Molecular and functional analyses of rice NHX-type Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter genes

We previously cloned a vacuolar Na⁺/H⁺ antiporter gene (OsNHX1) from rice (Oryza sativa). Here we identified four additional NHX-type antiporter genes in rice (OsNHX2 through OsNHX5) and performed molecular and functional analyses of those genes. The exon–intron structure of the OsNHX genes and the phylogenetic tree of the OsNHX proteins suggest that the OsNHX proteins are categorized into two subgroups (OsNHX1 through OsNHX4 and OsNHX5). OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na⁺, Li⁺, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K⁺ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA. When we studied the expression of β-glucuronidase (GUS) driven by either the OsNHX1 or the OsNHX5 promoter, we observed activity in the stele, the emerging part of lateral roots, the vascular bundle, the water pore, and the basal part of seedling shoots with both promoters. In addition, each promoter had a unique expression pattern. OsNHX1 promoter–GUS activity only was localized to the guard cells and trichome, whereas OsNHX5 promoter–GUS activity only was localized to the root tip and pollen grains. Our results suggest that the members of this gene family play important roles in the compartmentalization into vacuoles of the Na⁺ and K⁺ that accumulate in the cytoplasm and that the differential regulation of antiporter gene expression in different rice tissues may be an important factor determining salt tolerance in rice.     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

H<Superscript>+</Superscript> translocation by weak acid uncouplers is independent of H<Superscript>+</Superscript> electrochemical gradient

According to the common view, weak acid uncouplers increase proton conductance of biological (and phospholipid bilayer) membranes, thus effecting H⁺ fluxes driven by their electrochemical gradients. Under certain conditions, however, uncouplers can induce unexpected effects opposite to the dissipation of H⁺ gradients. Results are presented here demonstrating CCCP-induced proton influx into Saccharomyces cerevisiae cytosol driven by the electrochemical potentials of CCCP and its CCCP^? anions, independent of electrochemical H⁺-gradient. Another view of week acid uncouplers’ action is proposed that is logically consistent with these observations.     

An Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene from wheat plays an important role in stress tolerance

A vacuole Na⁺/H⁺ antiporter gene TaNHX2 was obtained by screening the wheat cDNA library and by the 5′-RACE method. The expression of TaNHX2 was induced in roots and leaves by treatment with NaCl, polyethylene glycol (PEG), cold and abscisic acid (ABA). When expressed in a yeast mutant (Δnhx1), TaNHX2 suppressed the salt sensitivity of the mutant, which was deficient in vacuolar Na⁺/H⁺ antiporter, and caused partial recovery of growth of Δnhx1 in NaCl and LiC1 media. The survival rate of yeast cells was improved by overexpressing the TaNHX2 gene under NaCl, KCl, sorbitol and freezing stresses when compared with the control. The results imply that TaNHX2 might play an important role in salt and osmotic stress tolerance in plant cells.     

Salt tolerance of barley: Relations between expression of isoforms of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter and <Superscript>22</Superscript>Na<Superscript>+</Superscript> accumulation

Two barley cultivars (Hordeum vulgare L., cvs. Elo and Belogorskii) differing in salt tolerance were used to study ²²Na⁺ uptake, expression of three isoforms of the Na⁺/H⁺ antiporter HvNHX1-3, and the cellular localization of these isoforms in the elongation zone of seedling roots. During short (1 h) incubation, seedling roots of both cultivars accumulated approximately equal quantities of ²²Na⁺. However, after 24-h incubation the content of ²²Na⁺ in roots of a salt-tolerant variety Elo was 40% lower than in roots of the susceptible variety Belogorskii. The content of ²²Na⁺ accumulated in shoots of cv. Elo after 24-h incubation was 6.5 times lower than in shoots of cv. Belogorskii and it was 4 times lower after the salt stress treatment. The cytochemical examination revealed that three proteins HvNHX1-3 are co-localized in the same cells of almost all root tissues; these proteins were present in the tonoplast and prevacuolar vesicles. Western blot analysis of HvNHX1-3 has shown that the content of isoforms in vacuolar membranes increased in response to salt stress in seedling roots and shoots of both cultivars, although the increase was more pronounced in the tolerant cultivar. The content of HvNHX1 in the seedlings increased in parallel with the enhanced expression of HvNHX1, whereas the increase in HvNHX2 and HvNHX3 protein content was accompanied by only slight changes in expression of respective genes. The results provide evidence that salt tolerance of barley depends on plant ability to restrict Na⁺ transport from the root to the shoot and relies on regulatory pathways of HvNHX1-3 expression in roots and shoots during salt stress.     

Molecular characterization and expression analysis of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger gene family in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

One important mechanism plants use to cope with salinity is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~?20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl^? contents increased by 152 and 162%, respectively. Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration in roots under salt stress as compared to Cl^?. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na⁺ and K ⁺ . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na⁺ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.     

Over-expression of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na⁺/H⁺ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T₂ generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na⁺ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> and K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporters AtNHX1 and AtNHX3 from <Emphasis Type="Italic">Arabidopsis</Emphasis> improve salt and drought tolerance in transgenic poplar

The tonoplast and plasma membrane localized sodium (potassium)/proton antiporters have been shown to play an important role in plant resistance to salt stress. In this study, AtNHX1 and AtNHX3, two tonoplast Na⁺(K⁺)/H⁺ antiporter encoding genes from Arabidopsis thaliana, were expressed in poplar to investigate their biological functions in the resistance to abiotic stresses in woody plants. Transgenic poplar plants expressing either gene exhibited increased resistance to both salt and water-deficit stresses. Compared to the wild type (WT) plants, transgenic plants accumulated more sodium and potassium ions in the presence of 100 mM NaCl and showed reduced electrolyte leakage in the leaves under water stress. Furthermore, the proton-translocating and cation-dependent H⁺ (Na⁺/H⁺ or K⁺/H⁺) exchange activities in the tonoplast vesicles isolated from the leaves of transgenic plants were higher than in those isolated from WT plants. Therefore, constitutive expression of either AtNHX1 or AtNHX3 genetically modified the salt and water stress tolerance of transgenic poplar plants, providing a potential tool for engineering tree species with enhanced resistance to multiple abitotic stresses.     

Regulation of durum wheat Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger TdSOS1 by phosphorylation

We have identified a plasma membrane Na⁺/H⁺ exchanger from durum wheat, designated TdSOS1. Heterologous expression of TdSOS1 in a yeast strain lacking endogenous Na⁺ efflux proteins showed complementation of the Na⁺- and Li⁺-sensitive phenotype by a mechanism involving cation efflux. Salt tolerance conferred by TdSOS1 was maximal when co-expressed with the Arabidopsis protein kinase complex SOS2/SOS3. In vitro phosphorylation of TdSOS1 with a hyperactive form of the Arabidopsis SOS2 kinase (T/DSOS2∆308) showed the importance of two essential serine residues at the C-terminal hydrophilic tail (S1126, S1128). Mutation of these two serine residues to alanine decreased the phosphorylation of TdSOS1 by T/DSOS2∆308 and prevented the activation of TdSOS1. In addition, deletion of the C-terminal domain of TdSOS1 encompassing serine residues at position 1126 and 1128 generated a hyperactive form that had maximal sodium exclusion activity independent from the regulatory SOS2/SOS3 complex. These results are consistent with the presence of an auto-inhibitory domain at the C-terminus of TdSOS1 that mediates the activation of TdSOS1 by the protein kinase SOS2. Expression of TdSOS1 mRNA in young seedlings of the durum wheat variety Om Rabia3, using different abiotic stresses (ionic and oxidative stress) at different times of exposure, was monitored by RT–PCR.     

Vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter from barley: Identification and response to salt stress

One of the protective mechanisms used by plants to survive under conditions of salt stress caused by high NaCl concentration is the removal of Na+ from the cytoplasm. This mechanism involves a number of Na+/H+-antiporter proteins that are localized in plant plasma and vacuolar membranes. Due to the driving force of the electrochemical H+ gradient created by membrane H+-pumps (H+-ATPases and vacuolar H+-pyrophosphatases), Na+/H+-antiporters extrude sodium ions from the cytoplasm in exchange for protons. In this study, we have identified the gene for the barley vacuolar Na+/H+-antiporter HvNHX2 using the RACE (rapid amplification of cDNA ends)-PCR (polymerase chain reaction) technique. It is shown that the identified gene is expressed in roots, stems, and leaves of barley seedlings and that it presumably encodes a 59.6 kD protein composed of 546 amino acid residues. Antibodies against the C-terminal fragment of HvNHX2 were generated. It is shown that the quantity of HvNHX2 in tonoplast vesicles isolated from roots of barley seedlings remains the same, whereas the rate of Na+/H+ exchange across these membranes increases in response to salt stress. The 14-3-3-binding motif Lys-Lys-Glu-Ser-His-Pro (371-376) was detected in the HvNHX2 amino acid sequence, which is suggestive of possible involvement of the 14-3-3 proteins in the regulation of HvNHX2 function.     

New isoform HvNHX3 of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter in barley: Expression and immunolocalization

The gene HvNHX3 encoding a new isoform of vacuolar Na⁺/H⁺-antiporter was identified in barley. This gene is expressed in roots and leaves of barley seedlings, and it encodes a protein consisting of 541 amino acid residues with pre-dicted molecular weight 59.7 kDa. It was found that by its amino acid sequence HvNHX3 is closest to the Na⁺/H⁺-antiporter HbNHX1 of wild type from Hordeum brevisibulatum that grows on salt-marsh (solonchak) soils (95% homology). The expression of HvNHX3 during salt stress is increased several-fold in roots and leaves of barley seedlings. At the same time, the amount of HvNHX3 protein in roots does not change, but in leaves it increases significantly. It was shown using HvNHX3 immunolocalization in roots that this protein is present in all tissues, but in control plants it was clustered and in experimental plants after salt stress it was visualized as small granules. It has been proposed that HvNHX3 is converted into active form during declusterization. The conversion of HvNHX3 into its active form along with its quantitative increase in leaves during salt stress activates Na⁺/H⁺-exchange across the vacuolar membrane and Na⁺ release from cytoplasm, and, as a consequence, an increase of salt stress tolerance.     

Functional Identification of H<Superscript>+</Superscript>-ATPase and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H⁺-ATPase in the PM of the root cells of S. altissima. The addition of Na⁺ and Li⁺ ions to the outer medium resulted in dissipation of ΔpH preformed by the H⁺-ATPase, which indicates the presence in PM of the functionally active Na⁺/H⁺ antiporter. The results are discussed with regard to involvement of the Na⁺/H⁺ antiporter and the PM H⁺-ATPase in loading Na⁺ ions into the xylem of S. altissima roots.     

Metagenome Cloning and Functional Analysis of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Genes from Keke Salt Lake in China

Na⁺/H⁺ antiporters are ubiquitous membrane proteins and play a central role in cell homeostasis including pH regulation, osmoregulation, and Na⁺/Li⁺ tolerance in bacteria. The microbial communities in extremely hypersaline soil are an important resource for isolating Na⁺/H⁺ antiporter genes. A metagenomic library containing 35,700 clones was constructed by using genomic DNA obtained from the hypersaline soil samples of Keke Salt Lake in Northwest of China. Two Na⁺/H⁺ antiporters, K1-NhaD, and K2-NhaD belonging to NhaD family, were screened and cloned from this metagenome by complementing the triple mutant Escherichia coli strain KNabc (nhaA ⁻ , nhaB ⁻ , chaA ⁻) in medium containing 0.2 M NaCl. K1-NhaD and K2-NhaD have 75.5% identity at the predicted amino acid sequence. K1-NhaD has 78% identity with Na⁺/H⁺ antiporter NhaD from Halomonas elongate at the predicted amino acid sequence. The predicted K1-NhaD is a 53.5 kDa protein (487 amino acids) with 13 transmembrane helices. K2-NhaD has 73% identity with Alkalimonas amylolytica NhaD. The predicted K2-NhaD is a 55 kDa protein (495 amino acids) with 12 transmembrane helices. Both K1-NhaD and K2-NhaD could make the triple mutant E. coli KNabc (nhaA ⁻ , nhaB ⁻ , chaA⁻) grow in the LBK medium containing 0.2–0.6 M Na⁺ or with 0.05–0.4 M Li⁺. Everted membrane vesicles prepared from E. coli KNabc cells carrying K1-NhaD or K2-NhaD exhibited Na⁺/H⁺ and Li⁺/H⁺ antiporter activities which were pH-dependent with the highest activity at pH 9.5. Little K⁺/H⁺ antiporter activity was also detected in vesicles form E. coli KNabc carrying K1-NhaD or K2-NhaD.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

Theoretical study of isotope effects on the stereodynamics of H<Superscript>+</Superscript>+HD and its isotopic variant D<Superscript>+</Superscript>+HD

The stereodynamics for H⁺+HD and its isotopic variant D⁺+HD were studied with a quasi-classical trajectory (QCT) method at a collision energy of 0.7 eV on the ground 1¹A′ potential energy surface (PES). The polarization-dependent differential cross-sections (PDDCSs) in the center-of-mass frame are presented here. Furthermore, the distribution of the angle between k and j′, p(θ _r ) and the distribution of the dihedral angle p(ϕ _r ) were calculated and are discussed. The results indicate that isotopic substitution exerts substantial effects on the differential cross-section and the product’s rotational polarization.     

Computational study of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter from <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Sodium proton antiporters are ubiquitous membrane proteins that catalyze the exchange of Na⁺ for protons throughout the biological world. The Escherichia coli NhaA is the archetypal Na⁺/H⁺ antiporter and is absolutely essential for survival in high salt concentrations under alkaline conditions. Its crystal structure, accompanied by extensive molecular dynamics simulations, have provided an atomically detailed model of its mechanism. In this study, we utilized a combination of computational methodologies in order to construct a structural model for the Na⁺/H⁺ antiporter from the gram-negative bacterium Vibrio parahaemolyticus. We explored its overall architecture by computational means and validated its stability and robustness. This protein belongs to a novel group of NhaA proteins that transports not only Na⁺ and Li⁺ as substrate ions, but K⁺ as well, and was also found to miss a β-hairpin segment prevalent in other homologs of the Bacteria domain. We propose, for the first time, a structure of a prototype model of a β-hairpin-less NhaA that is selective to K⁺. Better understanding of the Vibrio parahaemolyticus NhaA structure-function may assist in studies on ion transport, pH regulation and designing selective blockers.     

Interactions Between Vacuolar H<Superscript>+</Superscript>-ATPases and Microfilaments in Osteoclasts

Vacuolar H⁺-ATPases (V-ATPases) are transported from cytosolic compartments to the ruffled plasma membrane of osteoclasts as they activate to resorb bone. Transport of V-ATPases is essential for bone resorption, and is associated with binding interactions between V-ATPases and microfilaments that are mediated by an actin-binding site in subunit B. This site is contained within 44 amino acids in the amino terminal domain, and requires a sequence motif that resembles an actin-binding motif found in mammalian profilin 1. Small alterations in the profilin-like sequence disrupt the actin-binding activity of subunit B. The interaction between V-ATPases and microfilaments in osteoclasts is regulated in response to changes in phosphatidylinositol-3 kinase activity. During internalization of V-ATPases from the plasma membrane of osteoclasts after a cycle of resorption, V-ATPases bind microfilaments that are in podosomes, dynamic actin-based structures, also present in metastatic cancer cells. Studies are ongoing to establish the physiological role of the microfilament-binding activity of subunit B in osteoclasts and in other cells.