The membrane topology of the human Na ؉ /H ؉ exchanger isoform 1 (NHE1) was assessed by substituted cysteine accessibility analysis. Eighty-three cysteine residues were individually introduced into a functional cysteineless NHE1, and these mutants were expressed in the exchanger-deficient PS120 cells. The topological disposition of introduced cysteines was determined by labeling with a biotinylated maleimide in the presence or absence of preincubation with the membrane-impermeable sulfhydryl reagent, 2-trimethylammoniumethylmethanethiosulfonate in streptolysin O-permeabilized or nonpermeabilized cells. We proposed a new model for the topology of NHE1 that is significantly different from the model derived from hydropathy analysis. In this model, NHE1 is composed of 12 transmembrane segments (TMs) with the N and C termini located in the cytosol. The large, last extracellular loop in the membrane domain of the original model was suggested to comprise an intracellular loop, a new transmembrane segment (TM11), and an extracellular loop in the new model. Interestingly, cysteines at 183 and 184 and at 324 and 325 mapped to intracellular loops connecting TMs 4 and 5 (IL2) and TMs 8 and 9 (IL4), respectively, were accessible to sulfhydryl reagents from the outside. Furthermore, exchange activities of two mutants, R180C and Q181C, within IL2 were markedly inhibited by external MTSET. These data suggest that part of IL2 or IL4 may be located in a pore-lining region that is accessible from either side of the membrane and involved in ion transport.

The Na ؉ /H ؉ exchangers (NHEs) comprise a family of transporters that catalyze cell functions such as regulation of the pH and volume of a cell and epithelial absorption of Na ؉ and bicarbonate. Ubiquitous calcineurin B homologous protein (CHP or p22) is co-localized and co-immunoprecipitated with expressed NHE1, NHE2, or NHE3 independently of its myristoylation and Ca 2؉ binding, and its binding site was identified as the juxtamembrane region within the carboxyl-terminal cytoplasmic domain of exchangers. CHP binding-defective mutations of NHE1-3 or CHP depletion by injection of the competitive CHP-binding region of NHE1 into Xenopus oocytes resulted in a dramatic reduction (>90%) in the Na ؉ /H ؉ exchange activity. The data suggest that CHP serves as an essential cofactor, which supports the physiological activity of NHE family members.The Na ϩ /H ϩ exchanger (NHE) 1 is an electroneutral plasma membrane transporter that catalyzes H ϩ -extrusion coupled to Na ϩ -influx (1, 2). Six identified NHE isoforms exhibit different tissue expression patterns (1-4): NHE1, in all tissues; NHE2-4, mostly in epithelial cells; NHE5, in brain; and NHE6, in mitochondria. These isoforms seem to have distinctive properties despite their overall structural similarity. For example, NHE1 and NHE3, which have been the most intensively studied isoforms, are involved in regulation of intracellular pH and cell volume in all cell types and Na ϩ and bicarbonate absorption in the epithelial cells, respectively. These two isoforms exhibit very different modes of regulation by many physiological factors as well as a large difference in the sensitivity to inhibitors such as amiloride derivatives (1, 2). Furthermore, targeted gene disruption of NHE1, NHE2, or NHE3 produces remarkably different phenotypes in mice; epilepsy and seizure for NHE1, reduced acid secretion in stomach for NHE2, and reduced salt absorption in kidney and low blood pressure for NHE3, respectively (5-7). The functional diversity may suggest that NHE isoforms have fundamental differences in the regulatory mechanism.All NHE molecules comprise two major domains, aminoterminal transmembrane (ϳ500 amino acids) and carboxylterminal cytoplasmic domains (ϳ300 amino acids). The latter has been suggested to function as a regulatory domain involving multiple accessory factors (1, 2). For example, calmodulin (8, 9) and the NHE3 regulatory factor (10) have been suggested to regulate NHE1 and NHE3 by interacting with their cytoplasmic domains, respectively. Five years ago, Lin and Barber (11) identified a novel Ca 2ϩ -binding protein CHP that interacts with NHE1 and exerts regulatory influences on its activity. CHP is ubiquitously expressed and homologous to the calcineurin B subunit (11). This same protein has been identified independently as a factor (called p22) required for the vesicular transport of proteins (12). More recently, this protein has been reported to inhibit the calcineurin phosphatase activity (13) or to associate with microtubules (14). Lin and Barber (11) reported t...

We studied the effect of point mutation within the putative 11th transmembrane domain (TM11) of the Na + /H + exchanger NHE1 on the plasma membrane expression. Of the 19 mutants tested, two mutants (Tyr454 or Arg458 replaced by Cys) were retained in the endoplasmic reticulum. Interestingly, Y454C was expressed on the cell surface when one of the endogenous cysteine residues at position 8, 133, 421, or 477 was substituted with alanine. Random mutagenesis at Cys8 and its surrounding residues in the cytosolic N-tail revealed that replacement of Cys8 with Ala was the only identified single residue mutation that rescued Y454C. These results suggest that the abnormal conformation of the region of TM11 containing the Y454C mutation is compensated by the second mutation within other domains such as the N-tail. This approach may provide evidence for the interdomain interaction in NHE1. ß

We studied hyperosmolarity-induced changes in cell volume and cytoplasmic pH in PS120 cells expressing Na(+)/H(+) exchanger (NHE) isoforms and their mutants. Change in cell volume was estimated by measuring change in cell height by means of confocal microscopy. Regulatory volume increase (RVI) and cytoplasmic alkalinization were observed in cells expressing NHE1 but not in cells expressing NHE2 or NHE3. Studies using chimeric exchangers revealed that the membrane domain of the exchanger is responsible for the difference in volume sensitivity between NHE1 and NHE2. Although deletion or point mutation within the first extracellular loop of NHE1 did not affect RVI and alkalinization, point mutations within the corresponding region of NHE2, particularly a region containing aa 41-53, as well as replacement of the N-terminus of NHE2 with the corresponding region of NHE1, rendered NHE2 responsive to the activating effect of cell shrinkage. Thus, the membrane domain plays an important role in the response of the exchanger to cell shrinkage. The data suggest that the putative first extracellular loop of NHE2, but not that of NHE1, may exert an inhibitory influence on hyperosmolarity-induced activation of the exchanger and thereby block RVI.

In response to Clostera anachoreta larvae attack, poplar (Populus simonii 9 P. pyramidalis 'Opera 8277') leaves produced a high level of hydrogen peroxide (H 2 O 2 ). Histochemical localization revealed that H 2 O 2 was mainly localized in herbivore-wounded zones and might spread through the veins. The activities of three H 2 O 2 -scavenging enzymes, i.e., peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT), were also enhanced in herbivore-wounded leaves, and exhibited an opposite pattern to the accumulation of H 2 O 2 . It was found that diphenylene iodonium chloride (DPI, a special inhibitor of NADPH oxidase) treatment significantly inhibited the accumulation of H 2 O 2 induced by herbivory damage. Moreover, DPI treatment led to an obvious decrease in the activities of POD, APX, and CAT. The results indicated that NADPH oxidase contributed to the accumulation of H 2 O 2 and the increase in activities of H 2 O 2 -scavenging enzymes in poplar leaves induced by herbivory damage. The balance between H 2 O 2 -production pathway and H 2 O 2 -scavenging enzymes led to the tolerable level of H 2 O 2 acting in P. simonii 9 P. pyramidalis 'Opera 8277' cuttings in response to herbivory damage.