Attentional requirements of postural control in people with spinal cord injury: the effect of dual task.

STUDY DESIGN: Cross-sectional study. OBJECTIVES: To investigate the attentional requirements for maintaining standing balance in people with spinal cord injury (SCI) using a dual-task paradigm and to compare standing balance performance between SCI and able-bodied (AB) controls. SETTING: LaboratoryMethods:Nine adults with incomplete SCI, who were able to stand unassisted were recruited, along with eight AB controls. Subjects performed a dual task involving counting backwards by 3 s out loud while standing with eyes open or closed. The primary outcome measures were the differences between SCI and control groups for movement reinvestment and the change in performance between single task and dual task for: (i) maximum standing time (STime); (ii) error ratio and total number of words uttered; and (iii) center of pressure measures. Perceptual measures included perceived mental workload, fear and confidence. RESULTS: SCI subjects stood for shorter duration during dual task (stand and count) than single task (stand) compared with controls during eyes closed. Significant differences between groups were observed for movement reinvestment, center of pressure, perceived mental effort, fear and confidence. No significant effects were observed for math-task performance. CONCLUSIONS: Total STime during eyes closed is adversely affected by the addition of a math task for SCI subjects. Perceptual measures appear to correspond to increases in postural sway and conscious control of standing in subjects with SCI. Individuals who can stand for >60 s with eyes closed do not appear to be significantly affected by the addition of a concurrent secondary task of minimal mental workload.

Nucleoside transport in human colonic epithelial cell lines: evidence for two Na+-independent transport systems in T84 and Caco-2 cells.

RT-PCR of RNA isolated from monolayers of the human colonic epithelial cell lines T84 and Caco-2 demonstrated the presence of mRNA for the two cloned Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2, but not for the cloned Na+-dependent concentrative nucleoside transporters, CNT1 and CNT2. Uptake of [3H]uridine by cell monolayers in balanced Na+-containing and Na+-free media confirmed the presence of only Na+-independent nucleoside transport mechanisms. This uptake was decreased by 70-75% in the presence of 1 microM nitrobenzylthioinosine, a concentration that completely inhibits ENT1, and was completely blocked by the addition of 10 microM dipyridamole, a concentration that inhibits both ENT1 and ENT2. These findings indicate the presence in T84 and Caco-2 cells of two functional Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2.

Passive and carrier-mediated permeation of different nucleosides through the reconstituted nucleoside transporter.

When reconstituted into proteoliposomes, the human erythrocyte nucleoside transporter catalysed nitrobenzylthioguanosine (NBTGR)-sensitive zero-trans influx of three different nucleosides at broadly similar rates (inosine, uridine greater than adenosine). However, proteoliposomes also exhibited high rates of NBTGR-insensitive uptake of adenosine, making this nucleoside unsuitable for reconstitution studies. Equivalent high rates of adenosine influx were observed in protein-free liposomes, establishing that this permeability pathway represents simple diffusion of nucleoside across the lipid bilayer. In contrast to adenosine, inosine and uridine exhibited acceptable rates of NBTGR-insensitive uptake. Of the two, inosine is the more attractive permeant for reconstitution experiments, having a 2.5-fold lower basal membrane permeability. Studies of nucleoside transport specificity in reconstituted membrane vesicles should take account of the widely different passive permeabilities of different nucleosides.

Evidence for the asymmetrical binding of p-chloromercuriphenyl sulphonate to the human erythrocyte nucleoside transporter.

Nucleosides cross the human erythrocyte membrane by a facilitated-diffusion process which is selectively inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR). The chemical asymmetry of the transporter was investigated by studying the effects of p-chloromercuriphenyl sulphonate (PCMBS) on uridine transport and high-affinity NBMPR binding in inside-out and right-side-out membrane vesicles, unsealed erythrocyte ghosts and intact cells. PCMBS was an effective inhibitor of the transporter (50% inhibition at 30 microM), but only when the organomercurial had access to the cytoplasmic membrane surface. PCMBS inhibition of NBMPR binding to ghosts was reversed by incubation with dithiothreitol. Both uridine and NBMPR were able to protect the transporter against PCMBS inhibition.

Purification and reconstitution studies of the nucleoside transporter from pig erythrocytes.

The pig erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 64,000) on the basis of photoaffinity labelling experiments with the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). This protein was purified 140-fold by treatment of haemoglobin-free erythrocytes 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with n-octyl-glucoside and subsequent gradient-elution ion-exchange chromatography on DEAE-cellulose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified material revealed the presence of only two detectable protein bands, one which co-migrated with the radiolabelled NBMPR-binding protein, and a lower molecular weight species with an Mr of 43,000. The latter protein may be a degradation product of the band 3 anion-exchange transporter. The overall purification of the NBMPR-binding protein with respect to the Mr 64,000 band was 350-fold. Reversible NBMPR-binding to the partially-purified band 4.5 preparation was saturable (apparent Kd 7.2 nM). Adjustment of the chromatography conditions to allow elution of the NBMPR-binding protein along with the majority of solubilised membrane phospholipid reduced the apparent Kd value to 3.0 nM. Purification of reversible NBMPR-binding activity during ion-exchange chromatography was paralleled by an increase in the specific activity of nitrobenzylthioguanosine (NBTGR) -sensitive uridine transport as assayed in proteoliposomes reconstituted by a freeze-thaw-sonication procedure.

Short-term regulation of NHE3 by EGF and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells and fibroblasts.

NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na(+)-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in Vmax of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)i of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number.

Glucose transport in fish erythrocytes: variable cytochalasin-B-sensitive hexose transport activity in the common eel (Anguilla japonica) and transport deficiency in the paddyfield eel (Monopterus albus) and rainbow trout (Salmo gairdneri).

Erythrocytes from individual common eels (Anguilla japonica Temminck and Schlegel) exhibited widely variable initial rates of cytochalasin-B-sensitive 3-O-methyl-D-glucose (3-OMG) zero-trans influx, in the range of 0-19.5 mmol l-cells-1 h-1 (5 mmol l-1 extracellular concentration at 20 degrees C, 50 animals tested). Storage of cells at 4 degrees C in a glucose-containing medium for up to 72 h had no effect on 3-OMG uptake, and there was no correlation between the sugar permeabilities of erythrocytes from different fish and intracellular ATP levels. Adrenaline and noradrenaline increased cytochalasin-B-sensitive 3-OMG transport activity; half-maximal stimulation occurred at catecholamine concentrations in the region of 1 mumol l-1. This catecholamine-induced stimulation of sugar transport appeared to be independent of the basal cytochalasin-B-sensitive 3-OMG permeability of the cells. Kinetically, catecholamines increased the Vm of transport without changing the apparent Km (approx. 1.4 mmol l-1). Saturable 3-OMG influx was inhibited by phloretin, D-glucose, D-deoxyglucose and D-galactose, but not by D-fructose and L-glucose. Transporter stereoselectivity was confirmed by direct measurements of D- and L-glucose uptake. Erythrocytes from two other fish species, Monopterus albus Richardson (paddyfield eel) and Salmo gairdneri Richardson (rainbow trout), unlike those from the common eel, were uniformly deficient with respect to cytochalasin-B-sensitive 3-OMG and D-glucose transport activity. Catecholamines had no effect on sugar uptake in these species.

Immunolocalisation of NHE3-like immunoreactivity in the gills of the rainbow trout (Oncorhynchus mykiss) and the blue-throated wrasse (Pseudolabrus tetrious).

Na+/H+ exchange has been implicated in models of ion transport across the branchial epithelium of marine and freshwater fishes. In this preliminary study, we present immunohistochemical data using a polyclonal antibody raised against NHE3 which show NHE3-like immunoreactivity (IR) in the gills from a freshwater and a marine teleost species. In both species, branchial epithelial cells demonstrating NHE3-like IR were localised predominantly to the junction between the filament and the secondary lamellae. However, there was a marked difference in the morphology of the NHE3-like immunoreactive epithelial cells between the species. This morphological difference between the species suggests functional differences in the exchanger, which may be related to marine versus freshwater environments.

Chimeric Na+/H+ exchangers: an epithelial membrane-bound N-terminal domain requires an epithelial cytoplasmic C-terminal domain for regulation by protein kinases.

All cloned members of the mammalian Na+/H+ exchanger gene family encode proteins that consist of two functionally distinct domains: a membrane-bound N terminus and a cytoplasmic C terminus, which are required for ion transport and regulation of transport, respectively. Despite their similarity in structure, three members of this family, designated NHE1, NHE2, and NHE3, exhibit different kinetic mechanisms in response to growth factors and protein kinases. For instance, growth factors stimulate NHE1 by a change in the affinity constant for intracellular H+, K'(Hi+), and regulate NHE2 and NHE3 by a change in Vmax. We have constructed chimeric Na+/H+ exchangers by exchanging the N and C termini among three cloned rabbit Na+/H+ exchangers (NHE1 to NHE3) to determine which domain is responsible for the above Vmax-vs.-K'(H(i)+) effect of the Na+/H+ isoforms. All of the chimeras had functional exchange activity and basal kinetic properties similar to those of wild-type exchangers. Studies with serum showed that the N terminus is responsible for the Vmax-vs.-K'(H(i)+) stimulation of the Na+/H+ exchanger isoforms. Moreover, phorbol 12-myristate 13-acetate and fibroblast growth factor altered Na+/H+ exchange only in chimeras that had an epithelial N-terminal domain matched with an epithelial C-terminal domain. Therefore, the protein kinase-induced regulation of Na+/H+ exchangers is mediated through a specific interaction between the N- and C-termini, whcih is restricted so that epithelial N- and epithelial N-and C-terminal portions of the exchangers are required for regulation.

Structure/function studies of mammalian Na-H exchangers--an update.

Four mammalian Na+/H+ exchangers have recently been cloned. Despite the structural similarity, these Na+/H+ exchanger isoforms differ in kinetic characteristics and their response to external stimuli. The present review deals with the recent developments in their functional characterization and their short-term regulation.

Use of membrane vesicles to estimate the numbers of system y+ and system L amino acid transporters in human erythrocytes.

We have used equilibrium values for L-leucine and L-lysine uptake by right-side-out vesicles to estimate the membrane abundance (sites/cell) of Na(+)-dependent amino acid transport systems L and y+ in human erythrocytes. All of the intravesicular space was accessible to L-leucine, as judged by comparisons with uridine uptake via the equilibrative nucleoside transporter (10(4) sites/cell). In contrast, only 28% of the total intravesicular space was accessible to L-lysine uptake via system y+. Since human erythrocyte membranes generate an average of approximately 1000 vesicles/cell, these data provide evidence that system L is a relatively high-abundance membrane transport protein in human erythrocytes, while system y+ is present in smaller amounts (approximately 300 copies/cell). Calculated turnover numbers for L-lysine transport by system y+ at 37 degrees C are 24 s-1 for zero-trans influx and 150 s-1 for equilibrium-exchange influx.

Kinetics and regulation of three cloned mammalian Na+/H+ exchangers stably expressed in a fibroblast cell line.

The kinetics and second messenger regulation of three cloned mammalian intestinal Na+/H+ exchangers were studied using fluorometric techniques. These exchangers, NHE1, NHE2, and NHE3, were stably expressed in PS120 fibroblasts, which lack an endogenous Na+/H+ exchanger. H+ kinetic data indicated cooperativity by internal protons, with Hill coefficients of approximately 2 for all three isoforms. In contrast, Na+ kinetic data fit Michaelis-Menten kinetics, with Km (Na+) 15-18 mM and a Hill coefficient of approximately 1. The exchangers were all activated by growth factors and thrombin; in NHE1 these agonists increased the apparent affinity for intracellular H+, but did not change Vmax, while for NHE2 and NHE3 the effect was on Vmax alone. Phorbol ester stimulated NHE1 and NHE2, but inhibited NHE3 with a decrease in Vmax. ATP-depletion decreased Vmax and the apparent affinity for H+ for all three isoforms, and reduced the Hill coefficient to approximately 1, suggesting that a basal level of phosphorylation was required for the cooperativity. The differences in kinetics and second messenger regulation suggest that the NHE isoforms may serve different cellular functions. The up- and down-regulation of NHE3 by kinases indicates that this isoform may be involved in a specialized function such as Na+ absorption.

Identification of PTH-responsive Na/H-exchanger isoforms in a rabbit proximal tubule cell line (RKPC-2).

Renal epithelial cells may express apical and basolateral Na/H exchangers which are different in their physiological regulation and different in their sensitivities to the inhibitor amiloride. In the present study RKPC-2 cells [a Simian virus 40 (SV-40) transformed cell line of rabbit S2 proximal tubular origin] were examined for localization (apical vs basolateral) and regulation of Na/H-exchange activity(ies) by parathyroid hormone (PTH). In addition, using specific cDNA probes we determined the expression of multiple isoforms of Na/H exchangers in RKPC-2 cells. By the use of BCECF [2',7',bis(2-carboxyethyl)-5,6-carboxyfluorescein intracellular pH (pHi) indicator] and single cell fluorescence microscopy, Na/H-exchange activities (defined as initial rate of Na-dependent pHi recovery) were found on the apical and basolateral membrane of RKPC-2 cells; apical and basolateral transport activities differed in sensitivity to dimethylamiloride, the basolateral being more sensitive. Northern blot analysis demonstrated the presence of a 5.2-kb transcript, related to Na/H-exchanger activity NHE-1, and a 3.2-kb transcript, related to Na/H-exchanger activity NHE-2. PTH (10(-8) M) inhibited apically and basolaterally located Na/H-exchanger activities. The inhibitory effect of PTH was mimicked by 8-bromo-adenosine 3'5'-cyclic monophosphate (cAMP); it was blunted in the presence of H-89 (inhibitor of protein kinase A) and was unaffected by calphostin C (inhibitor of protein kinase C). In contrast to 8-bromo-cAMP (and PTH), exposure of RKPC-2 cells to phorbol 12-myristate 13-acetate (TPA) caused a significant stimulation of both Na/H-exchange activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, tissue distribution, and functional analysis of the human Na+/N+ exchanger isoform, NHE3.

We previously isolated a 1.4-kb partial cDNA from a human kidney cortex library. Using both library screening and reverse transcription-polymerase chain reaction of human kidney RNA, we obtained the entire coding region of the human NHE3 cDNA. The human NHE3 cDNA encoded a protein of 834 amino acids with a calculated relative molecular weight of 92,906. It exhibited 89 and 88% amino acid identity with rat and rabbit NHE3, respectively. The stable transfection of a composite human NHE3 cDNA into Na+/H+ exchanger-deficient PS120 cells established Na+/H+ exchange. Functionally, human NHE3 was similar to the rabbit and rat NHE3 homologues, being relatively resistant to inhibition by amiloride, half-maximal inhibition (IC50) = 49.0 microM, and ethylisopropylamiloride, IC50 = 6.6 microM, and being stimulated by fibroblast growth factor but inhibited by phorbol 12-myristate 13-acetate. However, unlike the rabbit or rat NHE3, human NHE3 message was not restricted to kidney, intestine, stomach, and brain. Northern analysis of multiple human tissues detected NHE3 message, in descending order, as follows: kidney >> small intestine >> testes > ovary > colon = prostate > thymus > peripheral leukocyte = brain > spleen > placenta. Message in the kidney, small intestine, and colon was primarily of 6.7 kb, whereas both 6.7- and 8.9-kb bands were expressed nearly equivalently in the other tissues. No NHE3 message was detected in the human heart, lung, liver, skeletal muscle, or pancreas.

Cloning and sequencing of a rabbit cDNA encoding an intestinal and kidney-specific Na+/H+ exchanger isoform (NHE-3).

We previously cloned, sequenced, and expressed two distinct mammalian Na+/H+ exchanger isoforms (NHE-1 and NHE-2). We report here the cloning of a composite cDNA which encodes a third mammalian isoform (NHE-3), which is expressed specifically in intestine and kidney. The protein deduced from the longest open reading frame of this composite sequence has 832 amino acids with a calculated Mr of 92,747. The hydrophobicity plot of NHE-3 is very similar to that of NHE-1 and NHE-2. NHE-3 is also predicted to have 10-12 membrane-spanning domains and a long cytoplasmic domain which contains putative protein kinase phosphorylation motifs. NHE-3 exhibits overall 41% amino acid identity with NHE-1. NHE-3 is likely a glycoprotein as it has one potential N-linked glycosylation site, which is conserved in all NHEs identified. Northern blot analysis of poly(A+) RNA isolated from rabbit ileum using NHE-3 cDNA as a probe hybridized to a single 5.4-kilobase transcript. More detailed tissue distribution of message was performed by ribonuclease protection assay. It was found that NHE-3 message is only expressed in intestine and kidney, with the kidney cortex having the most abundant message, followed by intestine and kidney medulla. In intestine, ileum and ascending colon have the same amount of message, with much lesser amounts in jejunum. The message is absent from duodenum and descending colon, which lack the neutral NaCl absorptive process. Thus, NHE-3 might be involved in Na+ absorption in intestinal and renal epithelial cells.

C-terminal domains of Na(+)/H(+) exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger.

When expressed either in polarized epithelial cells or in fibroblasts, two Na(+)/H(+) exchanger isoforms, NHE1 and NHE3, have different subcellular distributions. Using a quantitative cell surface biotinylation technique, we found PS120 cells target approximately 90% of mature NHE1 but only 14% of NHE3 to the cell surface, and this pattern occurs irrespective of NHE protein expression levels. In this study, we examined surface fractions of NHE3 C-terminal truncation mutants to identify domains involved in the targeting of NHE3. Removing the C-terminal 76 amino acids doubled surface fractions to 30% of total and doubled the V(max) from 1300 to 2432 microM H(+)/s. Removal of another 66 amino acids increased surface levels to 55% of total with an increase in the V(max) to 5794 microM H(+)/s. Surface fractions did not change with a further 105 amino acid truncation. We postulated that inhibition of the basal recycling of NHE3 could result in the surface accumulation of the NHE3 truncations. Accordingly, we found that, unlike wild-type NHE3, the truncations were shown to internalize poorly and were not affected by PI3 kinase inhibition. However, while the truncations demonstrated reduced basal recycling, they retained the same serum response as full-length NHE3, with a mobilization of approximately 10% of total NHE to the surface. We conclude that basal recycling of NHE3 is controlled by endocytic determinants contained within its C-terminal 142 amino acids and that serum-mediated exocytosis is independently regulated through a different part of the protein.

Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. Ent2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine.

We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC(50), 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 microM) and dipyridamole (IC(50), 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [(3)H]NBMPR binds to ENT1 cells with a high affinity K(d) of 0.377 +/- 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7. 7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [(3)H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.

Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein.

A polyclonal antibody (Ab597) was produced in rabbit against a fusion protein of glutathione-S-transferase and the last 87 amino acids of the Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognized proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stably transfected with NHE2), and this antibody did not cross-react with NHE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/NHE2 cells, permeabilization of the cells was required for staining, confirming the putative membrane topology of NHE2 that the C-terminus is cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glycosylated as it contains one potential N-linked glycosylation site (N350VS), which is conserved among NHE1, NHE3, and NHE4. However, NHE2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 protein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase further shifted the mobility of the neuraminidase-treated 81 kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-alpha-D-galactosaminide (Bz alpha GalNAc), an O-glycosylation inhibitor, decreased the size of the 85 kDa protein to 81 kDa. This treatment had no effect on the initial rate of Na+/H+ exchange of PS120/NHE2 cells. The 75 kDa protein was not affected by the glycosidase treatment of PS120/NHE2 membranes or the Bz alpha GalNAc treatment of PS120/NHE2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation.

The cloned epithelial cell-specific Na+/H+ exchanger (NHE) isoform NHE2 is stimulated by fibroblast growth factor (FGF), phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA), and fetal bovine serum (FBS) through a change in maximal velocity of the transporter. In the present study, we used COOH-terminal truncation mutants to delineate specific domains in the COOH terminus of NHE2 that are responsible for growth factor and/or protein kinase regulation. Five truncation mutants (designated by the amino acid number at the truncation site) were stably expressed in NHE-deficient PS120 fibroblasts. The effects of PMA, FGF, OA, FBS, and W-13 [a Ca2+/calmodulin (CaM) inhibitor] were studied. Truncation mutant E2/660, but not E2/573, was stimulated by PMA. OA stimulated E2/573 but not E2/540. FGF stimulated E2/540 but not E2/499. The most truncated mutant, E2/499, was stimulated by FBS. W-13 stimulated the basal activity of the wild-type NHE2. However, W-13 had no effect on E2/755. By monitoring the emission spectra of dansylated CaM fluorescence, we showed that dansylated CaM bound directly to a purified fusion protein of glutathione S-transferase and the last 87 amino acids of NHE2 in a Ca2+-dependent manner, with a stoichiometry of 1:1 and a dissociation constant of 300 nM. Our results showed that the COOH terminus of NHE2 is organized into separate stimulatory and inhibitory growth factor/protein kinase regulatory subdomains. This organization of growth factor/protein kinase regulatory subdomains is very similar to that of NHE3, suggesting that the tertiary structures of the putative COOH termini of NHE2 and NHE3 are very similar despite the minimal amino acid identity in this part of the two proteins.