Aluminum interaction with calmodulin. Evidence for altered structure and function from optical and enzymatic studies

The interaction of aluminum ions with bovine brain calmodulin has been examined by fluorescence spectroscopy, circular dichroic spectrophotometry and equilibrium dialysis, and by the calmodulin-dependent activation of 3',5'-cyclic nucleotide phosphodiesterase. These experiments show that aluminum binds stoichiometrically and cooperatively to calmodulin. Binding of aluminum at a molar ratio of 2:1 to calmodulin suffices to induce a major structural change. Estimates from spectroscopic data indicate that the binding affinity for the first mol of aluminum bound to the protein is about one order of magnitude stronger than that of calcium to its comparable site. These estimates agree with a dissociation constant of 0.4 microM derived from equilibrium dialysis experiments. Interaction of aluminum with calmodulin induces a helix-coil transition and enhances the hydrophobic surface area much more than calcium does. A molar ratio of 4:1 for [aluminum]/[calmodulin] is sufficient to block completely the activity of the calcium-calmodulin-dependent phosphodiesterase. Highly hydrated aluminum ions apparently promote solvent-rich, disordered polypeptide regions in calmodulin which, in turn, profoundly influence the protein's flexibility.     

S100A1 is a novel molecular chaperone and a member of the Hsp70/Hsp90 multichaperone complex

Although calmodulin is known to be a component of the Hsp70/Hsp90 multichaperone complex, the functional role of the protein remains uncertain. In this study, we have identified S100A1, but not calmodulin or other S100 proteins, as a potent molecular chaperone and a new member of the multichaperone complex. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments indicated the formation of stable complexes between S100A1 and Hsp90, Hsp70, FKBP52, and CyP40 both in vitro and in mammalian cells. S100A1 potently protected citrate synthase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and rhodanese from heat-induced aggregation and suppressed the aggregation of chemically denatured rhodanese and citrate synthase during the refolding pathway. In addition, S100A1 suppressed the heat-induced inactivation of citrate synthase activity, similar to that for Hsp90 and p23. The chaperone activity of S100A1 was antagonized by calmodulin antagonists, such as fluphenazine and prenylamine, that is, indeed an intrinsic function of the protein. The overexpression of S100A1 in COS-7 cells protected transiently expressed firefly luciferase and Escherichia coli beta-galactosidase from inactivation during heat shock. The results demonstrate a novel physiological function for S100A1 and bring us closer to a comprehensive understanding of the molecular mechanisms of the Hsp70/Hsp90 multichaperone complex.     

Calmodulin dependent formation of membrane potential in barley root plasma membrane vesicles: A biochemical model of aluminum toxicity in plants

Micromolar concentrations of aluminum ions interfere with calmodulin-stimulated, membrane bound ATPase activity which plays a role in the maintenance of the transmembrane potential of plasma membrane enriched vesicles isolated from barley roots. Calmodulin appears to be the major target for aluminum interaction resulting in pronounced changes in the exposure of a large, hydrophobic surface on this protein as determined with a fluorescent, hydrophobic surface probe. At a molar ratio of 3:1 [aluminum]/[calmodulin], the calmodulin stimulated enzymatic activity, probably associated with a Ca²⁺+ Mg²⁺ATPase, is about 95% inhibited. Aluminum induced changes in calmodulin structure are reflected in reduced formation of the membrane potential when assayed with a fluorescent potential probe, oxonol VI. We hypothesize that the aluminum caimodulin complex represents a primary lesion in toxic responses of plants to this metal.     

Involvement of altered cytoskeletal protein phosphorylation in aluminum-induced CNS dysfunction

In the present investigation, we have studied the effects of aluminum (10 mg/kg of body weight per day, i.p.) and desferrioxamine (6 mg/kg of body weight per day, i.p.) alone and in combination for 4 weeks on the regulation of phosphorylation of neuronal proteins. A marked decrease in the biological activity of calmodulin was observed after aluminum treatment; however, in combination with desferrioxamine, a reversal in the levels of calmodulin, in terms of nmol cAMP hydrolyzed/min/mg protein, was observed. Exogenous addition of calmodulin had an inhibitory effect on calmodulin-mediated synaptosomal protein phosphorylation in aluminum-exposed animals. An almost complete reversal of this inhibition was observed following coexposure to aluminum and desferrioxamine. Cyclic AMP-dependent synaptosomal protein phosphorylation was, however, stimulated following aluminum exposure. Coadministration of desferrioxamine along with aluminum was found to mitigate the neurotoxic effect of aluminum. © 1997 John Wiley & Sons, Inc.     

Characterization of Al-responsive citrate excretion and citrate-transporting MATEs in Eucalyptus camaldulensis

Many plant species excrete organic acids into the rhizosphere in response to aluminum stress to protect sensitive cells from aluminum rhizotoxicity. When the roots of Eucalyptus camaldulensis, a major source of pulp production, were incubated in aluminum-toxic medium, citrate released into the solution increased as a function of time. Citrate excretion was inducible by aluminum, but not by copper or sodium chloride stresses. This indicated that citrate is the major responsive organic acid released from the roots of this plant species to protect the root tips from aluminum damage. Four genes highly homologs to known citrate-transporting multidrugs and toxic compounds exclusion proteins, named EcMATE1–4, were isolated using polymerase chain reaction-based cloning techniques. Their predicted proteins included 12 membrane spanning domains, a common structural feature of citrate-transporting MATE proteins, and consisted of 502–579 amino acids with >60 % homology to orthologous genes in other plant species. One of the homologs, designated EcMATE1, was expressed in the roots more abundantly than in the shoots and in response to both Al and low pH stresses. Ectopic expression of EcMATE1 and 3 in tobacco hairy roots enhanced Al-responsive citrate excretion. Pharmacological characterization indicated that Al-responsive citrate excretion involved a protein phosphorylation/dephosphorylation process. These results indicate that citrate excretion through citrate-transporting multidrugs and toxic compounds exclusion proteins is one of the important aluminum-tolerance mechanisms in Eucalyptus camaldulensis.     

Al3+ versus Ca2+ ion binding to methionine and tyrosine spin-labeled bovine brain calmodulin.

Bovine calmodulin analogues, spin-labeled at either methionine or tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies to investigate possible calmodulin interactions with aluminum ion. The study attempts to clarify a previous report in the literature (H. Siegel, R. Coughlin, and A. Haug, Biochem. Biophys. Res. Commun. 115, 512 (1983)) which indicated, on the basis of EPR experiments on methionine spin-labeled protein, significant interaction between calmodulin and aluminum ion at pH = 6.5. In EPR metal ion titration experiments we have found that the signal line-shape (from both methionine and tyrosine spin labels) changed dramatically with the addition of calcium ion, but was virtually unchanged with the addition of aluminum ion at pH = 6.5. Experiments performed at pH = 5.5, where significantly more "free" aluminum ion (i.e., Al(H2O)6(3+) = Al3+) is present, also failed to produce the line-narrowing effect observed in the earlier study. Based on our EPR experiments, in the pH range 5.5 to 6.5, we find no evidence for significant interaction between calmodulin and aluminum ion.     

Aluminum deposition in the central nervous system. Preferential accumulation in the hippocampus in weanling rats

Simultaneous administration of 1,25-dihydroxyvitamin-D3, citrate, and aluminum-containing phosphate binders is frequently used in patients with chronic renal failure. In order to investigate whether citrate may represent a risk factor of aluminum intoxication, 16 Sprague-Dawley weanling rats were randomly assigned to four groups: 1,25-dihydroxyvitamin-D3 at 16 ng/kg/day was given to all groups except the control; in addition, two groups received either aluminum hydroxide at 160 mg elemental aluminum/kg/day, or aluminum citrate at 160 mg elemental aluminum/kg/day, respectively. The control group received only the vehicle. Extremely high aluminum concentrations were detected in the hippocampus of rats receiving aluminum compounds. This content of aluminum (microgram/g dry weight) was far higher than that found in other brain areas of the same animals (146.40 +/- 51.23 versus 4.49 +/- 0.62, P less than 0.001) as well as that detected in the hippocampus of the control animals (2.73 +/- 0.40). Thus, in non-uremic, weanling rats supplemented with 1,25-dihydroxyvitamin-D3, the administration of aluminum favors selective accumulation in the hippocampus. No differences between aluminum hydroxide and aluminum citrate administration were observed.     

Lipid bilayer permeation by neutral aluminum citrate and by three alpha-hydroxy carboxylic acids

Several groups have proposed that aluminum (Al) may permeate biological membranes as a neutral complex with citrate. We tested this hypothesis by measuring aluminum citrate flux across unilamellar phospholipid vesicles (liposomes). Results from two independent procedures show that lipid bilayer permeation by the neutral aluminum-citrate complex is slow (P approximately equal to 1 x 10(-11) cm.s-1). We then compared aluminum-citrate permeation with permeation by a series of alpha-hydroxy carboxylic acids and by trimethylcitrate. This comparison showed that the aluminum-citrate flux is limited by diffusion across the water/lipid interface. This is due to hydrogen bonding between water and the citrate carboxyl groups, and by hydration of the bound metal in the aqueous phase. By analogy with citric acid, steric hindrance of diffusion within the bilayer does not affect the permeation rate of aluminum citrate. Elevated tissue levels of Al in subjects fed a diet supplemented with citric acid and Al(OH)3 cannot be explained by lipid bilayer permeation of the neutral complex.     

13C heteronuclear NMR studies of the interaction of cultured neurons and astrocytes and aluminum blockade of the preferential release of citrate from astrocytes

Citrate has been identified as a major tricarboxylic acid (TCA) cycle constituent preferentially released by astrocytes. We undertook the present study to examine further the nature of metabolic compartmentation in central nervous system tissues using ¹³C-labeled glucose and to provide new information on the influence of aluminum on the metabolic interaction between neurons and astrocytes. Metabolites released into the culture medium from astrocytes and neuron-astrocyte coculture, as well as the perchloric acid extracts of the cells were analyzed using 2D ¹H and ¹³C NMR spectroscopy. Astrocytes released citrate into the culture medium and the released citrate was consumed by neurons in coculture. Citrate release by astrocytes was blocked in the presence of aluminum, with progressive accumulation of citrate within the cells. We propose citrate supply is a more efficient energy source than lactate for neurons to produce ATP, especially in the hypoglycemic state on account of it being a direct component of the TCA cycle. Astrocytes may be the cellular compartment for aluminum accumulation as a citrate complex in the brain.     

Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.     

Characterization of Arabidopsis thaliana AtFKBP42 that is membrane-bound and interacts with Hsp90

The twisted dwarf1 (twd1) mutant from Arabidopsis thaliana was identified in a screen for plant architecture mutants. The TWD1 gene encodes a 42 kDa FK506-binding protein (AtFKBP42) that possesses similarity to multidomain PPIases such as mammalian FKBP51 and FKBP52, which are known to be components of mammalian steroid hormone receptor complexes. We report here for the first time the stoichiometry and dissociation constant of a protein complex from Arabidopsis that consists of AtHsp90 and AtFKBP42. Recombinant AtFKBP42 prevents aggregation of citrate synthase in almost equimolar concentrations, and can be cross-linked to calmodulin. In comparison to one active and one inactive FKBP domain in FKBP52, AtFKBP42 lacks the PPIase active FKBP domain. While FKBP52 is found in the cytosol and translocates to the nucleus, AtFKBP42 was predicted to be membrane-localized, as shown by electron microscopy.     

One Novel Mitochondrial Citrate Synthase from <Emphasis Type="Italic">Oryza sativa</Emphasis> L. can Enhance Aluminum Tolerance in Transgenic Tobacco

Rice exhibits the greatest aluminum (Al) tolerance compared with other cereals such as wheat, barley, maize, etc. A full-length gene, OsCS1, encoding citrate synthase, which is highly induced by aluminum toxicity in rice (Oryza sativa L.), was isolated. Sequence analysis and the sub-cellular localization of OsCS1 in yeast revealed that it is a mitochondrial citrate synthase. OsCS1 was induced by Al toxicity. Several independent transgenic tobacco lines expressing OsCS 1 exhibitted increased citrate efflux and extraordinary Al tolerance. Possible outlook for OsCS1 to be applied to enhance plant tolerance to Al toxicity was also discussed.     

Aluminum speciation studies in biological fluids. Part 2. Quantitative investigation of aluminum-citrate complexes and appraisal of their potential significance in vivo

Because of the recent implications of aluminum in the pathogenesis of various disease states, its in vivo chemistry has been receiving growing attention from bioinorganic chemists over the last few years. In this context, the elucidation of the main factors that govern aluminum bioavailability constitutes an urgent objective. Clearly, prevention measures require that mechanisms of aluminum absorption be definitely characterized, whereas specific sequestering agents are needed to detoxify patients with high-aluminum-body burdens. In particular, speciation studies are necessary to discriminate among the chemical forms under which aluminum predominates in vivo. Low molecular weight (LMW) species, which are the most active in terms of bioavailability, cannot be assessed by analytical techniques, and so computer simulations must be used. In recent clinical studies as well as in preliminary simulations dealing with aluminum distribution in blood plasma, citrate has been recognized as the most important LMW ligand of aluminum. The present paper thus reports a quantitative investigation of aluminum-citrate equilibria, carried out at 37 degrees C in NaCl 0.15 mol dm-3 in accordance with the experimental protocol defined in our previous study on aluminum hydrolysis. The ML, MLH, ML2, M3L3H-4, M2L2H-2, ML2H-1, and ML2H-2 species have been characterized over the whole physiological pH range using as large reactant concentration ratios as possible. Corresponding formation constants have then been used to investigate the role of citrate towards aluminum bioavailability. Blood plasma simulations reveal that citrate can promote aluminum urinary excretion, which substantiates recent clinical observations made on mice. However, the higher plasma aluminum concentrations are, the less effective citrate is to be expected. Gastrointestinal simulations confirm that the electrically neutral ML complex does represent an important risk of aluminum absorption in the upper region of the gastrointestinal tract at usual therapeutic doses. At moderate- and low-aluminum concentrations, citrate is also capable of dissolving the aluminum trihydroxide precipitate, which may combine with the capacity of other ligands to complex Al3+ into absorbable complexes at less acidic pH.     

Calmodulin content and distribution in six human melanoma cell lines

Calmodulin content and distribution between soluble and particulate fractions were determined by radioimmunoassay in six human melanoma cell lines exhibiting differences in tumor origin (primary or metastatic), degree of tumorigenicity and of pigmentation (amelanotic or melanotic). The results indicate that a) total, soluble and particulate calmodulin levels expressed as ng/10(6) cells or ng/micrograms of proteins remained constant for five out of six cell lines when cells grew from subconfluency to confluency. For IGR 37 line, derived from metastatic melanoma, the calmodulin content decreases from 2.39 to 1.27 ng/micrograms protein for total calmodulin, from 2.17 to 1.52 ng/micrograms protein for soluble calmodulin and from 2.61 to 1.02 ng/micrograms protein for particulate calmodulin, b) total, soluble and particulate calmodulin levels expressed as ng/microgram proteins were twofold (at confluency) to fourfold (at subconfluency) higher in the two cell lines from metastatic origin, IGR 37 and IPC 167. As for example, for total calmodulin, values in IGR 37 and IPC 167 cell lines, were, respectively at subconfluency, 2.39 and 2.31 ng/micrograms protein as compared with the four other cell lines: 0.76 to 0.96 ng/micrograms protein and at confluency: 1.27 and 1.98 ng/micrograms protein as compared with the four other cell lines: 0.76 to 0.90 ng/micrograms protein, c) ratio of calmodulin between soluble and particulate fractions was about 1 for the two autologous cell lines IGR 37 and IGR 39 and varies from 2 to 3 for the four other cell lines.     

The distribution of aluminum into and out of the brain

The extent, rate and possible mechanism(s) by which aluminum enters and is removed from the brain are presented. Introduction of Al into systemic circulation as Al.transferrin, the predominant Al species in plasma, resulted in about 7 x 10(-5) of the dose in the brain 1 day after injection. This brain Al entry could be mediated by transferrin-receptor-mediated endocytosis (TfR-ME). When Al.citrate, the predominant small molecular weight Al species in blood plasma, is introduced systemically, Al rapidly enters the brain. The rate of Al.citrate brain influx suggests a more rapid process than mediated by diffusion or TfR-ME. The question has been raised: "Is the brain a 'one-way sink' for aluminum?". Clinical observations are a basis for this suggestion. Rat brain 26Al concentrations decreased only slightly from 1 to 35 days after systemic 26Al injection, in the absence or presence of the aluminum chelator desferrioxamine, suggesting prolonged brain Al retention. However, studies of brain and blood extracellular Al at steady state, using microdialysis, suggest brain Al efflux exceeds influx, suggesting carrier-mediated brain Al efflux. The predominant brain extracellular fluid Al species is probably Al.citrate. The hypothesis that brain Al efflux, presumably of Al.citrate, is mediated by the monocarboxylate transporter was tested and supported. Although some Al that enters the brain is rapidly effluxed, it is suggested that a fraction enters brain compartments within 24 h from which it is only very slowly eliminated.     

慢性铝暴露对大鼠海马神经元PKC、CaMKⅡ、Ng 的影响

通过研究慢性铝暴露对大鼠学习记忆和海马长时程增强 (long-term potentiation, LTP) 的影响,并检测海马神经元蛋白激酶Ｃ(protein kinase c, PKC) 活性及Ca²⁺钙调蛋白激酶Ⅱ(Ca²⁺calmodulin dependent protein kinase Ⅱ, CaMK Ⅱ) 和神经颗粒素(neurogranin, Ng) 蛋白表达的变化,探讨铝暴露损害学习记忆的作用机制.选用断乳后 Wistar 大鼠,以含有不同浓度 AlCl₃ 的蒸馏水进行饲养.3 个月后,测定铝暴露组大鼠脑内和血中的铝含量;测量记录大鼠海马群体峰电位(population spike,PS)LTP;用改良 Takai 法测定海马神经元 PKC 活性变化;Western 印迹法检测 CaMK Ⅱ和Ng的蛋白表达.结果显示,与对照组相比,铝暴露组的 PKC 活性降低,差异有统计学意义 (P<0.01);与对照组相比,铝暴露组的CaM Ⅱ蛋白表达降低,差异有统计学意义(P<0.05);与对照组相比,铝暴露组的 Ng 蛋白表达降低,且差异有统计学意义(P<0.05).实验结果说明：慢性铝暴露可以降低大鼠海马神经元 PKC 的活性及 Ng 和 CaMKⅡ 的蛋白表达,可能影响 Ng 磷酸化水平,从而影响 CaM 与 Ng 之间的亲和性,也影响 Ca²⁺CaM 对 CaMKⅡ 的调节,抑制 LTP 的形成,损害学习记忆的功能.     

Identification of a Ca2+/calmodulin-binding domain within the carboxyl-terminus of the angiotensin II (AT1A) receptor.

To identify regulators of the type 1A angiotensin II receptor (AT1A), we investigated the interaction of cellular proteins with a fusion protein containing the rat AT1A receptor carboxyl-terminus. An approximately 20 kDa cytoplasmic protein interacted with the fusion protein in a Ca2+-dependent manner and was identified as calmodulin. A control peptide with high affinity for Ca2+/calmodulin and a peptide corresponding to a membrane proximal portion of the AT1A receptor carboxyl-terminus with analogy to known calmodulin-binding sequences were synthesised and tested for calmodulin-binding. Using in vitro binding assays combined with gel shift analysis, we demonstrated the formation of complexes between calmodulin and both peptides, which were Ca2+-dependent and of 1:1 stoichiometry. Affinity gels produced from these peptides also purified calmodulin from cell extracts. These results suggest a novel feedback regulation of the AT1A receptor by Ca2+/calmodulin and identify the membrane proximal region of the carboxyl-terminus as a focal point for interactions important for AT1A receptor function.     

Brush-border calmodulin. A major component of the isolated microvillus core

Calmodulin is present in brush borders isolated from intestinal epithelial cells and is one of the major components of the microvillar filament bundle. Calmodulin was purified from either demembranated brush borders or microvilli by a simple boiling procedure. The boiled supernate derived from the microvillus cores contained one major polypeptide of 20,000 daltons.The supernate from the brush-border preparation contained the 20,000-dalton subunit and a second protein of 30,000 daltons. The 20,000-dalton subunit has been identified as calmodulin by several criteria: (a) heat resistance, (b) comigration with brain calmodulin on alkaline urea gels and SDS gels, both cases in which the 20,000-dalton protein, like calmodulin, exhibits a shift in electrophoretic mobility in the presence of Ca++, and (c) 4--5-fold activation of 3',5'-cyclic nucleotide phosphodiesterase in the presence but not the absence of Ca++. With a cosedimentation assay it was determined that brush-border calmodulin does not bind directly to actin. In the presence of Ca++ (greater than 5 x 10(-7) M) there was a partial release of calmodulin from the microvillus core, along with a substantial conversion of microvillus actin into a nonpelletable from. The dissociation of calmodulin was reversed by removal of Ca++. If microvillus cores were pretreated with phalloidin, the Ca++-induced solubilization of actin was prevented, but the partial dissociation of calmodulin still occurred. The molar ratio of calmodulin:actin is 1:10 in the demembranated brush border and 1:2-3 in the microvillus core. No calmodulin was detected in the detergent-solubilized brush-border membrane fraction.     

Probing the role of calmodulin in Al toxicity in maize

The role of calmodulin on Al toxicity was studied in two maize (Zea mays L.) inbred lines, Cat 100-6 (Al-tolerant) and S 1587-17 (Al-sensitive). Increasing levels of Al induced the release of malate at similar rate by roots of both genotypes, while the exudation of citrate, a stronger Al-binding compound, was 3.5 times higher in Cat 100-6 seedlings exposed to 16.2x10(-6) Al(3+) activity. The calmodulin inhibitor trifluoperazine significantly reduced the root growth in both genotypes, mimicking the main effect of Al. However, when Cat 100-6 and S 1587-17 seedlings were challenged with Al in conjunction with trifluoperazine, no further reduction in root growth or any other effect of Al toxicity was observed. The rate of Al-induced citrate exudation by both genotypes was not affected by treatment with trifluoperazine or calmidazolium, another calmodulin inhibitor. The Al(3+) interaction with cytoplasmic CaM was estimated using models for the binding of Al(3+) and Mg(2+) with CaM and physiological concentrations of citrate, CaM, InsP(3), ATP, ADP, Al(3+) and Mg(2+). In this simulation, Al(3+) associated with citrate and InsP(3), but not with CaM. We conclude that calmodulin is not relevant to the physiological processes leading to the Al tolerance in maize, nor is it a primary target for Al toxicity.     

Not all ALMT1-type transporters mediate aluminum-activated organic acid responses: the case of ZmALMT1 – an anion-selective transporter

The phytotoxic effects of aluminum (Al) on root systems of crop plants constitute a major agricultural problem in many areas of the world. Root exudation of Al-chelating molecules such as low-molecular-weight organic acids has been shown to be an important mechanism of plant Al tolerance/resistance. Differences observed in the physiology and electrophysiology of root function for two maize genotypes with contrasting Al tolerance revealed an association between rates of Al-activated root organic acid release and Al tolerance. Using these genotypes, we cloned ZmALMT1 , a maize gene homologous to the wheat ALMT1 and Arabidopsis AtALMT1 genes that have recently been described as encoding functional, Al-activated transporters that play a role in tolerance by mediating Al-activated organic acid exudation in roots. The ZmALMT1 cDNA encodes a 451 amino acid protein containing six transmembrane helices. Transient expression of a ZmALMT1 ::GFP chimera confirmed that the protein is targeted to the plant cell plasma membrane. We addressed whether ZmALMT1 might underlie the Al-resistance response (i.e. Al-activated citrate exudation) observed in the roots of the Al-tolerant genotype. The physiological, gene expression and functional data from this study confirm that ZmALMT1 is a plasma membrane transporter that is capable of mediating elective anion efflux and influx. However, gene expression data as well as biophysical transport characteristics obtained from Xenopus oocytes expressing ZmALMT1 indicate that this transporter is implicated in the selective transport of anions involved in mineral nutrition and ion homeostasis processes, rather than mediating a specific Al-activated citrate exudation response at the rhizosphere of maize roots.