Structural and functional analysis of transmembrane XI of the NHE1 isoform of the Na+/H+ exchanger

Abstract

The Na(+)/H(+) exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H(+) in exchange for one extracellular Na(+). We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys(447)-Lys(472)) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp(447)-Tyr(454) and Phe(460)-Lys(471) at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly(455)-Gly(459). TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na(+)/H(+) exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu(465) contributing to the pore.

FIGURE 1.

Models of the Na⁺/H⁺ exchanger. A, simplified topological model of the transmembrane domain of the NHE1 isoform of the Na⁺/H⁺ exchanger as described earlier (11). EL, extracellular loop; IL, intracellular loop. B, model of amino acids present in TM XI.

FIGURE 2.

Analysis of wild type and mutant NHE1 proteins. A, Western blot of cell extracts of AP-1 cells containing stably transfected Na⁺/H⁺ exchanger mutants or control. In all mutations the amino acid indicated was changed to cysteine. 100 μg of total protein was loaded in each lane. Numbers below the lanes indicate the values obtained from densitometric scans of both the 110- and 95-kDa bands relative to wild type NHE. Results are typical of three to five measurements. AP1 refers to mock transfected AP-1 cells. Wt, refers to cells stably transfected with wild type Na⁺/H⁺ exchanger protein. B, subcellular localization of control and TM XI mutants in AP-1 cells. Sulfo-NHS-SS-biotin-treated cells were lysed and streptavidin-agarose beads were used to bind labeled proteins as described under “Experimental Procedures.” Equal amounts of total cell lysate (T) and unbound intracellular lysate (I) were run on SDS-PAGE and Western blotting with anti-HA antibody identified NHE1 protein. Wt, refers to cells stably transfected with wild type Na⁺/H⁺ exchanger protein. Ct refers to a control experiment in which nonspecific binding to streptavidin-agarose beads was carried out following the standard procedure but without labeling cells with biotin. The percent of the total NHE1 protein found within the plasma membrane is indicated for each mutant. For Ct the numbers indicate the amount of nonspecific binding to streptavidin-agarose beads. Results are the mean ± S.E. of at least three determinations. C, rate of recovery from an acid load by AP-1 cells stably transfected with cNHE, and TM XI Na⁺/H⁺ exchanger mutants. Na⁺/H⁺ exchanger activity was measured after transient induction of an acid load as described under “Experimental Procedures.” The activity of cNHE1 stably transfected with NHE1 was 0.036 Δ pH/s, and this value was set to 100%. All mutations to cysteine were done in the background of the cysteine-less NHE1 and activities are percent of those of cNHE. Mutations were in the cNHE1 and results are expressed as mean ± S.E. of 8-16 determinations. Results are shown for mean activity of both uncorrected (black) and normalized for surface processing and expression levels (hatched). ^* indicates mutants with uncorrected activity that is less than 20% of cNHE1.

FIGURE 3.

Effect of MTSET and MTSES on activity of cNHE1 and single cysteine TM XI mutant NHE1 containing cell lines. A, summary of results of Na⁺/H⁺ exchanger assays of active TM XI mutants. Activity was measured after two pulses of transient induction of an acid load as described under “Experimental Procedures.” The first activity was in the absence of MTSES or MTSET, the second was when cells were treated with 10 mm reagent before transient acidification. Results illustrated represent the % of activity of the second acid load, in comparison to the first. ^* indicates that the second recovery from acid load was significantly lower than the first at p < 0.01, Mann-Whitney U test. Solid filled bars represent MTSES treatments and hatched bars represent MTSET treatments. B, example of results of effect of MTSET on activity of wild type and L465C mutant. Wild type (Wt) and L465C NHE1 protein activity was assayed in stably transfected AP-1 cells as described under “Experimental Procedures.” Activity was measured after two acid pulses as described in A. The first pulse in the absence of MTSET is shown. For ease of viewing, only the recovery from acidosis is shown for the second pulse, in which cells were treated with MTSET. NH₄Cl, treatment with ammonium chloride; sodium free, treatment with Na⁺ free buffer to induce acidosis; NaCl, recovery from acidosis in NaCl containing buffer, for the second pulse this contained MTSET and cells were pretreated with MTSET for 10 min prior to NH₄Cl-induced acid load.

FIGURE 4.

Characterization of site-specific mutants of Leu⁴⁶⁵. A, Western blot of cell extracts of AP-1 cells containing stably transfected Na⁺/H⁺ exchanger mutants or control. Samples were analyzed as described in the legend to Fig. 2A. Numbers below the lanes indicate the values obtained from densitometric scans of both the 110- and 95-kDa bands relative to wild type NHE. Results are typical of three to five measurements. cNHE refers to cysteine-less NHE1. A, D, and K refer to mutant cell lines containing cNHE1 with the Leu⁴⁶⁵ mutations to Ala, Asp, and Lys respectively. B, subcellular localization of TM XI mutants in AP-1 cells. Mutants contained the NHE1 Leu⁴⁶⁵ mutations to Ala, Asp, and Lys as indicated. Sulfo-NHS-SS-biotin-treated cells were lysed and streptavidin-agarose beads were used to bind labeled proteins as described under “Experimental Procedures.” Equal amounts of total cell lysate (T) and unbound intracellular lysate (I) were run on SDS-PAGE and Western blotting with anti-HA antibody identified the NHE1 protein. The percent of the total NHE1 protein found within the plasma membrane is indicated for each mutant. Results are the mean ± S.E. of at least three determinations. C, rate of recovery from an acid load by AP-1 cells stably transfected with cNHE, and TM XI Na⁺/H⁺ exchanger Leu⁴⁶⁵ mutations to Ala, Asp, and Lys. Na⁺/H⁺ exchanger activity was measured after transient induction of an acid load as described in the legend to Fig. 2C. All mutations to cysteine were done in the background of the cysteine-less NHE1 and activities are percent of those of cNHE. Mutations were in the cNHE1 and results are expressed as mean ± S.E. of 8-16 determinations. Results are shown for mean activity of both uncorrected (black) and normalized for surface processing and expression levels (hatched).

FIGURE 5.

Dihedral angle order parameters for the final 40 ensemble members. Order parameters are calculated as in Ref. . An order parameter of 1 indicates the particular angle is the same for all members in the ensemble, and is 0 when the angles are completely random.

FIGURE 6.

NMR structure of TM XI in DPC micelles. Superimposition of structurally conserved regions of the TM XI peptide structure. Superimposition of the backbone atoms of the structurally conserved regions (A) Lys⁴⁴⁷-Gly⁴⁵⁴ and (B) Gly⁴⁶⁰-Lys⁴⁷¹. The backbone is shown in black, side chains are shown in color. C, two views of a single ensemble member with MTSET-sensitive residues labeled.

FIGURE 7.

Comparison of TM XI of NHE1 with TM XI of NhaA. A, SEQSEE-based (40) alignment of the amino acid sequences of TM XI of NhaA and NHE1. B, alignment of the sequences based on optimal structural alignment of the extended regions (highlighted) of NHE1 TM XI (Gly⁴⁵⁵-Gly⁴⁵⁹) and NhaA TM XI. Colon indicates replacement by similar amino acid and asterisk indicates amino acid identity. MTSET-sensitive residues, and the corresponding residues in NhaA are shown in bold. C, comparison of the NMR structure of TM XI of NHE1 in DPC micelles with that of TM XI in the x-ray structure of NhaA. Amino acids 447-472 of NHE1 and amino acids 327-352 of NhaA are included. The ensemble member of the NMR structures chosen for comparison had the lowest backbone r.m.s. deviation compared with the corresponding region in NhaA for the superimposition of the extended regions, and with arbitrary rotation of the two helices around the flexible Gly⁴⁵⁵ and Gly⁴⁵⁹ to match the helices of NhaA.