Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.

[Cloning and characterization of Na+ / H+ antiporter gene (nhaA) from Pseudomonas sp. cn4902].

According to the sequences of the gene nhaA coding for Na+ / H+ antiporter,a structural gene was cloned from Pseudomonas sp. cn4902 by PCR reaction with a set of primers. It was 1089 bp in length and codes for 362 amino acids sharing homology with the gene nhaA of E. coli K12 as high as 97.0%. It was inserted into plasmid pBV220 to form a high level expression reconstruction plasmid pBVA. So an overexpression 41 kD protein band could be found in the lane of transformant harbored with pBVA after SDS-PAGE electrophoresis. The detection of growth curve showed that the biomass of the transformant was 2.3 times over that of the control in the medium containing 1.0 mol/L NaCl. It was found that Na+ concentration in cytoplasm of the transformant was low to 60.4% of the control by the detection of atomic absorption spectrum. Evidence of SDS-PAGE electrophoresis of membrane proteins also showed that the NhaA was located in membrane. Purified NhaA was harvested and digested by FXa proteinase. The sequence of eight amino acids in N termination of NhaA protein was entirely identical with the polypeptide deduced from the nhaA gene. Then ten strains of transformant were continuously cultivated for 18 generations under 42 degrees C hot shock condition,all of their reconstructed plasmids were lost with the result that salt-tolerant-level went back to the original standard. In summary, all the experiments proved that the cloned gene is nhaA gene. The gene has been accepted in GenBank by the accession number AY643494.

The cotton GhNHX1 gene encoding a novel putative tonoplast Na(+)/H(+) antiporter plays an important role in salt stress.

A cDNA clone was isolated from cotton (Gossypium hirsutum) cDNA library and characterized with regard to its sequence, regulation in response to salt stress and functions in yeast mutants and transgenic tobacco plants. The clone, designated as GhNHX1, contains 2485 nucleotides with an open reading frame of 1629 nucleotides, and the deduced amino acid sequence showed high identities with other plant vacuolar-type Na(+)/H(+) antiporters. Northern blot analysis indicated that the mRNA accumulation of GhNHX1 was strongly induced by salt stress and abscisic acid in cotton seedlings. The expression of GhNHX1 in yeast Na(+)/H(+) antiporter mutant showed function complementation. The transgenic tobacco plants overexpressing GhNHX1 also had higher salt tolerance than the wild-type plants. The salt-induced mRNA level of GhNHX1 was 3 and 7 times higher in the salt-tolerant cotton cultivar ZM3 than those in the salt-sensitive cotton cultivars ZMS17 and ZMS12, respectively. Together, these results suggest that the products of the novel gene, GhNHX1, function as a tonoplast Na(+)/H(+) antiporter and play an important role in salt tolerance of cotton.

A new sperm-specific Na+/H+ exchanger required for sperm motility and fertility.

It has long been speculated that intracellular pH is a critical regulator of both invertebrate and vertebrate sperm motility, and sodium-hydrogen exchange has been suggested as a mediator of such pH(i) regulation in various instances. Two sodium-hydrogen exchangers (NHE1 and NHE5) are expressed in spermatozoa. However, elimination of the NHE1 gene fails to cause infertility, suggesting that normal sperm function is maintained in NHE1-null animals. Here, we used a functionally unbiased signal peptide trap screen to identify a novel sperm-specific NHE. The NHE contains 14 predicted transmembrane segments, including a potential voltage sensor and a consensus cyclic nucleotide-binding motif. Testis histology, sperm numbers and morphology were normal, but NHE-null males were completely infertile with severely diminished sperm motility. The addition of ammonium chloride, which elevates intracellular pH, partially rescued the motility and fertility defects. Surprisingly, cyclic AMP analogues almost completely rescued the motility and infertility phenotypes. The existence of this new sperm NHE provides an attractive contraceptive target, given its cell-specific expression and absolute requirement for fertility.

Roles of the Na,K-ATPase alpha4 isoform and the Na+/H+ exchanger in sperm motility.

The Na,K-ATPase generates electrochemical gradients that are used to drive the coupled transport of many ions and nutrients across the plasma membrane. The functional enzyme is comprised of an alpha and beta subunit and families of isoforms for both subunits exist. Recent studies in this laboratory have identified a biological role for the Na,K-ATPase alpha4 isoform in sperm motility. Here we further investigate the role of the Na,K-ATPase carrying the alpha4 isoform, showing again that ouabain eliminates sperm motility, and in addition, that nigericin, a H+/K+ ionophore, and monensin, a H+/Na+ ionophore, reinitiate motility. These data, along with the observation that the K+ ionophore valinomycin has no effect on the motility of ouabain-inhibited sperm, suggest that ouabain may change intracellular H+ levels in a manner that is incompatible with sperm motility. We have also localized NHE1 and NHE5, known regulators of intracellular H+ content, to the same region of the sperm as the Na,K-ATPase alpha4 isoform. These data highlight the important role of the Na,K-ATPase alpha4 isoform in regulating intracellular H(+) levels, and provide evidence suggesting the involvement of the Na+/H+ exchanger, which is critical for maintaining normal sperm motility.

Kinetic properties of NhaB, a Na+/H+ antiporter from Escherichia coli.

NhaB, a Na+/H+ antiporter from Escherichia coli, was overproduced, purified, and reconstituted in a functional state, demonstrating that a single polypeptide, the product of the nhaB gene, can catalyze full activity. NhaB is a minor protein that accounts for less than 0.1% of the total membrane protein. The use of proteoliposomes made possible the determination of important kinetic and pharmacological properties in the absence of passive and mediated leaks. The activity of NhaB was found to have some pH dependence; the apparent Km for Na+ changes by 10-fold from 1.55 mM at pH 8.5 to 16.66 mM at pH 7.2, while the Vmax remains constant. It was demonstrated that NhaB is electrogenic and translocates more H+ than Na+ per cycle; the rate of sodium efflux from proteoliposomes was accelerated by a membrane potential, negative inside, and NhaB activity generated a membrane potential as monitored by two techniques. The stoichiometry of NhaB was estimated by a thermodynamic method in which the magnitude of delta psi generated by NhaB was measured at various Na+ gradients. A kinetic method, in which the electrophoretic movement of 86Rb+ (in the presence of valinomycin) was monitored in parallel with measurements of NhaB-mediated 22Na+ uptake, allowed us to determine a stoichiometry of 3H+/2Na+. The significance of the existence of two antiporters with different stoichiometries, NhaA and NhaB, active in the same cell, is discussed.

NHE3: a Na+/H+ exchanger isoform of renal brush border.

Na+/H+ exchangers in the brush-border (luminal, apical) membrane of renal proximal tubules are responsible for active, transcellular reabsorption of NaHCO3 and NaCl. Although well characterized kinetically, the protein that mediates Na+/H+ exchange in the renal brush border has not been identified. Several Na+/H+ exchanger genes, including NHE1, NHE2, NHE3, and NHE4, are expressed in the kidney. To identify the NHE3 gene product and to determine its cellular and subcellular localization in the rabbit kidney, an NHE3-isoform-specific antibody was prepared. Guinea pigs were immunized with purified fusion protein containing the carboxy-terminal 40 amino acids of NHE3 (fpNHE3-C40). After affinity purification, immune sera demonstrated specific reactivity to the NHE3 sequence within the fusion protein as well as to an 80-kDa polypeptide expressed in NHE3-transfected LAP1 cells. Western blot analysis showed that anti-fpNHE3-C40 specifically reacted with an 80-kDa protein that is relatively enriched in renal brush-border membrane compared with basolateral membrane. Immunocytochemical studies confirmed that the Na+/H+ exchanger isoform NHE3 is expressed along the microvillar membrane of the brush border of proximal tubule cells in the rabbit kidney.