Structural and functional analysis of extracellular loop 4 of the Nhe1 isoform of the Na(+)/H(+) exchanger

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein. It regulates intracellular pH by removing a single intracellular H(+) in exchange for one extracellular Na(+). The membrane domain of NHE1 comprises the 500 N-terminal amino acids and is made of 12 transmembrane segments. The extracellular loops of the transmembrane segments are thought to be involved in cation coordination and inhibitor sensitivity. We have characterized the structure and function of amino acids 278-291 representing extracellular loop 4. When mutated to Cys, residues F277, F280, N282 and E284 of EL4 were sensitive to mutation and reaction with MTSET inhibiting NHE1 activity. In addition they were found to be accessible to extracellular applied MTSET. A peptide of the amino acids of EL4 was mostly unstructured suggesting that it does not provide a rigid structured link between TM VII and TM VIII. Our results suggest that EL4 makes an extension upward from TM VII to make up part of the mouth of the NHE1 protein and is involved in cation selectivity or coordination. EL4 provides a flexible link to TM VIII which may either allow movement of TM VII or allow TM VIII to not be adjacent to TM VII.     

Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchanger

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.     

Structural and functional characterization of transmembrane segment IX of the NHE1 isoform of the Na+/H+ exchanger

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by removing one intracellular H(+) in exchange for one extracellular Na(+). It has a large N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the putative transmembrane segment IX (residues 339-363). Each residue was mutated to cysteine in a functional cysteineless NHE1 protein. Of 25 amino acids mutated, 5 were inactive or nearly so after mutation to cysteine. Several of these showed aberrant targeting to the plasma membrane and reduced expression of the intact protein, whereas others were expressed and targeted correctly but had defective NHE1 function. Of the active mutants, Glu(346) and Ser(351) were inhibited >70% by positively charged [2-(trimethylammonium)-ethyl]methanethiosulfonate but not by anionic [2-sulfonatoethyl]methanethiosulfonate, suggesting that they are pore lining and make up part of the cation conduction pathway. Both mutants also had decreased affinity for Na(+) and decreased activation by intracellular protons. The structure of a peptide representing amino acids 338-365 was determined by using high resolution NMR in dodecylphosphocholine micelles. The structure contained two helical regions (amino acids Met(340)-Ser(344) and Ile(353)-Ser(359)) kinked with a large bend angle around a pivot point at amino acid Ser(351). The results suggest that transmembrane IX is critical with pore-lining residues and a kink at the functionally important residue Ser(351).     

Topogenic properties of transmembrane segments of Arabidopsis thaliana NHX1 reveal a common topology model of the Na+/H+ exchanger family

The membrane topology of the Arabidopsis thaliana Na(+)/H(+) exchanger isoform 1 (AtNHX1) was investigated by examining the topogenic function of transmembrane (TM) segments using a cell-free system. Even though the signal peptide found in the human Na(+)/H(+) exchanger (NHE) family is missing, the N-terminal hydrophobic segment was efficiently inserted into the membrane and had an N-terminus lumen topology depending on the next TM segment. The two N-terminal TM segments had the same topology as those of TM2 and TM3 of human NHE1. In contrast, TM2 and TM3 of human NHE1 did not acquire the correct topology when the signal peptide (denoted as TM1) was deleted. Furthermore, there were three hydrophobic segments with the same topogenic properties as the TM9-H10-TM10 segments of human NHE1, which has one lumenal loop (H10) and two flanking TM segments (TM9 and TM10). These data indicate that the plant NHX isoforms can form the common membrane topology proposed for the human NHE family, even though it does not have a signal peptide.     

Structural and functional characterization of transmembrane segment IV of the NHE1 isoform of the Na+/H+ exchanger

The Na(+)/H(+) exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals. We characterized the structural and functional aspects of the critical transmembrane (TM) segment IV. Each residue was mutated to cysteine in cysteine-less NHE1. TM IV was exquisitely sensitive to mutation with 10 of 23 mutations causing greatly reduced expression and/or activity. The Phe(161) --> Cys mutant was inhibited by treatment with the water-soluble sulfhydryl-reactive compounds [2-(trimethylammonium)ethyl]methanethiosulfonate and [2-sulfonatoethyl]methanethiosulfonate, suggesting it is a pore-lining residue. The structure of purified TM IV peptide was determined using high resolution NMR in a CD(3)OH:CDCl(3):H(2)O mixture and in Me(2)SO. In CD(3)OH: CDCl(3):H(2)O, TM IV was structured but not as a canonical alpha-helix. Residues Asp(159)-Leu(162) were a series of beta-turns; residues Leu(165)-Pro(168) showed an extended structure, and residues Ile(169)-Phe(176) were helical in character. These three structured regions rotated quite freely with respect to the others. In Me(2)SO, the structure was much less defined. Our results demonstrate that TM IV is an unusually structured transmembrane segment that is exquisitely sensitive to mutagenesis and that Phe(161) is a pore-lining residue.     

Mutations of Arg440 and Gly455/Gly456 oppositely change pH sensing of Na+/H+ exchanger 1

To identify important amino acid residues involved in intracellular pH (pH(i)) sensing of Na(+)/H(+) exchanger 1, we produced single-residue substitution mutants in the region of the exchanger encompassing the putative 11th transmembrane segment (TM11) and its adjacent intracellular (intracellular loop (IL) 5) and extracellular loops (extracellular loop 6). Substitution of Arg(440) in IL5 with other residues except positively charged Lys caused a large shift in pH(i) dependence of (22)Na(+) uptake to an acidic side, whereas substitution of Gly(455) or Gly(456) within the highly conserved glycine-rich sequence of TM11 shifted pH(i) dependence to an alkaline side. The observed alkaline shift was larger with substitution of Gly(455) with residues with increasing sizes, suggesting the involvement of the steric effect. Interestingly, mutation of Arg(440) (R440D) abolished the ATP depletion-induced acidic shift in pH(i) dependence of (22)Na(+) uptake as well as the cytoplasmic alkalinization induced by various extracellular stimuli, whereas with that of Gly(455) (G455Q) these functions were preserved. These mutant exchangers did not alter apparent affinities for extracellular transport substrates Na(+) and H(+) and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride. These results suggest that positive charge at Arg(440) is required for normal pH(i) sensing, whereas mutation-induced perturbation of the TM11 structure may be involved in the effects of Gly mutations. Thus, both Arg(440) in IL5 and Gly residues in the conserved segment of TM11 appear to constitute important elements for proper functioning of the putative "pH(i) sensor" of Na(+)/H(+) exchanger 1.     

Mitogen-activated protein kinase-dependent activation of the Na+/H+ exchanger is mediated through phosphorylation of amino acids Ser770 and Ser771

We investigated regulation of the type 1 isoform of the Na(+)/H(+) exchanger by phosphorylation. Four specific groups of serine and threonine residues in the regulatory carboxyl-terminal tail were mutated to alanine residues: group 1, S693A; group 2, T718A and S723A/S726A/S729A; group 3, S766A/S770A/S771A; and group 4, T779A and S785A. The proteins were expressed in Na(+)/H(+) exchanger-deficient cells, and the activity was characterized. All of the mutants had proper expression, localization, and normal basal activity relative to wild type NHE1. Sustained intracellular acidosis was used to activate NHE1 via an ERK-dependent pathway that could be blocked with the MEK inhibitor U0126. Immunoprecipitation of (32)P-labeled Na(+)/H(+) exchanger from intact cells showed that sustained intracellular acidosis increased Na(+)/H(+) exchanger phosphorylation in vivo. This was blocked by U0126. The Na(+)/H(+) exchanger activity of mutants 1 and 2 was stimulated similar to wild type Na(+)/H(+) exchanger. Mutant 4 showed a partially reduced level of activation. However, mutant 3 was not stimulated by sustained intracellular acidosis, and loss of stimulation of activity correlated to a loss of sustained acidosis-mediated phosphorylation in vivo. Mutation of the individual amino acids within mutant 3, Ser(766), Ser(770), and Ser(771), showed that Ser(770) and Ser(771) are responsible for mediating increases in NHE1 activity through sustained acidosis. Both intact Ser(770) and Ser(771) were required for sustained acidosis-mediated activation of NHE1. Our results suggest that amino acids Ser(770) and Ser(771) mediate ERK-dependent activation of the Na(+)/H(+) exchanger in vivo.     

Membrane surface charge dictates the structure and function of the epithelial Na+/H+ exchanger

The Na(+)/H(+) exchanger NHE3 plays a central role in intravascular volume and acid-base homeostasis. Ion exchange activity is conferred by its transmembrane domain, while regulation of the rate of transport by a variety of stimuli is dependent on its cytosolic C-terminal region. Liposome- and cell-based assays employing synthetic or recombinant segments of the cytosolic tail demonstrated preferential association with anionic membranes, which was abrogated by perturbations that interfere with electrostatic interactions. Resonance energy transfer measurements indicated that segments of the C-terminal domain approach the bilayer. In intact cells, neutralization of basic residues in the cytosolic tail by mutagenesis or disruption of electrostatic interactions inhibited Na(+)/H(+) exchange activity. An electrostatic switch model is proposed to account for multiple aspects of the regulation of NHE3 activity.     

Functional analysis of acidic amino acids in the cytosolic tail of the Na+/H+ exchanger

The mammalian Na(+)/H(+) exchanger is a membrane protein with a C-terminal regulatory cytosolic domain and an N-terminal membrane domain. Na(+)/H(+) exchanger isoform 1 (NHE1) possesses a conserved amino acid sequence of seven consecutive acidic residues in the distal region of the cytosolic tail. We examined the structural and functional role of this acidic sequence. In human NHE1, varying mutations of the sequence (753)EEDEDDD(759) resulted in defective NHE1 activity. Mutation of the core acid sequence, (755)DED(757), or of the entire sequence caused a decrease in the activity of NHE1 in response to acute acid load. This was not due to changes in Na(+) affinity but rather due to decreased maximum velocity of the protein and delayed activation. Mutation of the target sequence did not affect the ability of the cytoplasmic domain to bind carbonic anhydrase II or tescalcin but did affect calmodulin binding. Mutation of the acidic domain also caused altered sensitivity to trypsin and changes in size of the protein in gel-filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results demonstrate that the acidic sequence is critical in maintaining proper conformation of the cytosolic domain, calmodulin binding, and in maintenance of Na(+)/H(+) exchanger activity.     

The yeast Na+/H+ exchanger Nhx1 is an N-linked glycoprotein. Topological implications

Nhx1, the endosomal Na(+)/H(+) exchanger of Saccharomyces cerevisiae represents the founding member of a newly emerging subfamily of intracellular Na(+)/H(+) exchangers. These proteins share significantly greater sequence homology to one another than to members of the mammalian Na(+)/H(+) exchanger (NHE) family encoding plasma membrane Na(+)/H(+) exchangers. Members of both subtypes are predicted to share a common organization, with an N-terminal transporter domain of transmembrane helices followed by a C-terminal hydrophilic tail. In the present study, we show that Nhx1 is an asparagine-linked glycoprotein and that the sites of glycosylation map to two residues within the C-terminal stretch of the polypeptide. This is the first evidence, to date, for glycosylation of the C-terminal region of any known NHE isoform. Importantly, the mapping of N-linked glycosylation to the C-terminal domain of Nhx1 is indicative of an unexpected membrane topology, particularly with regard to the orientation of the tail region. Although one recent study demonstrated that certain epitopes in the C-terminal domain of NHE3 were accessible from the exoplasmic side of the plasma membrane (Biemesderfer, D., DeGray, B., and Aronson, P. S. (1998) J. Biol. Chem. 273, 12391-12396), numerous other studies implicate a cytosolic disposition for the hydrophilic C-terminal tail of plasma membrane NHE isoforms. Our analysis of the glycosylation of Nhx1 is strongly indicative of residence of at least some portion of the hydrophilic tail domain within the endosomal lumen. These findings imply that the organization of the tail domain may be more complex than previously assumed.     

Structure and function of the NHE1 isoform of the Na+/H+ exchanger.

The Na+/H+ exchanger is a ubiquitous, integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion. There are seven known isoforms of this protein that are the products of distinct genes. The first isoform discovered (NHE1) is ubiquitously distributed throughout the plasma membrane of virtually all tissues. It plays many different physiological roles in mammals, including important functions in regulation of intracellular pH, in heart disease, and in cytoskeletal organization. The first 500 amino acids of the protein are believed to consist of 12 transmembrane helices, a membrane-associated segment, and two reentrant loops. A C-terminal regulatory domain of approximately 315 amino acids regulates the protein and mediates cytoskeletal interactions. Studies are underway to determine the amino acid residues important in NHE1 function. At present, it is clear that transmembrane segment IV is important in NHE1 function and that transmembrane segments VII and IX are also involved in transport. Further experiments are required to elucidate the mechanism of transport and regulation of this multifunctional protein.     

Cloning and characterization of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene of the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus aidingensis</Emphasis> AD-6<Superscript>T</Superscript>

A gene encoding a Na(+)/H(+) antiporter was obtained from the genome of Halobacillus aidingensis AD-6(T), which was sequenced and designated as nhaH. The deduced amino acid sequence of the gene was 91% identical to the NhaH of H. dabanensis, and shared 54% identity with the NhaG of Bacillus subtilis. The cloned gene enable the Escherichia coli KNabc cell, which lack all of the major Na(+)/H(+) antiporters, to grow in medium containing 0.2 M NaCl or 10 mM LiCl. The nhaH gene was predicted to encode a 43.5 kDa protein (403 amino acid residues) with 11 putative transmembrane regions. Everted membrane vesicles prepared from E. coli KNabc cells carrying NhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with the highest activity at pH 8.0, and no K(+)/H(+) antiporter activity was detected. The deletion of hydrophilic C-terminal amino acid residues showed that the short C-terminal tail was vital for Na(+)/H(+) antiporter activity.     

Intracellular trafficking of FXYD1 (phospholemman) and FXYD7 proteins in Xenopus oocytes and mammalian cells

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.     

NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell

The NHE6 protein is a unique Na(+)/H(+) exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.     

Structural and functional analysis of critical amino acids in TMVI of the NHE1 isoform of the Na+/H+ exchanger

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) resides on the plasma membrane and exchanges one intracellular H(+) for one extracellular Na(+). It maintains intracellular pH and regulates cell volume, and cell functions including growth and cell differentiation. Previous structural and functional studies on TMVI revealed several amino acids that are potentially pore lining. We examined these and other critical residues by site-directed mutagenesis substituting Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp, Gln; Glu248→Asp, Gln. Mutant NHE1 proteins were characterized in AP-1 cells, which do not express endogenous NHE1. All the TMVI critical amino acids were highly sensitive to substitution and changes often lead to a dysfunctional protein. Mutations of Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp; Glu248→Gln yielded significant reduction in NHE1 activity. Mutants of Asn227 demonstrated defects in protein expression, targeting and activity. Substituting Asn227→Arg and Ile233→Ala decreased the surface localization and expression of NHE1 respectively. The pore lining amino acids Ile233 and Leu243 were both essential for activity. Glu247 was not essential, but the size of the residue at this location was important while the charge on residue Glu248 was more critical to NHE1 function. Limited trypsin digestion on Leu243→Ala and Glu248→Gln revealed that they had increased susceptibility to proteolytic attack, indicating an alteration in protein conformation. Modeling of TMVI with TMXI suggests that these TM segments form part of the critical fold of NHE1 with Ile233 and Leu465 of TMXI forming a critical part of the extracellular facing ion conductance pathway.     

The Na+/H+ exchanger cytoplasmic tail: structure,function, and interactions with tescalcin

We characterized the regulatory cytoplasmic tail of the Na(+)/H(+) exchanger using a histidine-tagged protein containing the C-terminal 182 amino acids (His182). Both tescalcin and calmodulin, two Na(+)/H(+) exchanger binding proteins, bound to the His182 protein. Cascade blue was used to label the His182 protein. Calcium caused an increase in fluorescence, suggesting exposure of the label on the protein to a more hydrophilic environment. Decreasing external pH caused a transient increase in cascade blue fluorescence, followed by a decrease in fluorescence of the cascade blue labeled Na(+)/H(+) exchanger C-terminus. Tescalcin caused a decrease in fluorescence by labeled His182 protein, and calcium reversed this effect. Expression of tescalcin in vivo inhibited activity of the Na(+)/H(+) exchanger when there was an intact C-terminus of the protein. We examined the CD spectra of His182 in the presence and absence of tescalcin. The C-terminal amino acids demonstrated a very small amount of alpha-helical structure and much more beta-sheet and beta-turn. This was not greatly affected by the presence of tescalcin, but calcium caused an increase in the amount of beta-structure and a decrease in the unstructured proportion of the protein. Sedimentation equilibrium analysis demonstrated that the C-terminal 182 amino acids exist predominantly as a monomer. The results suggest that the C-terminus of the Na(+)/H(+) exchanger exists primarily as a monomeric protein that binds regulatory tescalcin and can change conformation depending on pH and calcium. Conformation changes in this region of the protein may be responsible for altering the pH sensitivity of the intact Na(+)/H(+) exchanger.     

Structural and functional analysis of extracellular loop 4 of the Nhe1 isoform of the Na+/H+ exchanger

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein. It regulates intracellular pH by removing a single intracellular H⁺ in exchange for one extracellular Na⁺. The membrane domain of NHE1 comprises the 500 N-terminal amino acids and is made of 12 transmembrane segments. The extracellular loops of the transmembrane segments are thought to be involved in cation coordination and inhibitor sensitivity. We have characterized the structure and function of amino acids 278–291 representing extracellular loop 4. When mutated to Cys, residues F277, F280, N282 and E284 of EL4 were sensitive to mutation and reaction with MTSET inhibiting NHE1 activity. In addition they were found to be accessible to extracellular applied MTSET. A peptide of the amino acids of EL4 was mostly unstructured suggesting that it does not provide a rigid structured link between TM VII and TM VIII. Our results suggest that EL4 makes an extension upward from TM VII to make up part of the mouth of the NHE1 protein and is involved in cation selectivity or coordination. EL4 provides a flexible link to TM VIII which may either allow movement of TM VII or allow TM VIII to not be adjacent to TM VII.     

Functional characterization of a NapA Na(+)/H(+) antiporter from Thermus thermophilus

Na(+)/H(+) antiporters are ubiquitous membrane proteins and play an important role in cell homeostasis. We amplified a gene encoding a member of the monovalent cation:proton antiporter-2 (CPA2) family (TC 2.A.37) from the Thermus thermophilus genome and expressed it in Escherichia coli. The gene product was identified as a member of the NapA subfamily and was found to be an active Na(+)(Li(+))/H(+) antiporter as it conferred resistance to the Na(+) and Li(+) sensitive strain E. coli EP432 (DeltanhaA, DeltanhaB) upon exposure to high concentration of these salts in the growth medium. Fluorescence measurements using the pH sensitive dye 9-amino-6-chloro-2-methoxyacridine in everted membrane vesicles of complemented E. coli EP432 showed high Li(+)/H(+) exchange activity at pH 6, but marginal Na(+)/H(+) antiport activity. Towards more alkaline conditions, Na(+)/H(+) exchange activity increased to a relative maximum at pH 8, where by contrast the Li(+)/H(+) exchange activity reached its relative minimum. Substitution of conserved residues D156 and D157 (located in the putative transmembrane helix 6) with Ala resulted in the complete loss of Na(+)/H(+) activity. Mutation of K305 (putative transmembrane helix 10) to Ala resulted in a compromised phenotype characterized by an increase in apparent K(m) for Na(+) (36 vs. 7.6 mM for the wildtype) and Li(+) (17 vs. 0.22 mM), In summary, the Na(+)/H(+) antiport activity profile of the NapA type transporter of T. thermophilus resembles that of NhaA from E. coli, whereas in contrast to NhaA the T. thermophilus NapA antiporter is characterized by high Li(+)/H(+) antiport activity at acidic pH.     

Functional analysis of polar amino-acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na+/H+ exchanger.

The NHE1 isoform of the Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells. Site-specific mutagenesis was used to examine the functional role of conserved, polar amino-acid residues occurring in segments of the protein associated with the membrane. Seventeen mutant proteins were assessed by characterization of intracellular pH changes in stably transfected cells that lacked an endogenous Na+/H+ exchanger. All of the mutant proteins were targeted correctly to the plasma membrane and were expressed at similar levels. Amino-acid residues Glu262 and Asp267 were critical to Na+/H+ exchanger activity while mutation of Glu391 resulted in only a partial reduction in activity. The Glu262-->Gln mutant was expressed partially as a deglycosylated protein with increased sensitivity to trypsin treatment in presence of Na+. Substitution of mutated Glu262, Asp267 and Glu391 with alternative acidic residues restored Na+/H+ exchanger activity. The Glu262-->Asp mutant had a decreased affinity for Li+, but its activity for Na+ and H+ ions was unaffected. The results support the hypothesis that side-chain oxygen atoms in a few, critically placed amino acids are important in Na+/H+ exchanger activity and the acidic amino-acid residues at positions 262, 267 and 391 are good candidates for being involved in Na+ coordination by the protein.     

A novel carbonic anhydrase II binding site regulates NHE1 activity

Carbonic anhydrase II (CAII) binds to and regulates transport by the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. We localized and characterized the CAII binding region on the C-terminal tail of the Na(+)/H(+) exchanger. CAII did not bind to acidic sequences in NHE1 that were similar to the CAII binding site of bicarbonate transporters. Instead, by expressing a variety of fusion proteins of the C-terminal region of the Na(+)/H(+) exchanger, we demonstrated that CAII binds to the penultimate group of 13 amino acids of the cytoplasmic tail. Within this region, site-specific mutagenesis demonstrated that amino acids S796 and D797 form part of a novel CAII binding site. Phosphorylation of the C-terminal 26 amino acids by heart cell extracts did not alter CAII binding to this region, but phosphorylation greatly increased CAII binding to a protein containing the C-terminal 182 amino acids of NHE1. This suggested that an upstream region of the cytoplasmic tail acts as an inhibitor of CAII binding to the penultimate group of 13 amino acids. The results demonstrate that a novel phosphorylation-regulated CAII binding site exists in distal amino acids of the NHE1 tail.