817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), with and for the of which wild-type nha A, and other nha A are A mutation by at pH 7 in the presence of The mutation and as in The only the mutation growth in the presence of Na+ only at pH but not at pH The expression of the nha A as with the mutant to mutant mutant not show antiporter activity in everted membrane vesicles at pH its activity at pH 8.5 in rate but not in as with the mutant for the growth of the double mutant at pH 7 in the presence of Na+ as with the which not Interestingly, both show pH sensitivity at pH 7 and active at pH 8.5 The of mutation an insertion amino were between Lys-249 and Arg-250 of loop VIII–IX, cleavage site insertion as as the wild-type and to specifically a single split in NhaA in loop VIII–IX with at the site as The of the fragments were as for a split in loop VIII–IX not The Na+/H+ antiporter activity in everted membrane vesicles NhaA in its maximal activity to the wild-type However, the pH profile of the activity of the mutant by a pH unit acidic pH the insertion mutation into loop VIII–IX affects the pH sensitivity of The mutation three in a these were introduced to loop VIII–IX of NhaA by and were and fully of the three growth to that of the wild-type membrane vesicles isolated from the three maximal Na+/H+ activity in magnitude to that of the B and However, in contrast with the the pH profile of the Na+/H+ activity of by 1 pH unit acidic the activity of the wild-type protein drastically with activity at pH 7 and at pH the mutant and activity at the pH and only at pH is that the mutation in loop VIII–IX has a on the pH profile of mutant also acidic shift in its pH profile the double mutant E241C/V254C a shift in the pH profile basic pH Both (V254C) and (K249-IEG-R250) proteins were on Ni2+-NTA and the purified proteins to trypsin at pH The results in show that the acid shift in the pH of the activity of the proteins is in a acidic pH shift in the pH of the by trypsin of the purified proteins in and have that the regulation by pH of NhaA, the Na+/H+ antiporter of E. is essential for the of the cells to at alkaline pH in the presence of Na+ (15Gerchman Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar, A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have also that activation of NhaA by pH is by a conformational that trypsin as a both wild-type NhaA A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Gerchman Y. Padan E. Schuldiner S. 1997; PubMed Scopus Google Scholar) and its with pH are to only at the pH it is the at pH 7 to pH 8.5 A. Gerchman Y. Padan E. Schuldiner S. 1997; PubMed Scopus Google Scholar), the mutant H225R at pH A. Gerchman Y. Padan E. Schuldiner S. 1997; PubMed Scopus Google Scholar), and the mutant at the pH between and 8.5 A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). found in the with the and the mutations an acidic shift in the pH profile both of the and the cleavage by the many trypsin cleavage of NhaA A. Gerchman Y. Padan E. Schuldiner S. 1997; PubMed Scopus Google Scholar), in these only two products are the of one trypsin cleavage which is by pH. is of the NhaA trypsin pH In the the trypsin that are by pH and found that loop VIII–IX a pH Both in the purified protein in dodecyl maltoside as well as in in membrane vesicles His-tagged NhaA, Lys-249 of loop VIII–IX found to the site that is to trypsin in a pH-dependent In both the products are two fragments of a fragment with the N terminus of NhaA and a fragment with the N terminus at Arg-250 on the site of the split and the protein in both proteins the for the fragment is while and are for the fragments from the two respectively. that the of the protein as well as that of the fragments as in are than in is a common of many membrane proteins (12Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar). In with these two NhaA fragments were by of everted membrane vesicles the protein A. Gerchman Y. Padan E. Schuldiner S. 1997; PubMed Scopus Google Scholar). However, in it to the amino acid sequence of the N of the which were not and not results the as to loop VIII–IX of NhaA is involved in the pH response of NhaA or its pH-dependent conformational is a the important conformational of loop VIII–IX that loop VIII–IX is involved in the pH response of of mutations in loop VIII–IX the pH profile of the Na+/H+ activity of the protein acidic pH the maximal activity or expression of the insertion mutation the pH profile by a pH mutations by one pH and by 0.3 pH Interestingly, in the double mutant the and the pH profile basic pH to more basic than of the pH profile of the wild-type is that of the of NhaA is for a of these as to loop VIII–IX is involved in the pH response of it is to at least to of involvement of a protein domain in the pH response of a pH domain that the pH and a that the pH to a in activity of the two The with a mutant of of loop VIII–IX that the loop VIII–IX is involved at least in the the Na+/H+ activity of both and the pH response activity at alkaline in the is suggested while the in these the activation is The other in loop VIII–IX, which shift the pH profile with other an involvement in the pH that loop VIII–IX is cytoplasmic and a which is at its by a found to the pH response of NhaA Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar, A. Gerchman Y. Olami Y. Schuldiner S. Padan E. J. Biol. Chem. 1995; 270: 26813-26817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, Y. Rimon A. Gerchman Y. A. Padan E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. T. J. 1997; PubMed Scopus Google Scholar, and A. Padan E. Schuldiner S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google other and also residues the pH response of NhaA A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google a NhaA from T. S. T. PubMed Scopus Google Scholar). of an E. coli mutant of its antiporter with nha A the of the mutant and everted membrane vesicles isolated from these Na+/H+ antiporter activity in the pH profile from that of E. coli NhaA it active at acidic and pH, is the primary and the of and were for one found in sequence has amino acid residues at position to of of and the loop Taken these results that loop VIII–IX has an important role in the pH response of the role of has not been in E. coli have that the pH regulation of the antiporter is essential for growth at alkaline pH in the presence of Na+ with two properties of the regulation the activation of the antiporter at alkaline pH (15Gerchman Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar) and its to at acidic pH A. Gerchman Y. Padan E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of these properties are the cells at alkaline pH in the presence of Furthermore, as as these properties are a shift in the pH profile basic pH by mutation a growth (16Rimon A. Gerchman Y. Olami Y. Schuldiner S. Padan E. J. Biol. Chem. 1995; 270: 26813-26817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In the show that acidic shift in the pH profile by the loop VIII–IX and not the growth of the is that the pH-dependent conformational as by trypsin is by the purified NhaA in a solution of Furthermore, the two NhaA polypeptide products complexed in and on Ni2+-NTA column. the in a Biol. PubMed Scopus Google its conformational were reversibly in results are in with results that is for and in proteoliposomes of many including NhaA (12Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar). A. from the for in the for the N terminus of the NhaA