Role of transmembrane segment 5 of the plant vacuolar H-pyrophosphatase

Vacuolar H⁺-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.     

Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation

The Na⁺/H⁺ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na⁺/H⁺ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na⁺/H⁺ exchanger were examined for their ability to translocate protons as assessed by a cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na⁺/H⁺ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na⁺/H⁺ exchanger. These results confirm that polar amino acids are important in proton flux activity of Na⁺/H⁺ exchangers.     

Modulation of the Shaker K(+) channel gating kinetics by the S3-S4 linker

In Shaker K(+) channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3-S4 linker. To investigate the role of the S3-S4 linker in the ShakerH4Delta(6-46) (ShakerDelta) K(+) channel activation, we constructed S3-S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K(+) currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerDelta. The apparent number of gating charges was 12.6+/-0.6 e(o) in ShakerDelta, 12.7+/-0.5 in 10 aa linker, and 12.3+/-0.9 in 5 aa linker channels, but it was only 5.6+/-0.3 e(o) in the 0 aa linker mutant channel. The maximum probability of opening (P(o)(max)) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89x and 45x slower than that of the ShakerDelta K(+) channel, respectively. The initial lag of ionic currents when the prepulse was varied from -130 to -60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3-S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3-S4 linker plays an important role in setting ShakerDelta channel activation and deactivation kinetics.     

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.     

Mechanism of proton transport by plant plasma membrane proton ATPases

The mechanism of proton translocation by P-type proton ATPases is poorly defined. Asp684 in transmembrane segment M6 of the Arabidopsis thaliana AHA2 plasma membrane P-type proton pump is suggested to act as an essential proton acceptor during proton translocation. Arg655 in transmembrane segment M5 seems to be involved in this proton translocation too, but in contrast to Asp684, is not essential for transport. Asp684 may participate in defining the E₁ proton-binding site, which could possibly exist as a hydronium ion coordination center. A model of proton translocation of AHA2 involving the side chains of amino acids Asp684 and Arg655 is discussed.     

Translocation of Na(+),K(+)-ATPase is induced by Rho small GTPase in renal epithelial cells

The distribution of transmembrane proteins is considered to be crucial for their activities because these proteins mediate the information coming from outside of cells. A small GTPase Rho participates in many cellular functions through its downstream effectors. In this study, we examined the effects of RhoA on the distribution of Na(+),K(+)-ATPase, one of the transmembrane proteins. In polarized renal epithelium, Na(+),K(+)-ATPase is known to be localized at the basolateral membrane. By microinjection of the constitutively active mutant of RhoA (RhoA(Val14)) into cultured renal epithelial cells, Na(+),K(+)-ATPase was translocated to the spike-like protrusions over the apical surfaces. Microinjection of the constitutively active mutant of other Rho family GTPases, Rac1 or Cdcd42, did not induce the translocation. The translocation induced by RhoA(Val14) was inhibited by treatment with Y-27632, a Rho-kinase specific inhibitor, or by coinjection of the dominant negative mutant of Rho-kinase. These results indicate that Rho and Rho-kinase are involved in the regulation of the localization of Na(+),K(+)-ATPase. We also found that Na(+),K(+)-ATPase seemed to be colocalized with ERM proteins phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in cells microinjected with RhoA(Val14).     

Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+). Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues.     

Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase

Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.     

Collapse of conductance is prevented by a glutamate residue conserved in voltage-dependent K(+) channels

Voltage-dependent K(+) channel gating is influenced by the permeating ions. Extracellular K(+) determines the occupation of sites in the channels where the cation interferes with the motion of the gates. When external [K(+)] decreases, some K(+) channels open too briefly to allow the conduction of measurable current. Given that extracellular K(+) is normally low, we have studied if negatively charged amino acids in the extracellular loops of Shaker K(+) channels contribute to increase the local [K(+)]. Surprisingly, neutralization of the charge of most acidic residues has minor effects on gating. However, a glutamate residue (E418) located at the external end of the membrane spanning segment S5 is absolutely required for keeping channels active at the normal external [K(+)]. E418 is conserved in all families of voltage-dependent K(+) channels. Although the channel mutant E418Q has kinetic properties resembling those produced by removal of K(+) from the pore, it seems that E418 is not simply concentrating cations near the channel mouth, but has a direct and critical role in gating. Our data suggest that E418 contributes to stabilize the S4 voltage sensor in the depolarized position, thus permitting maintenance of the channel open conformation.     

Nitrophenylphosphate as a tool to characterize gill Na, K-ATPase activity in hyperregulating Crustacea

The kinetic properties of a gill Na(+), K(+)-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na(+), K(+)-ATPase hydrolyzed PNPP (K(+)-phosphatase activity) obeying Michaelis-Menten kinetics with K(M)=1.72+/-0.06 mmol l(-1) and V(max)=259.1+/-11.6 U mg(-1). ATP was a competitive inhibitor of K(+)-phosphatase activity with a K(i)=50.1+/-2.5 micromol l(-1). A cooperative effect for the stimulation of the enzyme by potassium (K(0.5)=3.62+/-0.18 mmol l(-1); n(H)=1.5) and magnesium ions (K(0.5)=0.61+/-0.02 mmol l(-1), n(H)=1.3) was found. Sodium ions had no effect on K(+)-phosphatase activity up to 1.0 mmol l(-1), but above 80 mmol l(-1) inhibited the original activity by approximately 75%. In the range of 0-10 mmol l(-1), sodium ions did not affect stimulation of the K(+)-phosphatase activity by potassium ions. Ouabain (K(i)=762.4+/-26.7 micromol l(-1)) and orthovanadate (K(i)=0.25+/-0.01 micromol l(-1)) completely inhibited the K(+)-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K(+)-phosphatase activity of the Na(+), K(+)-ATPase alone and suggest that the use of PNPP as a substrate to characterize K(+)-phosphatase activity may be a useful technique in comparative osmoregulatory studies of Na(+), K(+)-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme.     

The transmembrane domain 6 of vacuolar H(+)-pyrophosphatase mediates protein targeting and proton transport

Vacuolar H(+)-pyrophosphatase (V-PPase; EC 3.6.1.1) plays a significant role in the maintenance of the pH in cytoplasm and vacuoles via proton translocation from the cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. The topology of V-PPase as predicted by TopPred II suggests that the catalytic site is putatively located in loop e and exposed to the cytosol. The adjacent transmembrane domain 6 (TM6) is highly conserved and believed to participate in the catalytic function and conformational stability of V-PPase. In this study, alanine-scanning mutagenesis along TM6 of the mung bean V-PPase was carried out to identify its structural and functional role. Mutants Y299A, A306S and L317A exhibited gross impairment in both PP(i) hydrolysis and proton translocation. Meanwhile, mutations at L307 and N318 completely abolished the targeting of the enzyme, causing broad cytosolic localization and implicating a possible role of these residues in protein translocation. The location of these amino acid residues was on the same side of the helix wheel, suggesting their involvement in maintaining the stability of enzyme conformation. G297A, E301A and A305S mutants showed declines in proton translocation but not in PP(i) hydrolysis, consequently resulting in decreases in the coupling efficiency. These amino acid residues cluster at one face of the helix wheel, indicating their direct/indirect participation in proton translocation. Taken together, these data indicate that TM6 is crucial to vacuolar H(+)-pyrophosphatase, probably mediating protein targeting, proton transport, and the maintenance of enzyme structure.     

Evidence that Na+-pumping occurs through the D-channel in Vitreoscilla cytochrome bo

The operon (cyo) encoding the Na(+)-pumping respiratory terminal oxidase (cytochrome bo) of the bacterium Vitreoscilla was transformed into Escherichia coli GV100, a deletion mutant of cytochrome bo. This was done for the wild type operon and five mutants in three conserved Cyo subunit I amino acids known to be crucial for H(+) transport in the E. coli enzyme, one near the nuclear center, one in the K-channel, and one in the D-channel. CO-binding, NADH and ubiquinol oxidase, and Na(+)-pumping activities were all substantially inhibited by each mutation. The wild type Vitreoscilla cytochrome bo can pump Na(+) against a concentration gradient, resulting in a transmembrane concentration differential of 2-3 orders of magnitude. It is proposed that Vitreoscilla cytochrome bo pumps four Na(+) through the D-channel to the exterior and transports four H(+) through the K-channel for the reduction of each O(2).     

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.     

Influence of human tryptophan hydroxylase 2 N- and C-terminus on enzymatic activity and oligomerization

Tryptophan hydroxylase (TPH) catalyses the first and rate limiting step in the biosynthesis of the neurotransmitter serotonin. There are two TPH isoenzymes in humans, encoded by two different genes: TPH1 and the recently described TPH2. We have expressed both human enzymes and various deletion mutants of TPH2 (DeltaN44, DeltaC17, DeltaC19, DeltaC51) in COS7 cells. TPH1 and 2 displayed different kinetic properties with a lower K(m) value of TPH1. Removal of 44 amino acids from the N-terminus of TPH2 resulted in a 3-4-fold increased V(max), which indicates a strong inhibitory function of this part on the enzymes activity. TPH1 and 2 were able to form homooligomers and also heterooligomers with each other. The different deletion mutants (DeltaC17, DeltaC19 and DeltaC51), which lack the putative C-terminal leucine zipper tetramerization domain, existed as monomeric enzymes. While short deletions (DeltaC17 and DeltaC19) hardly changed V(max) values, the DeltaC51 mutant lost 99% of TPH activity. These data identify a region between the C-terminal oligomerization domain and the catalytic domain, which is indispensable for TPH2 activity.     

Protons block BK channels by competitive inhibition with K+ and contribute to the limits of unitary currents at high voltages

Proton block of unitary currents through BK channels was investigated with single-channel recording. Increasing intracellular proton concentration decreased unitary current amplitudes with an apparent pKa of 5.1 without discrete blocking events, indicating fast proton block. Unitary currents recorded at pH(i) 8.0 and 9.0 had the same amplitudes, indicating that 10(-8) M H(+) had little blocking effect. Increasing H(+) by recording at pH(i) 7.0, 6.0, and 5.0 then reduced the unitary currents by 13%, 25%, and 53%, respectively, at +200 mV. Increasing K(+)(i) relieved the proton block in a manner consistent with competitive inhibition of K(+)(i) action by H(+)(i). Proton block was voltage dependent, increasing with depolarization, indicating that block was coupled to the electric field of the membrane. Proton block was not described by the Woodhull equation for noncompetitive voltage-dependent block, but was described by an equation for cooperative competitive inhibition that included voltage-dependent block from the Woodhull equation. Proton block was still present after replacing the eight negative charges in the ring of charge at the entrance to the intracellular vestibule by uncharged amino acids. Thus, the ring of charge is not the site of proton block or of competitive inhibition of K(+)(i) action by H(+)(i). With 150 mM symmetrical KCl, unitary current amplitudes increased with depolarization, reaching 66 pA at +350 mV (pH(i) 7.0). The increase in amplitude with voltage became sublinear for voltages >100 mV. The sublinearity was unaffected by removing from the intracellular solutions Ca(2+) and Ba(2+) ions, the Ca(2+) buffers EGTA and HEDTA, the pH buffer TES, or by replacing Cl(-) with MeSO(3)(-). Proton block accounted for approximately 40% of the sublinearity at +200 mV and pH 7.0, indicating that factors in addition to proton block contribute to the sublinearity of the unitary currents through BK channels.     

Plant proton pumps

Chemiosmotic circuits of plant cells are driven by proton (H(+)) gradients that mediate secondary active transport of compounds across plasma and endosomal membranes. Furthermore, regulation of endosomal acidification is critical for endocytic and secretory pathways. For plants to react to their constantly changing environments and at the same time maintain optimal metabolic conditions, the expression, activity and interplay of the pumps generating these H(+) gradients have to be tightly regulated. In this review, we will highlight results on the regulation, localization and physiological roles of these H(+)- pumps, namely the plasma membrane H(+)-ATPase, the vacuolar H(+)-ATPase and the vacuolar H(+)-PPase.     

Rapid release of Mg(2+) from liver mitochondria by nonesterified long-chain fatty acids in alkaline media

Long-chain fatty acids induce a rapid release of Mg(2+) from both energized and nonenergized rat liver mitochondria suspended at pH 8 in isotonic saline but not sucrose media. The effect is observed only with fatty acids that possess protonophoric activity. The most active saturated fatty acids are myristic and palmitic, while the most active unsaturated acids are oleic, linolenic, and arachidonic. The rate of Mg(2+) release drastically decreases with decreasing medium pH to 7.2-7.6. However, at those pH values this rate is doubled by energization of mitochondria with respiratory substrates. Mg(2+) release is accompanied by cyclosporin A-insensitive large-amplitude swelling of mitochondria. This swelling is similar to that produced by the divalent metal ionophore A23187 and is interpreted as being due to activation of the inner membrane anion channel, the K(+) uniporter, and the K(+)/H(+) exchanger. In energized mitochondria, both swelling and Mg(2+) release are blocked by the exogenous K(+)/H(+) exchanger nigericin. It is proposed that fatty acids under conditions of alkaline mitochondrial matrix activate latent Mg(2+)-sensitive ion-conducting pathways in the inner mitochondrial membrane, which mediate swelling and Mg(2+) release. It is hypothesized that fatty acids activate an intrinsic Mg(2+)/H(+) exchanger that is related to, or identical with, the K(+)/H(+) exchanger.     

Brain Na+, K+-ATPase isoforms: different hypothalamus and mesencephalon response to acute desipramine treatment

We studied Na(+), K(+)-ATPase activity alpha isoforms by performing ouabain inhibition curves in rat hypothalamus and mesencephalon after acute administration of desipramine to rats. In hypothalamus, Ki values for high, intermediate and low affinity populations were 0.075x10(-9) M, 0.58x10(-6) M and 0.97x10(-3) M, with isoform distribution of 55%, 28% and 17%, respectively. In mesencephalon, Ki values for high, intermediate and low affinity populations were 1.80x10(-9) M, 0.56x10(-6) M and 0.21x10(-3) M, with isoform distribution of 28%, 46% and 21%, respectively. Three hours after acute administration of 10 mg/kg desipramine to rats, Na(+), K(+)-ATPase activity in hypothalamus increased significantly 54%, 39% and 51% as assayed respectively in the absence of ouabain or in the presence of 1x10(-9) M, or 5x10(-6) M ouabain, whereas only a trend was recorded in the presence of 1x10(-3) M ouabain. In such conditions, enzyme activity in mesencephalon increased significantly 73%, 54%, 30% and 271%, respectively. Present results showed that desipramine treatment enhances the activity of Na(+), K(+)-ATPase alpha isoforms in rat hypothalamus and mesencephalon, but the extent of this increase differs according to the isoform and the anatomical area studied, suggesting a differential enzyme regulation in response to noradrenergic stimulation.     

Arbuscular mycorrhizal fungi induce differential activation of the plasma membrane and vacuolar H+ pumps in maize roots

Roots undergo multiple changes as a consequence of arbuscular mycorrhizal (AM) interactions. One of the major alterations expected is the induction of membrane transport systems, including proton pumps. In this work, we investigated the changes in the activities of vacuolar and plasma membrane (PM) H(+) pumps from maize roots (Zea mays L.) in response to colonization by two species of AM fungi, Gigaspora margarita and Glomus clarum. Both the vacuolar and PM H(+)-ATPase activities were inhibited, while a concomitant strong stimulation of the vacuolar H(+)-PPase was found in the early stages of root colonization by G. clarum (30 days after inoculation), localized in the younger root regions. In contrast, roots colonized by G. margarita exhibited only stimulation of these enzymatic activities, suggesting a species-specific phenomenon. However, when the root surface H(+) effluxes were recorded using a noninvasive vibrating probe technique, a striking activation of the PM H(+)-ATPases was revealed specifically in the elongation zone of roots colonized with G. clarum. The data provide evidences for a coordinated regulation of the H(+) pumps, which depicts a mechanism underlying an activation of the root H(+)-PPase activity as an adaptative response to the energetic changes faced by the host root during the early stages of the AM interaction.