Squarks and gluinos searches at the TeVatron

Philippe Gris 1 on behalf on the CDF and D0 collaborations 1-Laboratoire de Physique Corpusculaire -IN2P3/CNRS 24 avenue des Landais -63177 Aubière cedex -France A search for squarks and gluinos performed by the CDF and DØ collaborations is presented. The sample taken during the RunII of the Fermilab TeVatron collider at √ s = 1.96 TeV is used.

Supersymmetry (SUSY) is one of the most promising ways to solve crucial problems of the Standard Model (SM). This spacetime symmetry links bosons to fermions by introducing supersymmetric partners (sparticles) to all SM particles: squarks and gluinos are SUSY partners of quarks and gluons. The mass difference between the squark mass eigenstates depend on the mass of the corresponding quark. Hence squarks are (to a good approximation) mass degenerate for the two first generations whereas a (potentially) large mixing may appear for the third family, leading to a light stop. The former case corresponds to squarks and gluinos searches whereas dedicated searches for stop are performed for the latter.

Squarks and gluinos searches

At the TeVatron, the production cross section of squarks and gluinos is of the order of few pb. Searches have been performed in the mSUGRA model with the lightest neutralino χ0

1 as the lightest supersymmetric particle (LSP). Three regimes may be distinguished leading to three different analyses (Tab. 1). All squark species were considered except the stop (CDF and DØ) and the sbottom (CDF). The triggers retained were dijet+E / T and multijet+E / T .

Regime main decay signature production mq << mg q q q → q χ0 1 2 jets+E /T "dijet" analysis mq ∼ mg qg q → q χ0 1 at least 3 jets+E /T g → q q χ0 1 "3 jets" analysis mg << mq gg g → q q χ0 1 n(≥ 4) jets+E /T "gluino" analysis Table 1: Final states considered in the squark and gluino searches by CDF and DØ.

The main background was composed of two parts: SM processes (W/Z +jets, dibosons, t t), estimated from Monte-Carlo simulations, and the instrumental background (mismeasured jets), evaluated from data.

Selections on E / T , jets requirements and lepton vetos were made at the first stages of the analyses, followed by specific cuts for each final state.

The ultimate selection was performed on E / T and H T (defined as the p T sum of the jets). The cut values were rather different for CDF and DØ.

A good agreement was observed between data and MC expectation (Table 2) in a sample of 2 fb -1 . The main systematics uncertainties considered were: the Jet Energy scale (JES)(6-15% DØ, 15-30% CDF), the background normalization (15% DØ, 2-10% CDF), the luminosity (6%) and the ISR/FSR (6% DØ, 3-8% CDF). These uncertainties were combined to the statistical DIS 2008 ones to draw limits in the plane (m q ,m g ) (Fig. 1). For tanβ= 3, A 0 = 0, and µ < 0, squark masses lower than 392 GeV/c 2 and gluino mass lower than 327 GeV/c 2 have been excluded at 95% C.L. by DØ [2]. These values decrease to 379 GeV/c 2 and 308 GeV/c 2 for squarks and gluino respectively if theoretical uncertainties related to the Parton Distribution Function (PDF's) choice and to the renormalisation and factorisation scales are included. CDF has obtained similar results: for tanβ= 5, A 0 = 0, and µ < 0, squark masses lower than 378 GeV/c 2 and gluino mass lower than 280 GeV/c 2 have been excluded at 95% C.L. These limits include the theoretical uncertainties. CDF and DØ results are the best constraints to date on the squarks and gluino masses. In some region of the SUSY parameter space, squarks may decay to the lightest chargino χ± 1 or the second neutralino χ0

Gluino Mass (GeV

2 , leading to final states with leptons. For high tanβ values, the stau τ1 may be the lightest slepton, and squark production dominates.

Final states wit τ 's are then favoured.

A search for squarks (stop excepted) with τ 's in the final state has been performed by DØ, in a 0.96 pb -1 data sample.

Acoplanar dijets and multijets triggers were used by two analyses which were then ored after optimisation.

Events were selected on the basis of their E / T values, and two jets were to be present. Suitable angular cuts were applied to remove part of the instrumental background. At least one hadronic τ (in one of the mode: π ± ν τ , π ± π 0 ν τ , π ± π ± π ± (π 0 )ν τ ) was requested. The final optimisation was performed with two variables: sum of the transverse energy of the leading τ and the jets, and on the E / T .

A good agreement between data and SM expectations has been interpreted as a limit in the plane (m 0 , m 1/2 ) [3].

For high tanβ values, the sbottom b1 may be light. The gg pair production cross section is quite high ( σ gg ≃ 10 σ b1 b1 ) if b1 and g are mass degenerate. In this configuration, the final state resulting from the gluino pair production gg → b1 b1 b1 b1 → b bb b χ0

1 χ0 1 χ0 1 χ0
1 is comprised of 4 bjets+E / T ( χ0 1 LSP). CDF has searched this signature in a data sample of 156 pb -1 . After selecting the events with a jets+E / T trigger, standard cuts have been applied to remove the background(W/Z+jets, t t , multijets): E / T > 80 GeV, ∆φ(E / T , jets) > 2π/9.

DIS 2008

Events with at least three jets were selected and at least two b-identified. 4 events remained in data whereas 2.6±0.7 were expected. A limit in the plane (m g , m b1 ) has thus been estimated (Fig. 2 At the TeVatron, scalar tops are pair produced through gluon-gluon fusion or q q annihilation. For a stop mass of 100 (160) GeV/c 2 , 9000 (1000) events are expected at √ s = 1.96 TeV. The number of possible stop decays may be numerous, depending on the model under consideration. Two channels were considered at the TeVatron: t1 → c χ0 1 and t1 → blν l . 1 -LSP. DØ and CDF studied this signature in data sample of a luminosity of 995 pb -1 and 295 pb -1 respectively. The jets+E / T trigger was used. The search strategy involved three steps which include the application of the selection criteria on kinematical variables, heavy flavor (HF) tagging and optimization of the final selection depending on the t1 and χ0

1 masses. Fifteen selection criteria are applied to remove the multijet and the W+jets backgrounds. At the final stage of the analysis, additional criteria on three kinematic variables: E / T , S=∆φ min + ∆φ max (∆φ min (∆φ max ) is the smallest(largest) azimuthal separation between a jet and E / T ), and H T (scalar sum of the p T of all jets), are optimized by maximizing the expected lower limit on the neutralino mass for a given m t1 . Systematic uncertainties, evaluated for each t1 and χ0

1 mass combination, included jet energy scale (1.7-2%), jet energy resolution (1%), trigger (6%), scale factor(5%), HF tagging (3-4%) and the normalization of the background (10%). A good agreement has been observed between data and SM expectation. A limit has been drawn in the plane (m t1 ,m χ0 1 ) assuming that t1 decays into a charm quark and a neutralino with a 100% branching ratio. The largest stop mass excluded is 155 GeV/c 2 , for a neutralino mass of 70 GeV/c 2 (Fig. 3) [START_REF] Abazov | FERMILAB-PUB-08-063E[END_REF].

In the t1 → blν l case, νl is considered to be the LSP. The most promising final state is DIS 2008 t1 t1 → b be ± µ ± νe νµ : the cross section times the branching ratio is twice the one of the same flavor case, and the SM background is also lower. The signal characteristics (lepton or b-jet p T 's, E / T spectrum) depend both on the stop mass and on the difference between the stop mass and the sneutrino mass ∆m = m t1 -m ν : the p T 's decrease with ∆m . For a given ∆m , E / T values increase with the stop mass. For a stop mass, E / T values increase with ∆m . A search for t1 t1 → b be ± µ ± νe νµ has been performed by DØ in a 1 fb -1 data sample.

A leptonic trigger (electron+muon) has been used. The SM background was composed of Z/γ * → τ + τ -, multijets, WW and t t. Events were selected with the following criteria: one electron and one muon with p T bigger than 15 GeV/c, E / T bigger than 30 GeV. Additional cuts were applied to remove Drell-Yan and instrumental background events. After the last selection, 61 events survived in data whereas 64±4 were expected (mostly WW and t t events). Limits were drawn in the plane (m t1 ,m ν ) assuming that the stop decays to blν l with a 100% branching ratio. A stop mass of 175 GeV/c 2 has been excluded if m ν = 45 GeV/c 2 (Fig. 3).

(GeV)

Conclusion

The most constraining direct limits on squark and gluino masses to date have been obtained at the TeVatron: squark masses lower than 392 GeV/c 2 and gluino mass lower than 327 GeV/c 2 have been excluded at 95% C.L. for tanβ= 3, A 0 = 0, and µ < 0. Stop pair production has been searched in the c χ0 1 and blν l channels. No excess with respect to the Standard Model expectations has been observed and mass regions have been excluded. CDF and DØ have performed an impressive set of SUSY research and further information may be found on the public web pages of the two experiments [6].

Figure 1 :

 1 Figure 1: Limits on squark and gluino masses for DØ (top) and CDF (bottom).

Figure 2 :

 2 Figure 2: CDF limit in the gluino decaying to sbottom search.

Figure 3 :

 3 Figure 3: Stop limits for the c χ0 1 case (top) and the blν l analysis (bottom).

  ). Systematic uncertainties (JES, luminosity, b-tagging efficiency and rate, cross sections) were taken into account. A gluino mass lower than 280 GeV/c 2 has been excluded for a sbottom mass of 230 GeV/c 2[4].

	2 Stop searches
			dijet		3 jets		gluino
		H T > 330 E / T >225	H T >375 E / T >175	H T > 400 E / T >100
	DØ	11	11.1±1.2	9	10.7±0.9	20	17.7±1.1
		H T > 330 E / T >180	H T >330 E / T >120	H T > 280 E / T >90
	CDF	18	16±5	38	37±12	45	47±17

Table 2 :

 2 Final selections for the squarks and gluino searches.

Acknowledgement

Special thanks to my colleagues from CDF and DØ for providing the results. I also thank the organizers of the conference for their hospitality.


Mauro Salvi

Valentina Battaglia

Mario Mancon

Sebastiano Colombatto

Carlo Cravanzola

Rita Calheiros

Maria Mp Marques

Maria A Grillo

Rita Calheiros #

Maria P M Marques #

Antonio Toninello
email: antonio.toninello@unipd.it

Agmatine is transported in liver mitochondria by a specific electrophoretic mechanism

Keywords: Mitochondria, agmatine, transport, polyamines

INTRODUCTION

Agmatine [1-(4-aminobutyl)guanidine], the biogenic amine formed by decarboxylation of arginine catalyzed by arginine decarboxylase (ADC), is known to bind to α 2 -adrenergic and imidazoline receptors, and to have properties as a neurotransmitter or neuromodulator (for a review, see [START_REF] Reis | Is agmatine a novel neurotransmitter in brain?[END_REF]).

Clinical properties have been suggested, such as a neuroprotective effect, counteraction of tolerance to opiates [START_REF] Kolesnikov | Modulation of opioid analgesia by agmatine[END_REF] and tumor suppression [START_REF] Satriano | An emerging role for agmatine[END_REF]. Many other biochemical effects have also been shown: for instance, agmatine induces the ornithine decarboxylase antizyme [START_REF] Satriano | Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels[END_REF][START_REF] Babal | Regulation of ornithine decarboxylase activity and polyamine transport by agmatine in rat pulmonary artery endothelial cells[END_REF] and spermidine/spermine acetyltransferase (SSAT) [START_REF] Vargiu | Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase[END_REF] and thus influences polyamine homeostasis; it inhibits nitric oxide synthase (NOS), most potently the inducible form [START_REF] Galea | Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formed by decarboxylation of arginine[END_REF]. It may also be a precursor of polyamines, when it is hydrolyzed to putrescine by agmatinase (AGMase) [START_REF] Mistry | Cloning of human agmatinase. An alternate path for polyamine synthesis induced in liver by hepatitis B virus[END_REF].

In mammals agmatine is not only formed "in situ" by ADC, but may also be taken up by exogenous sources, as it is known to be present in food and to be produced by the intestinal flora [START_REF] Raasch | Agmatine, the bacterial amine, is widely distributed in mammalian tissues[END_REF]. It follows that transport of agmatine across the plasma membrane may contribute to regulation of its cytosol concentration and therefore its biological action. Generally, after being absorbed from the stomach by means of an energy-dependent mechanism, it is taken up by several organs, but particularly by the liver [START_REF] Molderings | Exposure of rat isolated stomach and rats in vivo to [(14)C]agmatine: accumulation in the stomach wall and distribution in various tissues[END_REF]. The mechanism involved has been studied in various cell types such as hepatocytes in primary culture [START_REF] Cabella | Transport and metabolism of agmatine in rat hepatocyte cultures[END_REF], endothelial [START_REF] Babal | Interactions between agmatine and polyamine uptake pathways in rat pulmonary artery endothelial cells[END_REF] and kidney cells [START_REF] Valle | Agmatine uptake by cultured hamster kidney cells[END_REF], and several other mammalian cell types [START_REF] Satriano | Polyamine transport system mediates agmatine transport in mammalian cells[END_REF] which take up agmatine using the same transporter as polyamines. In contrast, agmatine and putrescine appear to be transported by different carriers in gliomal cells [START_REF] Molderings | Agmatine and putrescine uptake in the human glioma cell line SK-MG-1[END_REF] and in a cell line derived from human embryonic kidney, in which the organic cation transporter (OCT-2) and the extraneural monoamine transporter (EMT) are used [START_REF] Grundemann | Agmatine is efficiently transported by non-neuronal monoamine transporters extraneuronal monoamine transporter (EMT) and organic cation transporter 2 (OCT2)[END_REF].

Agmatine has recently been found to be present in neuronal mitochondria [START_REF] Gorbatyuk | Localization of agmatine in vasopressin and oxytocin neurons of the rat hypothalamic paraventricular and supraoptic nuclei[END_REF]. ADC and AGMase have also been located in mitochondria [START_REF] Regunathan | Characterization of arginine decarboxylase in rat brain and liver: distinction from ornithine decarboxylase[END_REF][START_REF] Horyn | Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine[END_REF][START_REF] Sastre | Agmatinase activity in rat brain: a metabolic pathway for the degradation of agmatine[END_REF] as well as the I 2 imidazoline receptor, which binds agmatine and, in particular, is located on monoamine oxidase (MAO) [START_REF] Tesson | Localization of I2-imidazoline binding sites on monoamine oxidases[END_REF]. All these observations, revealing close relationships between this amine and mitochondria, led us to examine whether agmatine could be transported in these organelles and, if so, to characterize the mechanism and establish if uptake is mediated by some known transporter, e.g., that of polyamines.

MATERIALS AND METHODS

Rat liver mitochondria (RLM) were isolated in 0.25 M sucrose and 5 mM Hepes (pH 7.4) by conventional differential centrifugation [START_REF] Schneider | Intracellular distribution of enzymes. V. Further studies on the distribution of cytochrome c in rat liver homogenates[END_REF]. Mitochondrial protein concentration was assayed by a biuret method with bovine serum albumin as a standard [START_REF] Gornall | Determination of serum proteins by means of the biuret reaction[END_REF].

Incubations were carried out at 20°C with 1 mg of mitochondrial protein/ml in the following standard medium: 250 mM sucrose, 10 mM Hepes-HCl (pH 7.4), 5 mM succinate, and 1.25 µM rotenone. Sodium salts were used. Other additions are indicated in the descriptions of specific experiments. A sucrose-based medium was chosen in order to compare obtained results with those of polyamine transport (for a review, see Toninello et al. [START_REF] Toninello | Interaction of biologically active amines with mitochondria and their role in the mitochondrial-mediated pathway of apoptosis[END_REF]). However, the effects of higher ion strength media are also reported (Fig. 2, inset B). Uptake of [ 14 C]agmatine and [ 14 C]spermine was determined by a centrifugal filtration method, as previously described [START_REF] Toninello | Uptake of spermine by rat liver mitochondria and its influence on the transport of phosphate[END_REF]. Uptake of [ 14 C]agmatine was also measured by an HPLC method [START_REF] Cabella | Transport and metabolism of agmatine in rat hepatocyte cultures[END_REF] (demonstrating a similar trend to that mentioned above) as well as the presence in RLM of [ 14 C]guanidobutyric aldehyde and [ 14 C]putrescine. Hydrogen peroxide was measured fluorometrically by the scopoletin method [START_REF] Loschen | Mitochondrial H 2 O 2 formation: relationship with energy conservation[END_REF].

Membrane potential (∆Ψ) was measured in an open, thermostatically controlled, stirred vessel by monitoring the distribution of the lipophilic cation tetraphenylphosphonium (TPP + ) across the mitochondrial membrane with a selective electrode, prepared in our laboratory according to published procedures [START_REF] Kamo | Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state[END_REF][START_REF] Toninello | On the mechanism of spermine transport in liver mitochondria[END_REF] and an Ag/AgCl reference electrode. ∆Ψ values were corrected as proposed by Jensen et al. [START_REF] Jensen | The efficiencies of the component steps of oxidative phosphorylation. II. Experimental determination of the efficiencies in mitochondria and examination of the equivalence of membrane potential and pH gradient in phosphorylation[END_REF]. Mitochondrial matrix volume was calculated from the distributions of [ 14 C]sucrose and 3 H 2 O according to Palmieri and Klingenberg [START_REF] Palmieri | Direct methods for measuring metabolite transport and distribution in mitochondria[END_REF]. ∆pH was calculated from the distribution of [ 14 C]5,5'-dimethyl-oxazolidine-2, 4-dione (DMO) across the mitochondrial membrane [START_REF] Rottenberg | The measurement of membrane potential and deltapH in cells, organelles, and vesicles[END_REF].

Kinetic parameters from initial rate measurements were estimated applying the analysis reported by Reich et al. [START_REF] Reich | A general strategy for parameter estimation from isosteric and allosteric kinetic data and binding measurements[END_REF].

Binding parameters were by applying thermodynamic treatment of ligand receptor interactions [START_REF] Via | Spermine binding to liver mitochondria[END_REF].

Scatchard analyses were performed using Eq. [START_REF] Reis | Is agmatine a novel neurotransmitter in brain?[END_REF].

[ ] [ ] { }         + ⋅ - = ∑ = ) ( 1 ] [ ] [ ) ( 1 , 1 max, F K B B F B i t i s i i i ε [1]
where:

∑ ∏ = = - = i n k k j j i k i t K F F 2 1 , 1 ) ( ] [ ) ( ε
represents the appropriate measure of the extent of multiple coordination on the i-th sites. [B max, i ] is the maximum concentration of i-th sites which may be bound by the ligand, [B i ] is the concentration of i-th sites bound by the ligand, [B max ] is the maximum receptor-bound ligand concentration, [F] is the free ligand concentration, K i,j (t) is the affinity constant of the ligand for the i-th site, j is the occupancy number, and t is time.

Fitting was performed as described earlier [START_REF] Via | Spermine binding to liver mitochondria[END_REF].

The distribution of total bound agmatine on its binding sites was calculated by parameter Xi(F), obtained by means of Eq. 2:

] [ 1 1 ] [ ] [ ] [ ] [ ) ( max max, F B B B B F X i i i i β + = - - = [2]
where β i , is a parameter describing the influence of the parallel filling of the other k-th sites in comparison with filling of the i-th site [START_REF] Via | Spermine binding to liver mitochondria[END_REF].

[ 14 C]agmatine was synthetized as reported in [START_REF] Cabella | Transport and metabolism of agmatine in rat hepatocyte cultures[END_REF].

RESULTS

At physiological pH, agmatine is a diamine with two net positive charges (Fig. 1A), since it has the lower pKa value of 9.07 [START_REF] Grundemann | Agmatine is efficiently transported by non-neuronal monoamine transporters extraneuronal monoamine transporter (EMT) and organic cation transporter 2 (OCT2)[END_REF]. It may thus be considered as a divalent cation. Fig. 1B shows the most stable of the monovalent forms, present at very high pH.

As shown in Fig. 2, RLM, incubated in standard medium, takes up about 50 nmol/mg prot of

[ 14 C
]agmatine in 30 min of incubation. In the presence of FCCP or antimycin A, which completely collapse ∆Ψ (Fig. 2, inset A), gradual transport of the diamine is completely inhibited. An almost identical effect is observed when the medium is deprived of succinate (results not reported). Indeed, in the presence of KCl, significant inhibition may also be observed. The inset B of Fig. 2 shows the dose-dependent effect of saline-sucrose media.

It should be emphasized that no oxidation products of agmatine such as H 2 O 2 or [ 14 C]guanidobutyric aldehyde were detected; nor was [ 14 C]putrescine, a catabolic product of agmatinase activity [START_REF] Mistry | Cloning of human agmatinase. An alternate path for polyamine synthesis induced in liver by hepatitis B virus[END_REF] (results not reported).

The inhibition of agmatine transport by de-energizing conditions indicates that it is energydependent and requires an electrochemical gradient (∆µ H + ). This observation led us to identify which component of ∆µ H + , ∆Ψ or ∆pH (or both) drives agmatine uptake, i.e., to verify if the mechanism is electrophoretic or electroneutral in nature.

Fig. 3 shows that, in the presence of phosphate (Pi), which raises the ∆Ψ value from 150 to 180 mV (Fig. 3, inset), the amount of agmatine taken up by mitochondria is about 80 nmol/mg prot in 30 min of incubation. Furthermore, if RLM are incubated with the K + /H + exchanger, nigericin, instead of Pi -a condition which enhances ∆Ψ up to 200 mV (Fig. 3, inset) and completely collapses ∆pHagmatine uptake is further increased in both initial rate and final extent. In contrast with the effect of Pi and nigericin, treatment of RLM with the ionophore valinomycin, in the presence of K + -a condition which completely collapses ∆Ψ (Fig. 3, inset) and raises ∆pH -completely inhibits the uptake of the diamine (Fig. 3), as in the de-energizing conditions of Fig. 2. Fig. 3 Copyright 2006 Biochemical Society addition of agmatine to RLM (Fig. 4), also demonstrating that agmatine transport occurs in conditions of high ∆µ H + and that the driving force is ∆Ψ.

The results of Fig 5A show the dependence of the initial rate of agmatine transport on ∆Ψ which exhibits non-ohmic conductance, comparable to that of divalent putrescine [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF]. A first consideration arising from this comparison is that agmatine exhibits significantly higher initial rates of transport than putrescine with increasing ∆Ψ values.

The equation for influx rate J into the mitochondrial matrix of a cation is:

RT F z e J J / 0 ∆Ψ = β [3]
where J 0 is the exchange flux (transport rate at ∆Ψ = 0), z is the amine valence, and β is a parameter giving the shape and position of the energy barrier(s) for cation transport. It should be noted that, for sharp barriers, β equals the fractional distance from the external side of the membrane to the peak of the first barrier [START_REF] Garlid | On the nature of ion leaks in energytransducing membranes[END_REF].

The observation of an apparently exponential relationship between agmatine transport and ∆Ψ means that flux-voltage analysis can be applied to its transport, as also previously done for the polyamines [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF] and here re-calculated also for putrescine for the best comparison. In this regard, (400 is the inner membrane surface area measured in cm 2 /mg prot [START_REF] Mitchell | Regulation of Metabolic Processes in Mitochondria[END_REF]). Assuming that agmatine can cross the membrane with its net charge, at pH 7.4, 1.98 (the pK values of agmatine are 9.07 and >13 [START_REF] Grundemann | Agmatine is efficiently transported by non-neuronal monoamine transporters extraneuronal monoamine transporter (EMT) and organic cation transporter 2 (OCT2)[END_REF]), and also as a monovalent cation, the value of β can be estimated by the relative slope zβ of the curves shown in Fig. 5B. All the above parameters for agmatine and putrescine are listed in Table I.

The β value of agmatine, 0.25, is very close to those of putrescine and the other polyamines [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF] and is the theoretical value for a channel [START_REF] Garlid | On the nature of ion leaks in energytransducing membranes[END_REF]. Instead, the value of β, 0.5, is more appropriate for leaks or carriers (see below).

The initial rate of agmatine transport was also measured at varying temperatures from a linear Arrhenius plot in the range 5-35 °C (Fig. 6). The resulting slope gives the activation enthalpy, E a , for agmatine transport. The obtained value of 22.5 kJ/mol is very close to that of putrescine (24 kJ/mol), previously calculated [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF]. Also of note is the fact that the activation enthalpy/mol per charge of agmatine (E a /z), which is 11.25 kJ, is very close not only to that of putrescine (12 kJ) but also to those of spermidine (10.6 kJ) and spermine (14.5 kJ) [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF].

In mitochondrial preparations with ∆Ψ values of about 180 mV (presence of P i ), agmatine transport exhibits saturation kinetics, as illustrated by the apparent hyperbolic curve obtained in a typical experiment (Fig. 7A). Estimation of kinetic parameters from initial rate measurements gives K m and V max of 0.7 mM and 6.32 nmol/min/mg prot, respectively. For comparison, the K m and V max of putrescine transport are 1 mM and 1.14 nmol/min/mg prot, respectively [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF]. All these observations indicate that the transporter of agmatine may be the same as that of polyamine [START_REF] Toninello | On the mechanism of spermine transport in liver mitochondria[END_REF][START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF].

The experimental results shown in Fig. 8 aim at solving this question and at verifying if agmatine also uses other transporters, e.g., those of basic aminoacids, monoamines, or other monovalent cations. In this regard, polyamines, and the above-mentioned cationic molecules were used as possible inhibitors of agmatine transport. Fig. 8A shows that putrescine and cadaverine, also divalent, at 1 mM concentration, the same used for agmatine, completely fail to inhibit the transport of agmatine. It should be noted that both polyamines inhibit the initial binding of agmatine. Fig. 8A also shows that tetravalent spermine, at the same 1 mM concentration, exhibits marked inhibition of this binding and transport. However, this later effect is most probably due to the drop in ∆Ψ caused by spermine transport (Fig. 8A, inset) rather than to direct interaction between the two amines. It should be noted that neither putrescine nor cadaverine affect the ∆Ψ value (Fig. 8A, inset). Fig. 8A shows that agmatine transport is also not affected by 1 mM ornithine, lysine, arginine. An identical lack of efficacy is exhibited by the monoamines tyramine and benzylamine, and the monovalent cation tetraethylammonium (TEA) (Fig. 8B). None of these molecules affect ∆Ψ (results not reported).

Fig. 9 shows that strong inhibition is observed with the propargylamines pargyline and clorgyline, inhibitors of MAO activity. It should be emphasized that they are completely ineffective in inhibiting spermine transport (Fig. 9, inset A). These inhibitors do not in fact affect ∆Ψ (Fig. 9, inset B). It is noteworthy that propargylamines have a single protonated amino group [START_REF] De Marchi | L-Deprenyl as an inhibitor of menadione-induced permeability transition in liver mitochondria[END_REF], so that their inhibition sustain the hypothesis that agmatine is transported as a monovalent rather than a divalent cation.

As agmatine is able to bind to the I 2 imidazoline receptor located on the mitochondrial membrane [START_REF] Tesson | Localization of I2-imidazoline binding sites on monoamine oxidases[END_REF], the experiment shown in Fig. 9 was also performed with the aim of verifying whether this receptor is involved in agmatine transport. Results show that the I 2 inhibitor idazoxan does not prevent its net transport but completely inhibits the initial binding of agmatine (see extrapolation of transport traces at zero time, Fig. 9).

The observation that propargylamines strongly inhibit the transport of agmatine led us to identify the type of inhibition induced. The results of Fig. 7A show that clorgyline, at 50 µM concentration, causes marked inhibition of the initial rate of agmatine transport and that this inhibition is of noncompetitive type as demonstrated by the double reciprocal plot of Fig. 7B. In this case K m is 0.71 mM and V max 2.01 nmol/mg prot/min.

All experiments reporting agmatine transport (e.g., see Figs. [START_REF] Kolesnikov | Modulation of opioid analgesia by agmatine[END_REF][START_REF] Satriano | An emerging role for agmatine[END_REF][START_REF] Mistry | Cloning of human agmatinase. An alternate path for polyamine synthesis induced in liver by hepatitis B virus[END_REF][START_REF] Raasch | Agmatine, the bacterial amine, is widely distributed in mammalian tissues[END_REF] show that the gradual phase of agmatine accumulation (in the absence of effectors) is preceded by a phase of very rapid uptake of approximately 15 nmol/mg prot, as calculated by rough extrapolation of the curves at zero time (see also description of results, Fig. 9). Indeed, as Fig. 2 shows, this uptake is sensitive to the presence of KCl. This almost instantaneous uptake is very probably due to the electrostatic binding of agmatine to the accessible surface of mitochondrial membranes, according to the following considerations: i) it also occurs in de-energizing conditions (see Fig. 2); ii) addition of 5 mM cold agmatine during the accumulation of the labeled diamine induces the very fast loss of an amount almost identical to that already bound at zero time (Fig. 2).

The observation that propargylamines inhibit agmatine transport with no significant inhibition of initial binding (see extrapolations at zero time, Fig. 9) and that idazoxan behaves in the opposite way, indicates more than one binding site for agmatine on the mitochondrial membrane. The experiments of Fig. 10 were performed in order to clarify this point. Fig. 10A shows the different amounts of agmatine that bind to the mitochondrial membranes at zero time, as a function of the total, external agmatine concentration. As shown, binding of the amine tends towards saturation.

Fig. 10B, showing results of Scatchard analysis of agmatine binding, demonstrate that the theoretical curve for agmatine that satisfactorily fits the experimental data (Fig. 10B) is typical for two binding sites, S 1 and S 2 , both with monocoordination. Calculated total concentration of bound agmatine (B), 83.20±0.04 nmol/mg prot, is distributed between S 1 (B max1 = 3.20±0.03 nmol/mg prot) and S 2 (B max2 = 80.05±0.02 nmol/mg prot) at 3.85% and 96.15%, respectively. Dissociation constants (K 1 = 25±3 nmol/mg prot and K 2 = 4937±8 nmol/mg prot) of S 1 and S 2 , respectively, demonstrate that S 1 has an affinity about 200 times higher than that of S 2 . Comparisons with the binding parameters of polyamines [START_REF] Via | Binding of spermidine and putrescine to energized liver mitochondria[END_REF] show that both sites have high binding capacity and low affinity.

As the targets of propargylamines and idazoxan, that is MAO and I 2 receptor respectively, are located on the outer membrane, in order to establish if their effects are related to these interactions, agmatine transport was also determined in mitoplasts compared with mitochondria. However, results (not reported) showed that agmatine is taken up by mitoplasts at the same rate and to the same extent as mitochondria, indicating that the effects of the inhibitors are not due to their interactions with the outer membrane and that the transport process involves neither MAO nor the I 2 receptor. The results reported here provide evidence that agmatine is capable of binding to mitochondrial membranes and of being taken up into the matrix space of RLM. This binding, most probably electrostatic in nature, takes place in two sites S 1 and S 2 (Fig. 10), is affected by natural polyamines (Fig. 8A), idazoxan (Fig. 9) and saline media (Fig. 2), and is unaffected by de-energizing agents (Fig. 2), cationic aminoacids (Fig. 8A), monoamines or monovalent cations (Fig. 8B). Agmatine binding is followed by slow, long-lasting uptake which is highly dependent on mitochondrial energization (Figs. 2,3) and electrophoretic in nature (Figs. 3,4). It also exhibits a non-linear current voltage relationship (Fig. 5A), fitting the general behavior of monovalent and polyvalent cations (e.g. polyamines) in mitochondria [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF][START_REF] Garlid | On the nature of ion leaks in energytransducing membranes[END_REF].

DISCUSSION

Considering flux-voltage analysis (Fig. 5), it should be noted that agmatine, depending on the valence of transported species, can exhibit two different β values (Table I). The calculation of β=0.25 for the divalent form, very close to those of putrescine (Fig. 5B, Table I) and the other polyamines, suggests that agmatine is transported by a channel with two energy barriers, like that of polyamines [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF].

This possibility is also supported by the calculation of the intrinsic permeability coefficient, P (Fig. 5, Table I). This value, 26.8 • 10 -10 cm/sec, is higher than that of putrescine (Table I) and those of spermidine and spermine [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF], but of their same order of magnitude. These permeabilities are very similar to that of the monovalent cation TEA -3.54•10 -10 cm/sec -which is transported by a leak pathway [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF]. However, taking into account the divalency of agmatine, its permeability is very high, suggesting the presence of a uniport transporter which may be a channel.

This hypothesis is also strengthened by the E a /z value of this uptake, which is 11.25 kJ/mol (Fig. 6),

i.e., very similar to that of polyamines, which have an average value of 12.6 kJ/mol [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF]. These values are much lower than that of TEA, which is 76 kJ/mol [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF], and are comparable to that of the Ca 2+ channel, which is 20 kJ/mol [START_REF] Bragadin | Activation energies and enthalpies during Ca 2+ transport in rat liver mitochondria[END_REF]. Indeed, the E a for agmatine, 22.5 kJ/mol, is much lower than those of several mitochondrial carriers, which range from 64 to 92 kJ/mol [START_REF] Mende | Reconstitution of the isolated phosphate-transport system of pig-heart mitochondria[END_REF][START_REF] Palmieri | Bioenergetics and Mitochondrial and Cellular Levels[END_REF].

Force-flux analysis also demonstrates that monovalent agmatine is taken up by a transport system having a β value of 0.5 [Fig. 5B,Table I]. Bearing in mind that activation enthalpy values as low as 20-30 kJ/mol have also been identified for secondary transporters in mitochondria [42][START_REF] Chan | Adenine nucleotide transport in hepatoma mitochondria. Characterization of factors influencing the kinetics of ADP and ATP uptake[END_REF][START_REF] Zhang | Effect of the lipid phase transition on the lactose permease from Escherichia coli[END_REF], this β value may be applied to a single-binding center-gated pore, of which a typical example is the ATP/ADP carrier [START_REF] Klingenberg | Ligand-protein interaction in biomembrane carriers. The induced transition fit of transport catalysis[END_REF]. The observation that the initial rate of agmatine transport is higher than that of putrescine (Fig. 5A) but also that of the other polyamines [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF] is ascribed to the fact that agmatine in its divalent form (Fig. 1A) has a dipole moment, µ=5.1 D [START_REF] Toninello | Structural characterization of agmatine at physiological conditions[END_REF], whereas the polyamines have µ=0. In this regard, it should be noted that the monovalent agmatine [Fig. 1B] has a very high dipole moment (µ=15.8 D) [START_REF] Toninello | Structural characterization of agmatine at physiological conditions[END_REF], suggesting that this form may be predominant in transport. As the pH value of the channel or gated pore environment is not known, the question of whether the monovalent or divalent form of agmatine is actually transported is not solved. Because this information is critical for determining parameter β, the nature of the transport system still remains open.

As previously demonstrated, the polyamine transporter is common to all natural polyamines, so that they reciprocally inhibit their transport in a competitive manner [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF]. Hence, putrescine, cadaverine, and spermine should exhibit the same type of inhibition on agmatine transport. Observations that the divalent putrescine and cadaverine are ineffective (Fig. 8A), indicate the existence of different transport systems for agmatine and polyamines, rather than a single common one. Indeed, the results of Fig. 8A clearly exclude the possibility that agmatine can use the electroneutral transporter of basic amino acids [47,[START_REF] Palmieri | The mitochondrial transporter family (SLC25): physiological and pathological implications[END_REF]. Fig. 8B shows that TEA, tyramine and benzylamine, like other monoamines (results not shown), fail to exhibit any inhibition, suggesting that the leak pathway for monovalent cations [START_REF] Toninello | Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter[END_REF] and the transporter for monoamines (A. Toninello, unpublished results), both electrophoretic, are not involved. Strong inhibition of agmatine transport is observable with clorgyline and pargyline, which act as non-competitive inhibitors of transport and independently of this action on MAO [Fig. 9]. Observations that some compounds, e.g., putrescine, cadaverine [Fig. 8A] and idazoxan [Fig. 9], reduce initial binding without affecting transport, whereas others, such as propargylamines, inhibit transport without inhibition of initial binding, indicate that there are at least two types of binding sites for agmatine on mitochondrial membranes. The results shown in Fig. 10 clearly confirm the existence of two binding sites, S 1 and S 2 which, as also observed for polyamine binding [START_REF] Via | Spermine binding to liver mitochondria[END_REF][START_REF] Via | Binding of spermidine and putrescine to energized liver mitochondria[END_REF], exhibit monocoordination, with high binding capacity and low binding affinity.

The non-competitive inhibition exhibited by clorgyline on agmatine transport (Fig. 7B) excludes the possibility that both molecules are taken up by the same transporter. The observation that clorgyline does not completely inhibit agmatine transport (Fig. 7A) is consistent with residual binding of agmatine to its transporter, putatively identified as binding site S 1 . This site, in fact, has the same characteristics as the polyamine binding site involved in transport [START_REF] Via | Spermine binding to liver mitochondria[END_REF].

In conclusion, we report the existence of a specific selective transport system for agmatine in RLM, which may be a channel or, alternatively, a single-binding center-gated pore.

These results are considered of physiological importance, since the agmatine concentrations used (0.1-1 mM) and the K m calculated for transport (0.7 mM) are compatible with the concentration of agmatine normally measured in liver cells, i.e., 0.5 mM [START_REF] Lortie | Bioactive products of arginine in sepsis: tissue and plasma composition after LPS and iNOS blockade[END_REF]. It should also be taken into account that fluctuations in this concentration may be observed in various physiological and pathological situations [START_REF] Galea | Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formed by decarboxylation of arginine[END_REF]. For example, it may increase by about 20 times, as observed in rat aorta after ischemic injury [50]. In particular, agmatine transport in RLM is able to induce apoptosis in hepatocytes by activating caspase 3 [START_REF] Gardini | Agmatine induces apoptosis in rat hepatocyte cultures[END_REF], due to release of cytochrome c from mitochondria promoted by agmatine, as a result of the mitochondrial permeability transition (MPT) induced in the presence of phosphate and high Ca 2+ concentration. It is generally believed that MPT is closely connected with programmed cell death [for recent reviews, see Refs. 24, 52], the main role of which is probably to counteract tumor growth.

Another important point to be considered in evaluating the role of agmatine transport in RLM is the presence in these organelles of both biosynthetic and catabolic agmatine enzymes, such as ADC [START_REF] Regunathan | Characterization of arginine decarboxylase in rat brain and liver: distinction from ornithine decarboxylase[END_REF][START_REF] Horyn | Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine[END_REF] (the presence of ADC is strongly debated [START_REF] Coleman | Putrescine biosynthesis in mammalian tissues[END_REF]), and AGMase [START_REF] Sastre | Agmatinase activity in rat brain: a metabolic pathway for the degradation of agmatine[END_REF]. In this regard, it is noteworthy that the presence of agmatine in cells is particularly high in liver [START_REF] Lortie | Bioactive products of arginine in sepsis: tissue and plasma composition after LPS and iNOS blockade[END_REF]. Thus, identification of AGMase in the matrix of RLM indicates the great importance of finding a transport system for agmatine located on mitochondria. AGMase may represent a mechanism for regulating agmatine signaling properties in RLM via changes in agmatine levels in the organelles [START_REF] Morris | Vertebrate agmatinases: what role do they play in agmatine catabolism?[END_REF].

The observation that agmatine is an inhibitor of nitric oxide (NO) synthesis [START_REF] Galea | Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formed by decarboxylation of arginine[END_REF] raises the question of the important function of agmatine and AGMase at mitochondrial level. NO is produced by a group of NO synthases (NOSs) including three isoforms [START_REF] Nathan | Nitric oxide as a secretory product of mammalian cells[END_REF][START_REF] Marletta | Nitric oxide synthase: aspects concerning structure and catalysis[END_REF]. Isoform mtNOS is constitutively present in the mitochondrial matrix, and is involved in altered mitochondrial regulation during hypoxia [START_REF] Lacza | Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia[END_REF]. Indeed, mitochondrial NO has been identified as a physiological regulator of electron flux and ATP synthesis by inhibiting cytochrome c oxidase (COX) [START_REF] Carreras | Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease[END_REF]. Increase in mitochondrial NO may follow the induction and activation of mtNOS, and is important in mitochondrial pathology, including the effects of aging, inflammation and cancer [START_REF] Carreras | Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease[END_REF]. In this regard, it should be emphasized that mtNOS activity may be regulated by changes in agmatine matrix concentration, as the result of concerted activity by agmatine transporter and agmatinase [START_REF] Carreras | Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease[END_REF]. In conclusion, the presence of agmatine in the mitochondrial matrix, if it inhibits mtNOS, favors ATP synthesis, whereas its fall in concentration by AGMase activity facilitates NO production, with consequent inhibition of ATP synthesis (see Fig. 11).

The work was made possible by the award of a COST (European Science Foundation) STSM to V.B., and a PhD fellowship from the Portuguese Foundation for Science and Technology to R.C. Amine uptake values are corrected for instantaneous electrostatic binding [START_REF] Toninello | On the mechanism of spermine transport in liver mitochondria[END_REF]. Uptake of agmatine and putrescine was linear over incubation period. A typical experiment is reported. Six experiments were carried out with each compound, yielding almost identical results. [ C]agmatine uptake (nmol/mg prot)

1 4

[ C]spermine uptake (nmol/mg prot)

1 4

[ C]spermine uptake (nmol/mg prot)

Fig. 5B shows

  Fig. 5B shows the semi-logarithmic plot of the data of Fig. 5A. This gives an estimate of rate constant k and intrinsic permeability coefficient P, since kc J = 0

Figure 1

 1 Figure 1 Agmatine structure as divalent (A) or monovalent (B) cationStructures determined by ab initio molecular orbital calculations, coupled to Raman spectroscopy[START_REF] Palmieri | Bioenergetics and Mitochondrial and Cellular Levels[END_REF].

Figure 2

 2 Figure 2 Agmatine uptake by rat liver mitochondria. Dependence on energized state RLM were incubated in standard medium, as described in Materials and Methods, with 1 mM [ 14 C]agmatine (50 µCi/mmol). When present in medium: 0.1 µg/mg prot FCCP, or 1 µg/mg prot antimycin A (Ant. A). KCl 50 mM, sucrose 150 mM. Where indicated, 5 mM cold agmatine was added. Dotted line and empty circle on ordinate axis: extrapolation of agmatine binding at zero time. Mean values ±SD of five experiments. Inset A: de-energizing effect of FCCP and Ant. A, obtained by incubating RLM in standard medium without agmatine. ∆E: electrode potential. Inset B: dose-depedent inhibition by KCl on agmatine transport. RLM were incubated in standard medium, final concentration together with sucrose was maintained at 250 mOsM. Results show amount of agmatine uptake after 30 min of incubation.

Figure 3

 3 Figure 3 Effect of phosphate, nigericin and valinomycin + K + on agmatine uptake and fluxvoltage relationships RLM were incubated in standard medium containing 1 mM [ 14 C]agmatine (50 µCi/mmol). When

Figure 5 Flux

 5 Figure 5 Flux-voltage relationship (A) and flux-voltage analyses (B) for agmatine and putrescine Panel A: Agmatine and putrescine fluxes (nmol/min/mg prot) (J) plotted versus ∆Ψ. RLM were incubated for 5 min in standard medium with 1 mM P i . [ 14 C]agmatine or [ 14 C]putrescine (50 µCi/mmol) at 1 mM concentration. ∆Ψ was manipulated by including limiting amounts (5-60 nM) of FCCP. Highest values of amine uptake were achieved by adding nigericin (0.33 µg/mg prot.).

Panel B :

 : Log-linear plots of data of panel A. Linear regression yielded values for slopes of curves representing product zβ (see equation 3). Intercepts of curves on ordinate axis gave values of ln J 0 .

Figure 6

 6 Figure 6 Arrhenius plot of agmatine transport RLM with ∆Ψ 180 mV were incubated for 5 min over temperature range of 5-35 °C in standard medium with 1 mM P i . [ 14 C]agmatine (50 µCi/mmol) added at concentration of 1 mM. Binding correction as in Fig. 5A. Uptake was linear over incubation period. A typical experiment is reported. Five other experiments gave almost identical E a values.

Figure 7

 7 Figure 7 Saturation kinetics (A) and double reciprocal plot (B) of agmatine uptake. Inhibitory effect by clorgyline Panel A: RLM were incubated for 5 min in standard medium with 1 mM Pi and [ 14 C]agmatine (50 µCi/mmol) at indicated concentrations. When present, clorgyline was at 1 mM concentration. Uptake of agmatine was linear over incubation period. Binding correction as in Fig. 5. Mean values ±SD of five experiments are reported. Panel B: Double reciprocal plot of data of panel A. Inset: values of apparent K m and V max calculated by computer simulation.

Figure 8 4 4

 84 Figure 8 Effect of polyamines and basic amino acids (A) and monoamines and TEA (B) on agmatine uptake

B 40 AGMFigure 11

 4011 Figure 9

Biochemical Journal Immediate Publication. Published on 2 Mar 2006 as manuscript BJ20060003

  clearly demonstrates that agmatine transport by RLM is electrophoretic and excludes the involvement of ∆pH. This statement is further confirmed by the gradual drop of about 20 mV after 20 min of incubation in the ∆Ψ value, paralleled by an identical increase in the 58 ∆pH value upon the

Journal Immediate Publication. Published on 2 Mar 2006 as manuscript BJ20060003 Copyright 2006 Biochemical Society and

  14 C]agmatine(50 µCi/mmol). When present, 1 mM phosphate (P i ), 0.33 µg/mg prot. nigericin (nig), 0.33 µg/mg prot. valinomycin (val) 10 mM KCl. Empty circle on ordinate axis: agmatine bound at zero time. Mean values ±SD of six experiments. Inset: results obtained without agmatine, showing changes in ∆Ψ due to different effectors.

	Biochemical Figure 4 Changes in ∆Ψ ∆Ψ ∆Ψ ∆Ψ and 58∆ ∆ ∆ ∆pH values induced by agmatine uptake
	RLM were incubated in standard medium, with 1 mM P i , 2 µM TPP + , 400 µM [ 14 C]DMO (1
	µCi/mmol), 5 mM [ 3 H]glycerol (100 µCi/mmol). Where indicated, 1 mM agmatine was added.
	Both measurements of ∆Ψ and ∆pH were performed on same sample. A typical experiment is
	reported. Six other experiments gave almost identical results.

RLM were incubated in standard medium with 1 mM [ 14 C]agmatine (50 µCi/mmol) and 1 mM P i .

When present, putrescine, cadaverine, spermine, ornithine, lysine, arginine, tyramine, benzylamine and TEA were at 1 mM concentration. Inset A: effect of polyamine transport (at 1 mM ) on ∆Ψ.

Empty circles on ordinate axis: agmatine bound at zero time.

Figure 10 Binding of agmatine to energized mitochondria

Panel A: Concentration-dependent zero-time binding. Mitochondria incubated for 3 min in standard medium, as described in Materials and Methods, with varying [ 14 Χ] agmatine concentrations, in range 100-3000 µM (0.05 µCi/ml), as indicated, and 1 mM Pi. B values obtained by measuring agmatine uptake (nmol/mg prot) at various times and extrapolating at zero time (on y axis) trend of curves, which is linear at all concentrations, in first 5 min of incubation (for a more complete description, see Fig. 1 in Ref. [START_REF] Reich | A general strategy for parameter estimation from isosteric and allosteric kinetic data and binding measurements[END_REF]). Results represent a typical experiment. Standard deviations of binding constants are reported in Table II.


Maria Mayer

Marek Dynowski

Uwe Ludewig
email: uwe.ludewig@zmbp.uni-tuebingen.de

Ammonium ion transport by the AMT/Rh homolog LeAMT1;1

Keywords: Ammonium transport, Methylammonium, ion channel, homology modeling

AMT/Rh ammonium transporters/channels are identified in all domains of life and fulfill contrasting functions related either to ammonium acquisition or excretion. Based on functional and crystallographic high-resolution structural data, it was recently proposed that the bacterial AmtB is a gas channel for NH 3 . Key residues, proposed to be crucial for NH 3 conduction, and the hydrophobic, but obstructed pore, were conserved in a homology model of LeAMT1;1 from tomato. Transport by LeAMT1;1 was affected by mutations that were predicted to constitute the aromatic recruitment site for NH 4 + at the external pore entrance.

Despite the structural similarities, LeAMT1;1 was shown to transport only the ion; each transported 14 C-MeA molecule carried a single, positive elementary charge. Similarly, NH 4 + (or H + /NH 3 ) was transported, but NH 3 conduction was excluded. It is concluded that related proteins and a similar molecular architecture can apparently support contrasting transport mechanisms.

Introduction

There is an ongoing debate as to whether some or all AMT/Rh proteins function as NH 3 channels or as NH [START_REF] Javelle | In vivo functional characterisation of the E. coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase[END_REF] + transporters [START_REF] Ludewig | Rhesus factors and ammonium: a function in efflux?[END_REF]. Based on the recently obtained high-resolution structure of a bacterial homolog, AmtB from E.coli, a channel-like transport of NH 3 in AMT/Rh proteins has been proposed [START_REF] Khademi | Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å[END_REF][START_REF] Zheng | The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli[END_REF]. Ammonium-mediated vesicular alkalinization by reconstituted AmtB [START_REF] Khademi | Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å[END_REF] and studies with bacterial strains compromised in ammonium assimilation and transport are in accordance with NH 3 transport [START_REF] Javelle | In vivo functional characterisation of the E. coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase[END_REF]. However, mechanistic interpretations from a static structure should be taken with care. Though probably within the rage of experimental error, some MeA accumulation by AmtB was detected, which was only to be expected if AmtB transports NH 4

+

. MeA accumulation would be also expected if, hypothetically, the ion occasionally "slipped" through the channel/transporter [START_REF] Javelle | In vivo functional characterisation of the E. coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase[END_REF]. Additional in vitro and in vivo experiments, including flux measurements against ammonia gradients and/or current recordings, are required to establish the NH 3 transport mechanism in AmtB.

According to its transcriptional and post-transcriptional regulation, the physiological role of AmtB appears to be the uptake of ammonium under low ammonium concentrations, but the NH 3 gradient is unfavorable for uptake under most conditions [START_REF] Javelle | In vivo functional characterisation of the E. coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase[END_REF].

The transport mechanism proposed for AMT/Rh proteins from the X-ray structures involves NH 4 + recruitment at the external pore mouth close to the aromatic residues F103, F107 and W148, de-protonation and conduction as NH 3 [START_REF] Khademi | Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å[END_REF][START_REF] Zheng | The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli[END_REF]. Hallmarks of the AMT/Rh family are Copyright 2006 Biochemical Society two histidines that line the central hydrophobic pore. It has been proposed that NH 4 + cannot pass these histidines and thus no NH 4 + transport and no ionic currents would be expected in

AmtB. An electroneutral NH 3 transport by AMT/Rh homologs is compatible with our results on human RhBG and RhCG. The studies suggest that ammonium transport by these proteins is electroneutral when heterologously expressed in oocytes and yeast [START_REF] Ludewig | Electroneutral Ammonium Transport by Basolateral Rhesus B Glycoprotein[END_REF][START_REF] Mayer | Different transport mechanism in plant and human AMT/Rh-type ammonium transporters[END_REF], in accordance with results from other groups [START_REF] Zidi-Yahiaoui | Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells[END_REF].

In contrast, specific, saturable, voltage-dependent, pH o -independent NH 4 + and MeA + currents have been recorded from two tomato AMT protein homologs expressed in oocytes [START_REF] Ludewig | Uniport of NH 4 + by the root hair plasma membrane ammonium transporter LeAMT1;1[END_REF][START_REF] Ludewig | Homoand hetero-oligomerization of ammonium transporter-1 NH 4 + uniporters[END_REF].

There is little doubt that ammonium uptake in plants is at least partially as NH

Experimental

Plasmid constructs, preparation and injection of oocytes-The plasmid pOO2-LeAMT1;1 and oocyte handling have been described in previous studies [START_REF] Ludewig | Uniport of NH 4 + by the root hair plasma membrane ammonium transporter LeAMT1;1[END_REF][START_REF] Ludewig | Electroneutral Ammonium Transport by Basolateral Rhesus B Glycoprotein[END_REF]. Mutations were introduced into LeAMT1;1 by using the Quickchange method from Stratagene and the complete coding region was verified by sequencing.

Electrophysiological measurements & radiotracer uptake & Intracellular pH changes (pH i )

Recordings were performed as described [START_REF] Ludewig | Electroneutral Ammonium Transport by Basolateral Rhesus B Glycoprotein[END_REF]. Means ± SEM are given. There was no special selection applied, but studies on ammonium transport in oocytes may be hampered by endogeous ion channels that are activated by high ammonium such as 10 mM [START_REF] Burckhardt | Pathways of NH 3 /NH 4 + permeation across Xenopus laevis oocyte cell membrane[END_REF]11].

Currents from oocytes used in this study were insensitive to ammonium and methylammonium (MeA) at concentrations of 1 mM or 10 mM, respectively [START_REF] Ludewig | Uniport of NH 4 + by the root hair plasma membrane ammonium transporter LeAMT1;1[END_REF][START_REF] Holm | NH 3 and NH 4 + permeability in aquaporin-expressing Xenopus oocytes[END_REF] and care was taken to verify that these endogenous ammonium-inducible currents were not present in the oocyte batches used. Oocytes used in our laboratory had a background conductance of <0.9 µS (between 0 mV and -100mV) and background currents as low as ~-80 nA at -140 -conductance was absent in the oocytes used in our study, native oocytes alkalinized upon addition of 10 mM ammonium (Fig. 5).

In the cases where currents "induced by ammonium" are given; these were obtained by subtracting the current in the absence of ammonium (background) from the current in the presence of ammonium at each voltage. The concentration dependence of currents/uptakes were fitted using the following equation:

I=I max /(1+K m /c), where I max is the maximal current at saturating concentration, K m is the substrate concentration permitting half-maximal currents, and c is the experimentally used concentration. 14 C-MeA uptake was done as in [START_REF] Ludewig | Electroneutral Ammonium Transport by Basolateral Rhesus B Glycoprotein[END_REF].

Intracellular pH changes (pH i ) -were monitored using the pH-sensitive dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and were calibrated as described [START_REF] Ludewig | Electroneutral Ammonium Transport by Basolateral Rhesus B Glycoprotein[END_REF].

Briefly, oocytes were incubated in uptake buffer (ND96, containing 5 µM BCECF-AM, from a 50 mM stock in DMSO) for 30 min protected from light. Fluorescence was monitored using an inverted Leica fluorescence microscope at maximal rate (5 sec) using a spinning wheel system with excitation at 410+30 and 470+20 nm and emission at 535+20 nm of BCECFloaded oocytes placed in a recording chamber and superfused with the standard solution (+ NH 4 Cl). In some experiments, oocytes were voltage clamped during fluorescence recording. Copyright 2006 Biochemical Society

Homology modeling-The primary sequence of LeAMT1;1 was aligned with AmtB using ClustalW, followed by manual inspection of functionally important residues. The alignment was identical to the alignment in [START_REF] Khademi | Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å[END_REF] for the first transmembrane helix (see supplementary figure). Structural fitting was done using MODELLER 7v7 (available at http://salilab.org/modeller/), a well-established structure prediction program [START_REF] Wallner | All are not equal: a benchmark of different homology modeling programs[END_REF]. The structures were evaluated using the WHATIF program (http://biotech.ebi.ac.uk:8400/) and Procheck (http://www.biochem.ucl.ac.uk/~roman/procheck/procheck.html). The LeAMT1;1 model used had 90.5% residues in the most favored regions in a Ramachandran plot (compared to 92.7% in the AmtB structure). Only 9 amino acids were identified that had "bad" stereochemistry. All of these were located outside of the core structure of the transmembrane helices. Further evaluation was done by energy minimization of the monomers of AmtB (PDB accession code: 1U7G) and the LeAMT1;1 model for 10 psec using NAMD2.5 (http://www.ks.uiuc.edu/Research/namd/), using the Amber7 force field.

The stability of the structure was used as a measure for the quality and was assessed by measuring the RMSD between backbone residues of the structures. Minor attention was given to reconstruction of loops, as these are expected to minimally influence the core structure and transport mechanism. The pore diameter was accessed using the program HOLE2 (http://d2o.biop.ox.ac.uk:38080//; [START_REF] Smart | The pore dimensions of gramicidin A[END_REF]). Monomeric bovine AQP1 (PDB accession code:

1J4N) was analyzed in parallel.

Expression in yeast -

The open reading frames of LeAMT1;1 and a translational LeAMT1;1-GFP fusion were cloned into plasmid pDR196 and heat shock-transfected in the ura - Saccharomyces cerevisiae wild type strain (23344c) or the ammonium transporter defective yeast strain (31019b; mep1;2;3) [START_REF] Marini | A family of ammonium transporters in Saccharomyces cerevisiae[END_REF]. The nitrogen deficient growth medium for strain 31019b was YNB (Difco), supplemented with 3% glucose and appropriate NH 4 Cl concentrations. The media was adjusted to pH 5.5 using 50 mM 2-[Nmorpholino]ethanesulfonic acid (MES). The MeA toxicity medium contained YNB, 3% glucose, 0.1% arginine and 125 mM MeA. Growth of yeast was not affected by expression of the different constructs under non-seletive conditions.

Results

Homology model of LeAMT1;1

To identify the molecular determinants for differences in the transport mechanism between 1A,B. Major deviations are seen in extrahelical loops, consistent with the low similarity in these regions. The model was less stable than AmtB after a minimization for 10 ps, which reduces "bad" contacts within the protein (Fig. 1C). Three monomers preferably assembled in a trimeric form (Fig. 1E).

AmtB does not form an "open" channel-like structure as, e.g. aquaporin channels. Several aquaporins transport ammonia in addition to water [START_REF] Loqué | Tonoplast aquaporins AtTIP2;1 and AtTIP2;3 facilitate NH3 transport into the vacuole[END_REF][START_REF] Jahn | Aquaporin homologues in plants and mammals transport ammonia[END_REF], which justifies the comparison between these protein classes. The substrate pore of aquaporin monomers is illustrated for bovine AQP1 (PDB code:1J4N) [START_REF] Sui | Structural basis of water-specific transport through the AQP1 water channel[END_REF] using the program HOLE2, a program that identifies putative ionic and solute pores in proteins based on the van der Waals radii of amino acid residues close to the pore [START_REF] Smart | The pore dimensions of gramicidin A[END_REF]. The AQP1 pore constriction does not go below 0.8 A (Fig. 1D) [START_REF] Sui | Structural basis of water-specific transport through the AQP1 water channel[END_REF], but the solute pathway in AmtB is blocked by residues F107 and F215 and partially by residue F31 (Fig. 1C,F). In accordance with this, preliminary comparative steered molecular dynamics studies did reveal solute flux in aquaporins, but not in AmtB (unpublished observation). The fundamental difference appears important, since the proposed channel-like mechanism was recently illustrated by a misleading "open-pore" cartoon-like model [START_REF] Knepper | Structural biology. The atomic architecture of a gas channel[END_REF]. At least minimal conformational changes are necessary to obtain a channel-like open pore structure. Another possibility is that transport-cycle dependent conformational changes are involved in ammonium transport.

The pore analysis using HOLE2 did not reveal a continuous solute pathway in the LeAMT1;1 homology model. Most residues, if not all, that have been suggested to be crucial for the NH 4 + recruitment and NH 3 conduction in the occluded pore of AmtB, are identical or similar in LeAMT1;1. The residues F103, F107, W148 and S219 in AmtB have been predicted to participate in the recruitment and coordination of the NH 4 + ion; the equivalent residues in LeAMT1;1 are at positions Y133, F137, W178 and S263, respectively. The aromatic residues F107 and F215 block the pore in AmtB, these residues are at positions F137 and F259 in LeAMT1;1. The model has a similar hydrophobic pore to AmtB, with two conserved histidines at positions H207 and H374 that line the putative pore lumen (Fig. 1G). Although the model cannot be a substitute for a crystal structure, its similarity to the NH 3 transporting AmtB is striking. Copyright 2006 Biochemical Society

The aromatic nature of two residues forming the ion binding cavity (Y133 and W178 in LeAMT1;1) is conserved between bacterial and plant homologs, but not in Rh glycoproteins.

In Rhs, non-polar small neutral residues occupy these positions (Isoleucine and Leucine in RhCG). These residues might thus be responsible for the different affinity that is observed in Rh glycoproteins [START_REF] Ludewig | Electroneutral Ammonium Transport by Basolateral Rhesus B Glycoprotein[END_REF][START_REF] Mayer | Different transport mechanism in plant and human AMT/Rh-type ammonium transporters[END_REF]. The overall quality of the LeAMT1;1 model was tested by mutational analysis of the residues Y133 and W178 that are putatively involved in substrate affinity.

Ammonium transport by LeAMT1;1 pore mutants

Initial attempts to functionally express EcAmtB in oocytes failed (data not shown), so we concentrated on the function of LeAMT1;1. Residues tyrosine 133 and tryptophan 178 were exchanged to the respective residues found in RhC glycoprotein (Y133I and W178L).

LeAMT1;1 and, to a lower extent, the two mutants functionally complemented the ammonium-transporter deficient yeast strain mep1,2,3 with ammonium as sole nitrogen source (Fig. 2). The mutations did not largely alter the expression pattern as assessed by confocal microscopy of translational c-terminal GFP-fusions in yeast. A similar cell-to-cell variability in fluorescence levels was observed in homogenous yeast cultures when analyzed with the identical microscopy settings at low magnification (Fig. 2E). LeAMT1;1 and the mutants showed the same subcellular localization when analyzed at higher magnification (Fig. 2E).

125 mM MeA are toxic to wild type yeast and expression of Rh glycoproteins has been shown

to rescue growth on toxic MeA. Rh glycoproteins probably export uncharged MeA along its chemical gradient, even though Rh glycoproteins are saturated well below these high MA concentrations [START_REF] Mayer | Different transport mechanism in plant and human AMT/Rh-type ammonium transporters[END_REF]. Both mutants and LeAMT1;1 showed increased sensitivity to MeA, which is most simply explained by MeA + import by LeAMT1;1 and the mutants (Fig. 2D). In all assays, the W178L mutant displayed less transport activity than mutant Y133I.

Altered saturation of NH 4 + transport in a LeAMT1;1 mutant

For a more detailed analysis, the ionic currents elicited by the mutant transporters were assayed using the two-electrode voltage clamp in oocytes. No significant 14 C-MeA uptakes and currents were detected with W178L, precluding further analysis. In contrast, the mutant Y133I displayed small, but reliable currents on exposure to ammonium. Currents were more than 10-fold smaller than in the wild type (Fig. 3), in accordance with its reduced overall transport rate or reduced functionality compared to wild type in the yeast growth experiments.

Importantly, the half maximal current, which is a measure that is independent of the expression level, saturated at very low concentrations. In contrast to wild type (K m = 12 µM), Copyright 2006 Biochemical Society the K m for NH 4 + in the Y133I mutant was only 1 µM at -120 mV. Similarly, MeA + currents saturated at a half maximal concentration of 584 µM at -120 mV in LeAMT1;1, while the small currents of the mutant were half maximal at 4.5 µM (Fig. 3).

Charge coupling of MeA + transport in LeAMT1;1

The ratio of charged vs. uncharged solute transport was assayed using the analog methylammonium labeled at its carbon ( 14 C-MeA). Transport and charge translocation were monitored simultaneously in native and LeAMT1;1-expressing oocytes from the same batch.

LeAMT1;1-expressing oocytes incubated in 1 mM 14 C-MA accumulated 9.0 + 1.5 pmol min - . The MeA + /MeA-induced current was specific to LeAMT1;1-expressing oocytes, was time-independent over several minutes and was strongly inwardly rectifying. Taking the error bar into account, the maximal slippage of uncharged H 3 14 C-NH 2 must be below 10%. Identical results were obtained with 3 independent batches of oocytes; one of these experiments is shown in Fig. 4. The results show that preferentially H 3 14 C-NH 3 + is transported but, at this resolution, the possible transport of H 3 14 C-NH 2 cannot be excluded (Fig. 4).

Ionic currents and acidification by LeAMT1;1

To determine if a residual flux of NH 3 is maintained by LeAMT1;1, current and pH i recordings were combined. Current-voltage relations from LeAMT1;1-expressing oocytes are shown in Fig. 5. The open symbols represent a current-voltage relation from LeAMT1;1expressing oocytes in the absence and the closed circles after addition of 200 µM ammonium (Fig. 5A). The ammonium-induced current was inward and the reversal potential of the total currents shifted to more positive membrane potentials. No pH i changes upon 500 µM ammonium exposure were observed in BCECF-loaded, LeAMT1;1-expressing oocytes and in water-injected controls (Fig. 5B). Although NH 4 + transport is almost saturated by 500 µM in LeAMT1;1 (Fig. 3) [START_REF] Ludewig | Uniport of NH 4 + by the root hair plasma membrane ammonium transporter LeAMT1;1[END_REF], 10 mM ammonium was also used to compare the results with older studies on native oocytes that used these excess ammonium concentrations. Only negligible Copyright 2006 Biochemical Society influx in voltage-clamped oocytes at a negative membrane potential. The negative membrane potential mimics the in vivo situation of LeAMT1;1 in tomato root epidermal cells [START_REF] Ayling | The effect of ammonium ions on membrane potential and anion flux in roots of barley and tomato[END_REF].

The pH i of water-injected and LeAMT1;1-expressing oocytes clamped to -80 mV is shown in The pH i -changes were not due to possible oocyte to oocyte variability, which is shown in a continuous recording from a single LeAMT1;1-expressing oocyte in Fig. 5D. Low ammonium induced acidification and -170 nA current during voltage-clamp, while no acidification was seen when the membrane potential was then allowed to change freely. Interestingly, the voltage-clamped LeAMT1;1 oocytes that acidified with NH 4 + uptake did not recover the pH i to the baseline level after the withdrawal of ammonium. This suggests that ammonium that passively exits the oocyte as NH 3 , is reabsorbed as NH 4 + and thus the cytosolic ammonium concentration, and pH i , are maintained.

Discussion

Prediction of the function of proteins based on their crystal structure is an exceptionally elegant method, but we cannot yet distinguish a channel from a transporter that couples conformational changes to transport on the basis of the crystal structure. The structure of the bacterial AMT/Rh homolog AmtB does not show a clearly open transmembrane pore (Fig. 1), which would be required for a channel but is unlikely in any well-coupled exchanger. This is also true for the model of LeAMT1;1, which shows that key residues are highly conserved between these proteins. Thus, even in AmtB, conformational changes or side chain movements are required to allow solute flow. If, however, AmtB undergoes a conformational change that is not detected by the crystallographic approach, then hydrogen bond acceptors may become available for interaction with ammonium.

Similar to the case of AMT/Rh proteins, the X-ray structures of a prokaryotic homolog of CLC anion channels did not reveal its mechanism, which was later identified as rapid 2Cl -/H + antiport [START_REF] Accardi | Secondary active transport mediated by a prokaryotic homologue of ClC Cl-channels[END_REF]. Although the pathway for anions is well apparent from the atomic CLC- passage, an unresolved pathway would exist for H + . This had already been discussed [START_REF] Ludewig | Rhesus factors and ammonium: a function in efflux?[END_REF] and explains the superb selectivity of NH 4 + transporters against alkali cations [START_REF] Ludewig | Uniport of NH 4 + by the root hair plasma membrane ammonium transporter LeAMT1;1[END_REF]. NH 4 + transport is equal at external pH o 5.5 and 8.5 [START_REF] Ludewig | Uniport of NH 4 + by the root hair plasma membrane ammonium transporter LeAMT1;1[END_REF] and exceeds that of NH 3 , which suggests that LeAMT1;1 can transport against NH 3 gradients.

Despite similar key residues in the structure, AmtB apparently uses a different transport mechanism, as it transports at least some NH 3 , shown by the rapid alkalinization of AmtB vesicles in ammonium solution [START_REF] Khademi | Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å[END_REF]. It is currently impossible to exclude the possibility that AmtB is sloppier and passes more NH 3 together with NH 4

+

. It remains to be shown that AmtB dissipates NH 3 gradients and thus acts as a channel. Even if AmtB would transport 95% in the form of NH 4 + and only 5% in the form of NH 3 , alkalinization would be expected due to a net inward NH 3 flux (at physiological pH i total ammonium is ~98.5% NH 4 + and ~1.5% NH 3 ).

The data on LeAMT1;1 presented here are in accordance with earlier data [START_REF] Ludewig | Uniport of NH 4 + by the root hair plasma membrane ammonium transporter LeAMT1;1[END_REF] and argue against the speculation that LeAMT1;1 currents may be endogenous to oocytes [START_REF] Khademi | Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å[END_REF]. The saturation of currents was altered by more than 10-fold (NH 4

+

) and 100-fold (MeA + ) in LeAMT1;1 mutants and differed from endogenous ammonium-induced oocyte currents [START_REF] Mayer | Different transport mechanism in plant and human AMT/Rh-type ammonium transporters[END_REF].

Transport of NH Copyright 2006 Biochemical Society expressed in oocytes [START_REF] Mayer | Role of AMT1;1 in NH4+-acquisition in Arabidopsis thaliana[END_REF]. Similar to the plant transporters, the Saccharomyces homologs (MEP1-3) are involved in re-uptake and accumulation of lost ammonium [START_REF] Marini | A family of ammonium transporters in Saccharomyces cerevisiae[END_REF].

Microorganisms and plants maintain a near neutral cytoplasmic pH i , but the acidic external pH o often requires the acquisition of NH 4 + /NH 3 against a NH 3 concentration gradient.

Small conformational changes that may represent "gating" of AmtB have already been documented in the different crystal structures [START_REF] Khademi | Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å[END_REF][START_REF] Zheng | The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli[END_REF], but more structures showing different conformations of AMT/Rhs are required to distinguish between channel and transporter-like mechanisms. In any case, the functional data suggest that the classical concept of clear discrimination between channels and transporters is diminishing on a molecular level: highly similar proteins from the AMT/Rh-family may function as equilibrative "gas" or ion channels/transporters.

3 +/CH 3 -

 33 NH 3 and NH 4 + concentrations were determined from total ammonium concentrations using the Henderson-Hasselbach equation: log 10 [NH 3 ]/[NH 4 + ] = pH -9.25. The pK a = 10.66 was used for calculations involving MeA (CH 3 -NH NH 2 ).

NH 4 + 4 +

 44 influx is expected from non-voltage clamped, LeAMT1;1-expressing oocytes at depolarized potentials (compare the current-voltage relation in Fig.5A), but significant NH Biochemical Journal Immediate Publication. Published on 27 Feb 2006 as manuscript BJ20060051

Fig. 5C&D . 4 +.

 5C&D4 Fig.5C&D. 500 µM ammonium induced -150 nA current in LeAMT1;1 and a persistentacidification, in accordance with NH 4 + influx, but in opposition to significant NH 3 flux. This acidification was not observed in water-injected control oocytes. It is necessary to recall that at the cytosolic pH i of 7.4, the pH i is highly sensitive to NH 3 influx, as almost every incoming NH 3 will bind H + to form NH 4 + . This situation is distinct with NH 4 + transport: most of the influxed NH 4 + will remain as NH 4 + and only a minor fraction (~1.5 %) will dissociate into NH 3 and H + , and thus acidify the cytosol.

Biochemical Journal Immediate Publication. Published on 27 Feb 2006 as manuscript BJ20060051

Biochemical Journal Immediate Publication. Published on 27 Feb 2006 as manuscript BJ20060051 Copyright 2006 Biochemical Society

	mV. This background current is about half of the endogenous current from oocytes used in the
	laboratories that have recorded and investigated NH 4	+ induced currents [11, 13]. Interestingly,
	seasonal variations were reported for the endogenous amine-inducible current [11]. Although
	activation of such endgenous oocyte currents at lower ammonium concentrations has been
	reported [13], we and others observed that ionic currents from oocytes are insensitive to
	ammonium at < 1 mM at physiological pH [5-7, 12, 14] (and data not shown). Although the
	nature of this discrepancy and the natural variance in oocyte currents is unclear, Xenopus
	oocytes with reduced or absent endogenous MeA + and NH 4	+ -currents have the great advantage
	that measurements on ammonium transport can be performed with minimal contamination
	from the endogenous ammonium-induced currents. The absence of endogenous MeA + and
	NH 4	+ -currents in oocytes used in this study demands that internal pH i will respond differently
	to ammonium than in previous studies on oocytes with large NH 4	+ -currents (Fig.5). Oocytes
	expressing large endogenous ammonium-activated currents had been reported to acidify
	slowly when measured with pH-sensitive electrodes [11, 13]. As the NH 4	+

Biochemical Journal Immediate Publication. Published on 27 Feb 2006 as manuscript BJ20060051

Biochemical Journal Immediate Publication. Published on 27 Feb 2006 as manuscript BJ20060051 Copyright 2006 Biochemical Society core

  (excluding the N-and-C-terminus). The most stable model (see methods) was obtained by using a slightly modified alignment from that published by Khademi et al., which is given as a supplementary figure and only differs in the first transmembrane region. A ribbon representation of the structural backbone of AmtB and the model of LeAMT1;1 is shown in Fig.

AMT/Rh homologs, a homology model of LeAMT1;1 with AmtB (PDB code 1U7G) as template was constructed. Both proteins are 25% identical and 44% similar within the protein

Biochemical Journal Immediate Publication. Published on 27 Feb 2006 as manuscript BJ20060051

Biochemical Journal Immediate Publication. Published on 27 Feb 2006 as manuscript BJ20060051

Biochemical Journal Immediate Publication. Published on 27 Feb 2006 as manuscript BJ20060051 Copyright 2006 Biochemical Society residues

  involved in sugar/H + co-transport in LacY transporters had long been known, the mechanism of H + co-transport was not readily apparent from the structure[START_REF] Abramson | Lactose permease as a paradigm for membrane transport proteins[END_REF]. Crystal structures provide a static view, and even if structures look alike in the presence and absence of substrate, this does not exclude the existence of other conformations, since the protein may preferably crystallize in one state. Solute transport in proteins needs to be viewed as dynamic,

	but crystallographic structures are, by nature, static. A full understanding of the transport
	mechanism will result from a resourceful combination of structural and functional
	biochemical approaches.
	The 3D model of LeAMT1;1 was successfully used to predict residues involved in NH 4	+
	recruitment (or binding). Though no attempt is made to derive mechanistic conclusions from
	the homology model and the hydrophobic pore of LeAMT1;1, the molecular modeling may
	be useful in guiding future experimental work on this transporter/channel class. A very similar
	AMT/Rh structure has recently been reported from a thermophilic archaeon [25], which
	emphasizes that AMT/Rhs adopt a similar structure. The functional data of LeAMT1;1 in
	Fig.4 and Fig.5 show that LeAMT1;1 is a net NH 4

structures, it has not yet been resolved how H + is transported in CLCs. Similarly, although + transporter (or "channel"). Based on the AmtB structure and the hydrophobic pore in the LeAMT1;1 model, one may speculate that the molecular mechanism is NH 3 /H + co-transport. If NH 4 + dissociates into NH 3 and H + during

Biochemical Journal Immediate Publication. Published on 27 Feb 2006 as manuscript BJ20060051

  4 + is not restricted to LeAMT1;1, since other plant homologs have now been identified in our laboratory which also induce saturable NH 4 + and MeA + currents when

Acknowledgements: We thank Binghua Wu for initial help with the constructs, Petra Neumann for excellent technical assistance and Felicity deCourcy for critically reading the manuscript. Support from the Deutsche Forschungsgemeinschaft to U.L is acknowledged. Biochemical Journal Immediate Publication. Published on 27 Feb 2006 as manuscript BJ20060051 Copyright 2006 Biochemical Society