Laurent M Arsac

Christophe Beuste

Sylvain Miraux

Véronique Deschodt-Arsac

Eric Thiaudière

Jean-Michel Franconi

Philippe H Diolez

In vivo Modular Control Analysis of Energy Metabolism in Contracting Skeletal Muscle

Keywords: Muscle energetics, elasticity coefficient, control coefficient, metabolic regulation, 31 P MR spectroscopy, Modular Control Analysis

SYNOPSIS

We used 31 P MR spectroscopy measurements of energetic intermediates (ATP, Pi, PCr) in combination with the analytical tools of metabolic control analysis to study in vivo energy metabolism in contracting skeletal muscle of anesthetized rats over a broad range of workload.

According to our recent Modular Control Analysis (MoCA) used to describe regulatory mechanisms in beating heart, we defined the energetic system of muscle contraction as two modules (PCr-Producer and PCr-Consumer) connected by the energetic intermediates.

Hypoxia and electrical stimulation were used in this in vivo study as reasonably selective modulations of Producer and Consumer respectively. As quantified by elasticity coefficients, the sensitivities of each module to PCr determine the control of steady state contractile activity and metabolite concentrations. The magnitude of the elasticity of the producer was high (4.3±0.6) at low workloads and decreased 5-fold (to 0.9±0.2) at high workloads. By contrast, the elasticity of the consumer remained low (0.5 to 1.2) over the range of metabolic rates studied. The control exerted by each module over contraction was calculated from these elasticities. The control of contraction was found on the consumer at low workloads then swung to the producer, due to the workload-dependent decrease in the elasticity of producer.

The workload-dependent elasticity and control pattern of energy production in muscle is a major difference with heart. Since module rate and elasticity depend on the concentrations of substrates and products, the absence of homeostasis of the energetic intermediates in muscle, by opposition to heart, may likely be the origin of the workload-dependent elasticity of the producer module.

INTRODUCTION

In skeletal muscle as in heart, contraction is powered up by the Gibbs free energy of ATP hydrolysis (∆Gp) available at actomyosin ATPases level. In the absence of changes in regulatory external effectors, both rates of ATP supply and ATP demand depend on the concentration of their substrates/products ATP, ADP and Pi. The concentration of ATP remains fairly constant during muscle contraction and [ADP] remains low and undetectable by non-invasive techniques like 31 P magnetic resonance spectroscopy. As far as the cytosolic creatine kinase reaction remains close to equilibrium, [PCr] reflects the status of energy balance within the entire myocyte and over all the fibres in the studied muscles [START_REF] Kushmerick | Energy balance in muscle activity: simulations of ATPase coupled to oxidative phosphorylation and to creatine kinase[END_REF][START_REF] Kushmerick | Regulation of oxygen consumption in fast-and slow-twitch muscle[END_REF]. Since energy production and energy consumption during muscle contraction are respectively responsible for increasing and decreasing PCr concentration, 31 P MRS assessments of PCr levels are representative of cellular energetic intermediates, and have been widely used to study the behaviour of the energetic system of contraction in heart as well as in muscle [START_REF] Hochachka | Cellular metabolic homeostasis during large-scale change in ATP turnover rates in muscles[END_REF][START_REF] Hogan | Human muscle performance and PCr hydrolysis with varied inspired oxygen fractions: a 31P-MRS study[END_REF][START_REF] Jeneson | Regulation analysis of contractile ATPase flux in skeletal muscle[END_REF][START_REF] Jeneson | A metabolic control analysis of kinetic controls in ATP free energy metabolism in contracting skeletal muscle[END_REF][START_REF] Jones | Muscle [phosphocreatine] dynamics following the onset of exercise in humans: the influence of baseline workrate[END_REF][START_REF] Matheson | Skeletal muscle metabolism and work capacity: a 31P-NMR study of Andean natives and lowlanders[END_REF][START_REF] Paganini | Linear dependence of muscle phosphocreatine kinetics on oxidative capacity[END_REF].

Recently, our group proposed a new experimental approach to describe control and regulatory mechanisms of energetics in intact beating hearts, based on the development of the Modular Control Analysis (MoCA). Heart energetics was defined as a two-module system.

Modules respectively produce (so-called Producer) and consume (Consumer) energy and are connected by PCr concentration [10]. Central to this approach are the elasticity coefficients of the ATP/PCr producing and ATP/PCr consuming modules to PCr concentration [10]. Indeed, elasticity values in such a modular system quantify how the system is maintained in a steady state. Elasticities also allow to quantify how the effects of an external effector signal/molecule that change the steady state are transmitted through the intermediate [10][11][START_REF] Brand | Regulation analysis of energy metabolism[END_REF][START_REF] Brand | Simplifying metabolic complexity[END_REF].

It is worth noting that heart is capable of dramatically altering its overall energy flux with only minimal changes in the concentrations of energetic intermediates [14]. Homeostasis of the intermediates in heart is likely the consequence of a perfectly balanced parallel activation of ATP supply and ATP demand [14,15] as demonstrated in intact hearts stimulated by either calcium [10] or adrenaline [16]. By comparison, an unbalanced activation could thus be suspected in skeletal muscle [START_REF] Jeneson | Regulation analysis of contractile ATPase flux in skeletal muscle[END_REF]15,17] since the transition from low to high work intensity is consistently accompanied by a decrease level of PCr and an increase in the connected intermediates (free creatine, ADP and Pi) [START_REF] Hochachka | Cellular metabolic homeostasis during large-scale change in ATP turnover rates in muscles[END_REF][START_REF] Hogan | Human muscle performance and PCr hydrolysis with varied inspired oxygen fractions: a 31P-MRS study[END_REF][START_REF] Jones | Muscle [phosphocreatine] dynamics following the onset of exercise in humans: the influence of baseline workrate[END_REF][START_REF] Paganini | Linear dependence of muscle phosphocreatine kinetics on oxidative capacity[END_REF][START_REF] Giannesini | New experimental setup for studying strictly noninvasively skeletal muscle function in rat using 1H-magnetic resonance (MR) imaging and 31P-MR spectroscopy[END_REF]19]. These changes in the energetic intermediates may in turn impact the kinetics of the modules. Therefore, it appears that the in vivo determination of the elasticities of ATP/PCr producing and consuming modules over a wide range of activity is of great interest in skeletal muscle. Plantar flexor muscles of intact anesthetized rats were interrogated by a two-probe ( 1 H and 31 P) setup providing muscle imaging at rest, and muscle content of phosphorylated intermediates during sustained contractions. The experimental design was defined in the frame of MoCA: the Consumer module contains all the reactions that consume ATP/PCr in contracting myocytes, and the Producer module grouped the reactions catalysed by mitochondria and glycolysis together with substrates delivery to contracting muscles. Under steady state conditions, the selective modulation of ATP/PCr production required by MoCA was achieved by changing the oxygen concentration in the gas mixture inhaled by the rat. The other required modulation -ATP/PCr consumption -was carried out by decreasing the electrical stimulation of plantar flexors. The energetic flux through this two-module system is represented by the ATP turnover rate. In contracting muscles during sustained submaximal exercise, the ATP turnover rate is a direct function of the work rate by contraction [START_REF] Arthur | Modeling the effects of hypoxia on ATP turnover in exercising muscle[END_REF]; therefore the later can be used as an index of the former [START_REF] Nioka | Substrate regulation of mitochondrial oxidative phosphorylation in hypercapnic rabbit muscle[END_REF].

We carried out in vivo the analytical study by MoCA of energy metabolism in contracting muscle over a broad range of metabolic rates. The full MoCA analysis provided a complete integrative description of the behaviour of muscle energetics for each individual animal tested. In agreement with previously modelled elasticities [START_REF] Jeneson | Regulation analysis of contractile ATPase flux in skeletal muscle[END_REF], we demonstrate here that the sensitivity of (mitochondrial) ATP/PCr production in vivo decreases as a function of activity in skeletal muscle. Rats were anesthetized by continuous inhalation of a gas mixture containing 1.5% isoflurane delivered in a face-mask during a 90-min period corresponding to the duration of the experiment into the magnet, then housed back in their environmentally controlled facility.

Muscle stimulation

Plantar flexor muscles of rats in supine position were stimulated (3Hz, pulse duration 220µs) directly with transcutaneous surface electrodes located at the knee and heel levels [START_REF] Giannesini | New experimental setup for studying strictly noninvasively skeletal muscle function in rat using 1H-magnetic resonance (MR) imaging and 31P-MR spectroscopy[END_REF] and connected to a Compex 2 stimulator (Compex Médical SA, Ecublens, Switzerland).

Current intensity was set to 4mA (low workload), 5-6mA (moderate workload) and 7-8mA (high workload). MRS acquisition was synchronized to electrical stimulation. Electrically stimulated regions of the plantar flexor group were assessed by T 2 -mapping MRI [START_REF] Giannesini | New experimental setup for studying strictly noninvasively skeletal muscle function in rat using 1H-magnetic resonance (MR) imaging and 31P-MR spectroscopy[END_REF].

Transverse relaxation times constants (T 2 ) were measured using an imaging CPMG (Carr-Purcell-Meiboom-Gill) pulse sequence (64 echoes, echo time: 7.3 ms, repetition time : 2000 ms). T 2 -maps indicated that the mixed-gastrocnemius muscle responded to electrical stimulation over the range of stimulations used (figure 2).

P magnetic resonance spectroscopy

Rats were placed in a supine position into a supraconducting magnet (4.7 Tesla, 47/50 Biospec Avance MR system (Brüker, Karlsruhe, Germany). The foot was positioned on the ergometer pedal and the hindlimb was immobilized so that the lower limb was centred inside a 30 mm diameter 1 H Helmoltz imaging coil tuned to the 1H frequency (200.3 MHz).

A circular homebuilt transmit-receive probe (18mm) was placed horizontally under the calf and was tuned to the 31P frequency (81.1 MHz). Field homogeneity was achieved locally in a 20x20x20mm 3 voxel by using the water proton resonance, with typical line widths of 30-35 Hz.

31 P free-induction decays (FIDs) (100µs rectangular pulse, 60° flip angle at the centre of the coil; 40 accumulations; 2.8s recovery time; 3.3 kHz spectral width; 1024 data points) were acquired in 111s blocks throughout the experimental protocol.

Contraction measurements

Contraction resulting from muscle electrical stimulation was measured with a homebuilt ergometer consisting in a foot pedal connected to a hydraulic piston. A hydraulic circuit filled with water connected the piston to a force transducer (MLT0699 + Powerlab, AD Instruments, Bella Vista, Australia), which was placed outside the magnet. The pedal was adjusted so that the foot was perpendicular to the leg. Changes in pressure induced by pedal stroke were recorded every 5 ms. The magnitude of strokes was computed as a function of time (Igor, Wavemetrics, Oregon, USA) to provide the contraction signal. Since only relative changes (in %) in contraction are required for MoCA, contraction force was expressed as the voltage delivered by the sensor (µvolts, see figure 3).

The modular system

We defined the system of contraction as two modules, Producer and Consumer connected by the phosphorylation potential (∆Gp) as represented by [PCr] in our conditions [10,16,22].

By grouping reactions and reactants into large modules connected by a small number of explicit intermediates, complex systems are thus simplified [START_REF] Brand | Simplifying metabolic complexity[END_REF][START_REF] Brown | A 'top-down' approach to the determination of control coefficients in metabolic control theory[END_REF]. The behaviour of the system was studied over a wide range of steady states achieved by sustained steady level of electrical stimulation. The elasticity coefficients of Producer and Consumer were determined under each given steady state conditions. The typical design of each experiment is described in figure 1. Two 31 P-MR spectra (spectra 1 and 2) were acquired before muscle stimulation began to assess phosphocreatine concentration at rest ([PCr] rest ). During spectra 3 to 12, muscle stimulation was progressively increased up to the desired current intensity, and hypoxia was gradually initiated by reduction of the FiO 2 of breathed gas to 9% O 2 (in N 2 ).

The system reached thus a hypoxic steady state where the producer is inhibited for several minutes by hypoxia (spectra 14 to 18). During spectrum 19, hypoxia was stopped; the rat then inspired air and the system achieved quickly the so-called 'reference' steady state (absence of modulation, spectra 20 to 24). The consumer elasticity was calculated from relative changes in steady levels of PCr and contraction between 'reference' and hypoxic conditions (figure 3).

During the last steady state period, the electrical stimulation was decreased by 1 mA to lessen energy consumption while F i O 2 was purposely maintained at 0.21 (ambient air). The steady

Statistics

Data are presented as mean ± SD. Elasticity and control coefficients at low, moderate and high work rates were compared using one-way analysis of variance. Significant differences were considered at P<0.05.

RESULTS AND DISCUSSION

In the present study, we experimentally apply for the first time metabolic control analysis in vivo to study non-invasively the behaviour of energy metabolism in contracting skeletal muscle on an anesthetized rat. The analysis was carried out using controlled electrical stimulation to provide a wide range of steady muscle activity. Our experimental two-probe setup ( 1 H and 31 P) also allowed the application of a specific MRI technique (T 2 -mapping) dedicated to identify the contracting regions inside the studied muscle group [START_REF] Giannesini | New experimental setup for studying strictly noninvasively skeletal muscle function in rat using 1H-magnetic resonance (MR) imaging and 31P-MR spectroscopy[END_REF]. Figure 2 shows typical axial T 2 -maps from the rat lower hindlimb obtained at rest (a) and immediately following low (b) and high (c) steady stimulated contraction. Although this technique did not indicate which precise fibres were contracting, the images in Figure 2 We may therefore consider that the present in vivo application of MoCA to skeletal muscle energetics might be a crude method and the experimentally determined values of the elasticity coefficient would be approximated. However, we assume that under our conditions, this would not change the general conclusions (see below) and the development of a quantitative approach in vivo, even based on a crude method seems yet to be advantageous [START_REF] Jeneson | Regulation analysis of contractile ATPase flux in skeletal muscle[END_REF][START_REF] Jeneson | A metabolic control analysis of kinetic controls in ATP free energy metabolism in contracting skeletal muscle[END_REF][START_REF] Wu | Oxidative ATP synthesis in skeletal muscle is controlled by substrate feedback[END_REF].

The intermediate. In the energy metabolism of contracting skeletal muscle, the true intermediate between production and consumption is the phosphorylation potential ∆Gp. A direct measurement of ∆Gp in vivo is difficult. Although ∆Gp can be calculated from MRS of phosphorylated intermediates and pH, errors in the detection of Pi, PCr, and each ATP peak sum up in this calculation. Thus, we based our analysis on [PCr] changes, taking into account the reliable signal-to-noise ratio obtained in vivo with our setup (figure 3). Furthermore, the phosphorylation potential depends on the concentrations of ATP, ADP, Pi and H + , which are all directly related to PCr concentration and therefore changes in PCr directly reflects changes show only very small changes in pH over the workload range tested. In conclusion, in vivo MRS assessments of PCr changes are relevant to study the kinetic response of the PCr Producer and PCr Consumer.

Experimental elasticities and muscle activity.

Quantifying elasticity coefficients in the frame of MoCA requires the simultaneous measurements of relative changes in [PCr] and in contraction power output. This was achieved here in vivo as illustrated by typical 31 P-MR spectra and contraction measurements presented in figure 3. Such a typical experiment was repeated under various levels of electrical stimulation, thus allowing the quantification of elasticity coefficients over a wide range of skeletal muscle activity. The activity has been scaled according to the steady-state level of [PCr] relatively to [PCr] at rest, since a strong correlation has been demonstrated between this parameter and the relative workload in human muscle [START_REF] Hogan | Human muscle performance and PCr hydrolysis with varied inspired oxygen fractions: a 31P-MRS study[END_REF]. The PCr level varied from 82% at the lowest workload to 38% [PCr]rest at the highest workload. Experimental values for [PCr] and contraction were arbitrary grouped as low, moderate and high level of muscle activity. Activity-specific averaged values of PCr and contraction are presented in table 1. These values are only indicative since the present study was designed to allow single-animal comprehensive MoCA analysis, so that elasticity coefficients were actually calculated for each animal. Therefore, between-animal variability, as indicated by standard deviation in table 1, does not interfere with the calculation of elasticities. Averaged individual elasticities measured experimentally are reported in table 2.

The statistical analysis indicates that the Producer elasticity to PCr decreased more than 5fold from low to high workloads (from -6.7 to -0.3). The Consumer elasticity to PCr was much lower than that of Producer and remained roughly unchanged (± 1.0) when the workload changed. The shift in kinetic control over contraction. The respective control coefficients of the producer and the consumer were calculated from the above elasticity coefficients as allowed by MoCA [10]. Therefore we can describe how control over contraction is distributed in our conditions. The major finding here is that the control distribution is not unique but depends on muscle activity, in agreement with previous predictions [START_REF] Jeneson | Regulation analysis of contractile ATPase flux in skeletal muscle[END_REF]. The change in the control distribution was almost completely accounted for by the decrease in the producer elasticity.

At low activity, the producer had a very high elasticity and consequently very low control (10%) over contraction (table 2 andfig 4b). Under these conditions, energy consumption mainly set by the activity of the AM-ATPases and SR-ATPases controls the contraction (90%), i.e. ATP turnover. At moderate activity, due to the elasticity decrease, the control of the producer module over the contraction increased but remained low (25%); whereas the control by consumer module shifted down to 75%. Under high activity conditions, where PCr depletion was above 50%, the control distribution changed dramatically: the producer rather than the consumer had dominant control over contraction. It was even observed in some animals that the energy production controlled almost totally (>80%) contraction (fig 4b).

Again, it is unlikely that approximations in the experimental determination of elasticity due to our in vivo method can modify the control pattern in a large extent.

CONCLUSION

For the first time, the behaviour of the energy metabolism in contracting skeletal muscles under steady state conditions has been quantified here experimentally in vivo in anesthetized rats. Despite the difficulty to apply strictly selective modulations of energy production and consumption in in vivo skeletal muscle, the values of the elasticity coefficients indicate clearly how the steady state of contraction is maintained in skeletal muscle over a wide range of metabolic rates. We highlighted here the low sensitivity of the energy-consuming ATPases to reactants at every metabolic rate as compared to energy production and concluded therefore that, over the range of activity studied here, the activation of myofibrils is strongly dependent on the direct stimulation by calcium. By contrast, the oxidative phosphorylation processes included in the Producer module appeared highly reactive to small changes in the intermediates at low skeletal muscle activity, so that feedback metabolic activation is one possible -although not exclusive -mechanism of activation for energy production. This mechanism is far less efficient in activating energy production at higher level of muscle As concentrations of all reactants remain constant, the local kinetic response of each connected module is constant as well. By contrast, the drop in [PCr] in skeletal muscle when activity increases illustrates substantial changes in all reactants likely due to unbalanced activation. We hypothesize here that the producer elasticity dependence on activity could be due to these changes in reactant concentrations highlighted by MRS assessments of PCr levels (table 1). By contrast with the absence of modification of consumer response to the intermediates, results indicates that producer module kinetics shows substrate saturation (probably by creatine) so that its response to substrate changes decreases at high workload and the elasticity drops to the low values observed.

As we still did not test the regulatory effect of any effector on the system -the effects responsible for a change in the steady state in the formalism of MoCA -no conclusion can be made about the regulation of muscle energetics in vivo. The magnitude of respective activation of energy supply and energy demand when muscle activity increases [16] cannot be detected by our measurements of elasticity and flux control coefficients. To date only approaches in silico have provided insights about intact skeletal muscle [15,17,25,29,[START_REF] Liguzinski | Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies[END_REF] while experimental evidences were obtained recently in intact heart [10,16].

We anticipate that the application of MoCA analysis, will be of great interest for integrative biology studies of healthy or pathological muscles, since it allows deciphering with the biochemistry of energy transfer into the physiology of muscle cells. At this stage, the application of MoCA to skeletal muscle energetics in vivo may be considered as a semiquantitative approach, because of methological limitations, but could well be an advance in relation to purely intuitive considerations. Other semi-quantitative approaches have been demonstrated very helpful in this area [START_REF] Jeneson | A metabolic control analysis of kinetic controls in ATP free energy metabolism in contracting skeletal muscle[END_REF]29]. MoCA has the potential to highlight defects/adaptations in the energy transfer system on the basis of elasticity changes. It is unknown to date whether physical activity, chronic hypoxia or pathologies have the potential to alter elasticities or whether only modules activities are affected. The design and setup of

Biochemical

  Journal Immediate Publication. Published on 22 May 2008 as manuscript BJ20080280 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080280 S t a g e 2 ( a ) P O S T -P R I N T For this purpose, we developed a non-invasive experimental setup providing simultaneous in vivo assessments of PCr content and contraction force from electrically stimulated rat muscle.

Biochemical

  Journal Immediate Publication. Published on 22 May 2008 as manuscript BJ20080280 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080280 S t a g e 2 ( a ) P O S T -P R I N T METHODS Animals Female Wistar rats weighing 280-340g were used according to the guidelines of the National Research Council Guide for the care and use of laboratory animals. Rats were housed in an environmentally controlled facility (12-h light 12-h dark cycle, 22°C), and received water and food ad libitum.

Biochemical

  Journal Immediate Publication. Published on 22 May 2008 as manuscript BJ20080280 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080280 S t a g e 2 ( a ) P O S T -P R I N T After Fourier transform, the NMR spectra were then routinely deconvolved into Lorentzian lines (Igor Pro, Wavemetrics, Oregon USA). The phosphocreatine, Pi, and ß-ATP peak areas were calculated and were converted to concentrations using [ATP] = 8.2 mM. Intracellular pH was determined by using the chemical shift of Pi relatively to phosphocreatine.

  Biochemical Journal Immediate Publication. Published on 22 May 2008 as manuscript BJ20080280 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080280 S t a g e 2 ( a ) P O S T -P R I N T values during this stage when compared to 'reference' were used for the calculation of the producer elasticity (figure 3). The elasticity coefficients in each individual animal were quantified by dividing relative change in contraction by relative changes in [PCr] [10]. The single-experiment determination of the elasticities developed here allows the determination of the control distribution for each animal studied. The contribution of each module to the control over the flux is quantified by a parameter called the flux control coefficient, defined as the ratio of the relative change in a given flux caused by a small relative change in the module activity. While the elasticity is a property of the module, the control coefficient is a property of the whole system. Control coefficients over the contraction energy flux (C p and Cc) were calculated from the experimental measurements of elasticities [10].

  figure3). However, the modulation of the consumer module by changing electrical stimulation may be less selective: some cytosolic factors, e.g. intracellular calcium pulses, might impact the producer module in some extent. It is yet of note that in contrast with cardiac muscle wherein direct activation of Producer by calcium has been demonstrated,[Ca 2+ ] changes may likely be of minor concern in the interpretation of skeletal muscle results. This is illustrated by the dynamics of [PCr] and oxygen consumption in perfused cat soleus (oxidative muscle) as compared to biceps brachii (dominantly glycolytic muscle)[START_REF] Kushmerick | Regulation of oxygen consumption in fast-and slow-twitch muscle[END_REF]: a postexercise overshoot of [PCr] was detectable only in soleus but not in biceps brachii, indicating the persistence of the activation of PCr production only in the electrically stimulated fully oxidative muscle. T 2 -maps indicate that only gastrocnemius muscle groups are stimulated in the present study. Since the rat gastrocnemius is not a fully aerobic muscle[START_REF] Bahi | Does ACE inhibition enhance endurance performance and muscle energy metabolism in rats?[END_REF], the direct activation of PCr production by slight changes in electrical stimulation is minimized. A strong additional argument comes from the integration of a broad range of experimental data from skeletal muscle into a dynamic computer model of oxidative phosphorylation and ATP usage[15, 17, 25]. Indeed, to fit with concerned data, the model predicts the absence of direct activation of PCr production in electrically stimulated muscle[17].

  in ∆Gp. Since only relative changes in the intermediate (and flux) are considered in the Biochemical Journal Immediate Publication. Published on 22 May 2008 as manuscript BJ20080280 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080280 S t a g e 2 ( a ) P O S T -P R I N T modular control analysis, PCr may be used as a representative of ∆Gp in this study [10]. Under our conditions, changes in [H + ] have negligible effects on ∆Gp (e.g. a drop in muscle pH by 0.15 only corresponds to less than 2% change in ∆Gp), and the negligible effect of pH on myofibrils has been demonstrated in skeletal muscle at physiological temperature [27]. At last, muscle pH was assessed in our MRS experiment in vivo and values reported in table 1

Figure 4

 4 shows the results from individual experiments and provides a clear picture of the activity-dependent value of the Producer elasticity to PCr and of the roughly constant Consumer elasticity to PCr. Taking into account these marked differences in elasticity values, and the strong effect of muscle activity on the producer elasticity, the even crude method developed here in vivo provides for the first time a clear picture of the behaviour of the energetic system in contracting skeletal muscle in the in vivo integrative context. Biochemical Journal Immediate Publication. Published on 22 May 2008 as manuscript BJ20080280 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080280 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 22 May 2008 as manuscript BJ20080280 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080280 S t a g e 2 ( a ) P O S T -P R I N T activity, as indicated by the activity-dependent decrease in the producer elasticity, not observed in the heart [10, 14, 28]. It sounds crucial to understand the mechanisms underlying the observed changes in the Producer elasticity with activity in skeletal muscle but not in heart. In heart, due to the perfectly balanced parallel activation of energy supply and energy demand by external effectors [10, 14, 16], PCr (as well as the other energetic metabolites and ∆Gp) does not change as a function of contractile activity.

Biochemical

  Journal Immediate Publication. Published on 22 May 2008 as manuscript BJ20080280 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080280 S t a g e 2 ( a ) P O S T -P R I N T

( 4 Table 2 .Figure 1 :

 421 Figure1: Experimental protocol. Two 31 P spectra were obtained from plantar flexor muscles at rest and then muscles were electrically stimulated for about one hour. After a 20-min period, modulations around the "reference" steady state were achieved, beginning with the steady state under hypoxic conditions (low supply, 9%O 2 ); then, after the switch to normal air, the reference conditions were maintained for 12 minutes. To achieve the "low demand" episode, the consumer was modulated by a slight decrease in electrical stimulation. Simultaneous measurements of 31 P spectra and muscle contraction during modulation of the steady state allowed calculation of the elasticity coefficient of each module. The value for n depends on the desired imposed workload (see Methods section).

Figure 2 :

 2 Figure 2 : 1 H-MRI study of the lower hindlimb of the anesthetized rat placed in the experimental setup. (a) typical T 2 -weighted image obtained from the axial view at rest. (b) T 2 -maps obtained immediately after a typical steady state under low electrical stimulation and (c) high electrical stimulation. The whiter parts (arrows) in the maps b and c as compared to resting conditions show the contracting regions of the muscles in response to electrical stimulation.

Figure 3 :

 3 Figure 3 : Typical PCr and contraction records carried out to calculate producer and consumer elasticities in a single animal. Muscle contraction and 31 P MRS spectra were measured simultaneously during modulations of the steady state. MR spectra were analyzed to assess steady values of energetic intermediates. The magnitude of the contraction signal was averaged over the corresponding period of time. Relative changes in contraction and [PCr] in response to modulations of the steady state provided all the required data for the calculation of the elasticities.

Figure 4 :Figure 1 BiochemicalFigure 2 BiochemicalFigure 3 BiochemicalFigure 4 Biochemical

 41234 Figure 4 : Individual elasticity (A) and control (B) coefficients over the range from low to high muscle workload as a function of relative PCr level. Resting phosphocreatine concentration [PCr] rest amounted to 27-28 mM. Over the range of selected electrical stimulations, the reference steady state [PCr] amounted to 19-20 mM at low workloads, 16-17 mM at moderate workloads and 11-12 mM at high workloads. Changes in elasticity and flux control coefficients as a function of muscle workload were approximated by mono-exponentialfunctions. Exponential functions were drawn from least-square fits of elasticity data and then used to fit control coefficients using equation[START_REF] Kushmerick | Regulation of oxygen consumption in fast-and slow-twitch muscle[END_REF]. Vertical dashed lines delimit the 3 sub-groups of data analysed as low, moderate and high workloads in table 1 and 2.

Table 1 . Average PCr (mM), pH and contraction (microvolts) values during steady state modulations as a function of muscle workload. Steady

 1

	PCr ref (% rest)	77 ± 3%	60 ± 4%	44 ± 4%
	Cytosolic pH	7.01 ± 0.04	6.98 ± 0.03	6.96 ± 0.04
	Modulations around the Reference steady state
	PCr-low supply	16.7 ± 2.1 * †	10.7 ± 2.4 * †	9.6 ± 2.5
	PCr-Reference	20.2 ± 1.8	16.3 ± 1.4	12.4 ± 1.0
	PCr-low demand	23.5 ± 2.5 ‡	19.1 ± 2.7 ‡	14.4 ± 1.3
	Contraction-low supply	279 ± 197	257 ± 122	273 ± 145
	Contraction-Reference	318 ± 172	323 ± 142	355 ± 162
	Contraction-low demand	168 ± 87	261 ± 138	318 ± 161

state conditions are described in figure 1. Values are ± SD. The different workload conditions were achieved by controlled electrical stimulation; respectively low

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgments

This work was supported by: Centre National de la Recherche Scientifique, Université Victor Segalen Bordeaux 2, Conseil Régional de la Région Aquitaine and Institut Fédératif de Recherche 4, Bordeaux, France. Biochemical Journal Immediate Publication. Published on 22 May 2008 as manuscript BJ20080280 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080280 S t a g e 2 ( a ) P O S T -P R I N T

the present experimental approach provide a useful tool to investigate non-invasively the primary target(s) responsible for these various alterations of muscle contraction.


Marek Dynowski

Gabriel Schaaf

Dominique Loque

Oscar Moran

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

Plant plasma membrane water channels conduct the signaling molecule H 2 O 2

Keywords: Hydrogen peroxide permeation Hydrogen peroxide, reactive oxygen species (ROS), aquaglyceroporins, aquaporin Biochemical

Hydrogen peroxide (H 2 O 2 ) is a relatively long-lived reactive oxygen species that signals between cells and organisms. H 2 O 2 signaling in plants is essential for response to stress, defense against pathogens, and the regulation of programmed cell death. Although H 2 O 2 diffusion across membranes is often considered as a passive property of lipid bilayers, native membranes represent significant barriers for hydrogen peroxide. We addressed the question of whether channels might facilitate H 2 O 2 conduction across plasma membranes. The expression of several plant plasma membrane aquaporins in yeast, including PIP2;1 from Arabidopsis, enhanced the toxicity of H 2 O 2 and increased the fluorescence of dye-loaded yeast when exposed to H 2 O 2 . The sensitivity of aquaporin-expressing yeast to hydrogen peroxide was altered by mutations that alter gating and the selectivity of the aquaporins. The conduction of water, H 2 O 2 and urea was compared, using molecular dynamics simulations based on the crystal structure of SoPIP2;1 from spinach. The calculations identify differences in the conduction between the substrates and reveal channel residues critically involved in H 2 O 2 conduction. The results of the calculations on tetramers and monomers are in agreement with the biochemical data. Taken together, the data strongly postulate that plasma membrane aquaporin pores determine the efficiency of H 2 O 2 -signaling between cells. Aquaporins are present in most species and their capacity to facilitate the diffusion of H 2 O 2 may be of physiological significance in many organisms and particularly in communication between different species.

Introduction

Water channels -aquaporins-are identified in most organisms, including plants, and have specific physiological functions related to water facilitation in many tissues [START_REF] Maurel | Plant aquaporins: novel functions and regulation properties[END_REF][START_REF] Kaldenhoff | Functional aquaporin diversity in plants[END_REF][START_REF] Chaumont | Regulation of plant aquaporin activity[END_REF]. Aquaporins are tetramers and each subunit forms a pore. Many plant aquaporin homologs are believed to specifically conduct water, while several homologs also conduct other small neutral solutes, such as glycerol, and are, therefore, called aquaglyceroporins [START_REF] Maurel | Plant aquaporins: novel functions and regulation properties[END_REF][START_REF] Kaldenhoff | Functional aquaporin diversity in plants[END_REF][START_REF] Chaumont | Regulation of plant aquaporin activity[END_REF]. The aromatic/arginine (ar/R) region and the highly conserved NPA region (for Asn, Pro, Ala) have been implicated in the selectivity of aquaporins. In the model plant Arabidopsis thaliana, 35 aquaporin homologs are encoded in the genome, and several of them conduct urea or ammonia in addition to water [START_REF] Liu | Urea Transport by Nitrogen-Regulated Tonoplast Intrinsic Proteins in Arabidopsis[END_REF][START_REF] Loqué | Tonoplast aquaporins AtTIP2;1 and AtTIP2;3 facilitate NH3 transport into the vacuole[END_REF]. For simplicity, we will designate the proteins of this family, which are also called major intrinsic proteins, as aquaporins throughout the article, despite the ability of several homologs to conduct other solutes. In several organisms, including mammals and plants, the expression of water channels is highly regulated at the transcriptional level. In addition, the conductance of several aquaporins can be post-transcriptionally regulated by different stimuli. Regulation by phosphorylation [START_REF] Johansson | Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation[END_REF][START_REF] Lee | Phosphorylation of nodulin 26 on serine 262 affects its voltage-sensitive channel activity in planar lipid bilayers[END_REF], calcium [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF], extracellular pH [START_REF] Zeuthen | Transport of water and glycerol in aquaporin 3 is gated by H(+)[END_REF] or intracellular pH [START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF] has been reported. Crystal structures have unambiguously proven that the plant plasma membrane aquaporin SoPIP2;1 exists in two different, phosphorylation dependent conformational states, an open and a closed state [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. These conformations mostly differ in their cytoplasmic pore openings, but the size and geometry of the narrowest part of the pore, the aromatic/arginine (ar/R) region, is nearly identical. H 2 O 2 is a relatively long-lived reactive oxygen species and can function as a signaling molecule in eukaryotes, leading to specific downstream responses [START_REF] Hancock | Role of reactive oxygen species in cell signalling pathways[END_REF][START_REF] Bienert | Membrane transport of hydrogen peroxide[END_REF]. H 2 O 2 is a small neutral solute and membranes appear to play an important role in protecting cells against its toxicity, which may derive from H 2 O 2 itself or from the more toxic hydroxyl radical derived from H 2 O 2 . This implies that native membranes restrict free H 2 O 2 diffusion and that significant H 2 O 2 gradients across membranes exist. Cellular H 2 O 2 redox chemistry is mainly restricted to compartments surrounded by membranes such as peroxisomes and mitochondria and may also be found in other endomembrane compartments [START_REF] Huckelhoven | Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation[END_REF]. In plants, H 2 O 2 is generated and released upon various stresses, e.g. during nutrient starvation [START_REF] Shin | Hydrogen peroxide mediates plant root cell response to nutrient deprivation[END_REF]. H 2 O 2 is a signal that controls plant programmed cell death [START_REF] Gechev | Hydrogen peroxide as a signal controlling plant programmed cell death[END_REF]. Specific H 2 O 2 generation in plants often involves plasma membrane NADPH oxidases that produce the superoxide anion, by enabling the transfer of electrons from the cytosolic NADPH across the membrane to external molecular oxygen. The spontaneous dismutation of the short-lived superoxide anion (that is very effective at the low apoplastic pH) will produce external hydrogen peroxide. This is followed by H 2 O 2 influx and the subsequent activation of Ca 2+channels [17]. Such NADPH-dependent processes have been identified in the plant defense against attack by pathogens ('oxidative burst'), root hair development [18] and the regulation of abscisic acid signaling and stomatal movements [START_REF] Hancock | Role of reactive oxygen species in cell signalling pathways[END_REF]17]. A recent study has shown that the human AQP8 and the plant tonoplast intrinsic proteins AtTIP1;1 and AtTIP1;2 conduct hydrogen peroxide when heterologously expressed in yeast [START_REF] Bienert | Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes[END_REF]. Earlier work had suggested that algae plasma membranes may contain pores for hydrogen peroxide [22]. In that work, theoretical calculations combined with measurements of the membrane permeability at very high concentrations (up to 350 mM) and the relative unspecific aquaporin inhibitor HgCl 2 were used [20]. In this study, we present evidence that several plant plasma membrane aquaporin homologs conduct the signaling-and defense-related small neutral solute H 2 O 2 . Furthermore, molecular simulations identify channel residues critically involved in H 2 O 2 conduction. The conservation of these residues suggests that many plasma membrane aquaporins from plants facilitate the diffusion of hydrogen peroxide. Although many aquaporins have a welldefined physiological function in water conduction, aquaporins may thus also participate in regulating and controlling H 2 O 2 -signaling.

Experimental

Yeast strains, media and growth conditions

The Saccharomyces cerevisiae strains BY4741 (MATa, his3 1, leu2 0, met15 0, ura3 0) (Euroscarf) and dur3 (MAT , dur3 , ura3 ) and 23344c (MAT , ura3 ) [START_REF] Liu | AtDUR3 encodes a new type of high-affinity urea/H + symporter in Arabidopsis thaliana[END_REF] were employed for growth tests. Uptake studies were performed in the yca1 yeast mutant (MATa, his3 1, leu2 0, met15 0, ura3 0, yor197w::kanMX4) (Euroscarf, Y02453). Yor197w encodes a putative cysteine protease that is similar to mammalian caspases and is involved in the regulation of apoptosis upon hydrogen peroxide treatment. For growth tests cells were grown up to stationary phase in liquid YNB medium (1.7 g / L yeast nitrogen base, 3.0% glucose, 1% arginine, supplemented with histidine, leucine, methionine when appropriate) at 28°C and 160 rpm. The cells were spotted as 5-fold dilution series on freshly prepared plates containing H 2 O 2 ranging from 0.25 mM up to 2.5 mM. The pictures were taken after 96 h.

Plasmid constructs for yeast expression

The open reading frames of AtPIP1;1 (At3g61430), AtPIP2;1 (At3g53420), AtPIP2;4 (At5g60660), AtNIP1;1 (At4g19030) and AtNIP1;2 (At4g18910) were amplified from an Arabidopsis thaliana (Col-0) cDNA library, cloned into the pGEM-T Easy vector (Promega) and then subcloned into pDR195. AtTIP2:3 was described earlier [START_REF] Loqué | Tonoplast aquaporins AtTIP2;1 and AtTIP2;3 facilitate NH3 transport into the vacuole[END_REF]. The point mutations were inserted by using the Quickchange kit from Stratagene. All constructs were verified by sequencing at GATC (Konstanz, Germany). The sequence encoding the N-terminal c-myc epitope was generated by PCR. SDS-PAGE analysis and western blotting followed standard protocols using a monoclonal anti-c-myc antibody (Sigma). The yeast oxysterol binding protein Kes1 was used as a normalization control and visualized with a monoclonal antibody (a kind gift of Vytas Bankaitis).

Fluorescence assays

To reduce endogenous production of H 2 O 2 , the yca1 yeast mutant was employed. This mutant is partially defective in H 2 O 2 induced apoptosis [START_REF] Liu | AtDUR3 encodes a new type of high-affinity urea/H + symporter in Arabidopsis thaliana[END_REF]. Transformants were grown in selective media to midlog phase, and successively supplied with the oxidant-sensitive fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA, Molecular Probes) to a final concentration of 20 µM and incubated at 28 °C and 550 rpm for 45 minutes to allow uptake of the dye. Cells were then washed in water and resuspended in an experimental solution consisting of 40 mM Na-phosphate pH 5.8 containing 3% glucose. The fluorescence was measured before and after addition of H 2 O 2 (final concentration 2 mM or 10 mM). The fluorescence was measured in 10 min. steps using a TECAN 96-well spectrofluorimeter, set at an excitation wavelength of 493 nm and an emission wavelength of 524 nm.

Structure of the open SoPIP2;1 aquaporin

A model of the "open conformation" SoPIP2;1 aquaporin was obtained by combining the open and closed pore crystal structures of SoPIP2;1. The closed structure (PDB accession number 1Z98; ALA24 -VAL179 and PRO201 -SER274) and the open structure (PDB accession 2B5F; PHE180 -LEU200) were combined [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF] and a structural alignment was done using VMD (Suppl. Fig. 2). Serines 115 and 274 were exchanged by phosphoserines to keep the channel in an open state [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. To avoid an artificial negative charge at the Cterminus (as in the partially resolved C-terminus in the structure), a phenylalanine residue, which is not resolved in the crystal, was added to this critical region. For the simulations, this "open conformation" SoPIP2;1 was embedded into a pre-equilibrated 70 x 70 Å (as a monomer, Suppl. Fig. 3) or 130 x 130 Å (as a tetramer) spanning 16:0/18:1c9-palmitoyloleylphosphatidyl-ethanolamin (POPE) lipid bilayer. The system was hydrated with TIP3P water molecules and counter ions were added to obtain a neutral net charge, using the module LEAP from the AMBER8 suite. The total tetrameric system included approximately 111.200 atoms. One substrate molecule was placed above each monomer (>-30 Å in z-direction from the C of valine 206, Fig. 6) and a fifth above the central cavity of the tetramer (Fig. 5). Within a tetramer, the starting position of the substrates differed by + 3 Å in lateral direction from the central axis of each monomer. These four different starting positions were identical for all substrates (i.e. for the center of mass of each substrate). Molecular dynamics simulations Molecular dynamics (MD) was carried out using the NAMD2 program [22] using the Amber03 force field. Force field parameters for hydrogen peroxide and the POPE lipid bilayer were estimated using the module ANTECHAMBER and the general AMBER force field (GAFF) of AMBER8. The bond angles, dipole moment and other properties matched the well-known properties of hydrogen peroxide [START_REF] Bienert | Membrane transport of hydrogen peroxide[END_REF]. The force field parameters for urea were provided with AMBER8 and the force field parameters for phosphoserine was obtained from the AMBER Parameter Database (pharmacy.man.ac.uk/amber/) [START_REF] Craft | An AMBER/DYANA/MOLMOL phosphorylated amino acid library set and incorporation into NMR structure calculations[END_REF]. The system was initially minimized for 1000 steps with the protein atoms fixed. Then another 1000 minimization steps were applied without any atoms fixed. The system was then equilibrated for 2 ns using a timestep of 2 fs with periodic boundary conditions (PBC). A pressure of 1 atm (101,3 KPa) and a temperature of 310 K were applied during equilibration and were kept constant using the Langevin Piston method. Non-bounded interactions were calculated with a cutoff of 12 Å, and the long range electrostatic forces where calculated using the Particle Mesh Ewald method (PME). To keep the protein in position, the C atoms of the protein backbone were constrained with a harmonic force of 0.5 kcal x mol -1 x Å -2 . For monomeric simulations, periodic boundary conditions were used, but the individual MD simulations on tetramers were carried out without PBC, PME and Langevin dynamics. To simulate the permeation across the aquaporin channels, the system was confined by applying a harmonic constraint of 200 kcal x mol -1 x Å -2 to the 3.5 Å outer layer of water. A second water layer of the same thickness below was constrained with 5 kcal x mol -1 x Å -2 . To keep simulation times reasonable, a constant force vector in z-direction was applied to the permeant molecule, either water, hydrogen peroxide or urea [24]. In initial trials we found that the constant force of ~41 pN was required that solutes entered the channel entrance within a few ns. The trajectories along the z-axis were calculated relative to the C atom of valine 206 as reference. The entrance point into the channel along the z-coordinate was defined as C of lysine 144 (on average -20.5 Å + 1.5 Å from valine) and the internal channel exit was defined as C of proline 33 (19.2 Å + 1.3 Å from the valine 206).

Residency time and energy profiles

To construct the overall histogram of the time at each position along the reference axis, the residence time measured for 0.2 Å intervals of each simulation was accumulated in a weighted sum, such that the biased potentials due to the applied force were discounted. The energy profiles were then estimated in 1 Å intervals along the channel axis from transition state theory as [START_REF] Johnson | The Theory of Rate Processes in Biology and Medicine[END_REF]: where is the residence time, G ‡ is the Gibbs free energy of activation, T is the absolute temperature, R is the universal gas constant, k b is Boltzmann's constant, and h is Planck's constant.

Results

Expression of AtPIP2;1 increased H 2 O 2 sensitivity and H 2 O 2 accumulation in yeast

We considered the possibility that aquaporins could mediate the conduction of H 2 O 2 across membranes, since some channel homologs have been shown to conduct several low molecular weight molecules. Initial studies focused on the Arabidopsis protein AtPIP2;1. When the Arabidopsis AtPIP2;1 was heterologously expressed in yeast, growth and doubling times were unaffected in standard liquid culture or solid media. However, expression of AtPIP2;1 increased sensitivity of yeast cells to H 2 O 2 (Fig. 1A). Growth of cells expressing AtPIP2;1 was affected in a dose dependent manner and impaired at 400 µM H 2 O 2 , while non-expressing controls, transformed with the vector pDR195 without the aquaporin coding insert, were not sensitive up to 1.6 mM H 2 O 2 (Fig. 1A). In a similar growth assay, the plant tonoplast aquaporin TIP1;1 was recently shown to conduct hydrogen peroxide [START_REF] Bienert | Membrane transport of hydrogen peroxide[END_REF][START_REF] Bienert | Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes[END_REF]. In principle, reduced or impaired growth may be due to factors, which perturb the metabolism in an unknown way. To further test whether the increased sensitivity of cells expressing AtPIP2;1 was due to increased uptake of H 2 O 2 , a carboxylated derivative of the oxidant sensitive probe 2',7'dichlorofluorescein (DCF) was employed. When cells are incubated with a di-ester derivative of the probe, it enters the cytosol by passive diffusion. Once inside the cell, the esters are cleaved by endogenous esterases, rendering the probe impermeable to the membrane. The DCF can be oxidized by reactive oxygen species such as H 2 O 2 resulting in a fluorescence increase [START_REF] Yurkow | Characterization of hypoxia-dependent peroxide production in cultures of Saccharomyces cerevisiae using flow cytometry: a model for ischemic tissue destruction[END_REF]. To reduce the production of internally generated reactive oxygen species (ROS) by H 2 O 2 -stimulated apoptosis, we used the yeast yca1 mutant that is partially defective in undergoing apoptosis [START_REF] Madeo | A caspase-related protease regulates apoptosis in yeast[END_REF]. Upon exposure to H 2 O 2 , the overall intracellular accumulation of reactive oxygen species that was measured with the fluorescent dye, was higher in AtPIP2;1-expressing yeast cells compared to cells transformed with empty vector alone. Consistent with these data, the c-myc epitope tagged AtPIP2;1 was stable in yeast and detected as a monomeric protein of ~30 kDa (Fig. 2A) and as an oligomeric protein (data not shown).

Sensitivity of yeast to H 2 O 2 is altered by a mutation that reduces PIP2 water conductance

A conserved cytosolic histidine residue has been shown to be involved in PIP2 regulation of water flux by cytosolic pH, a phenomenon that was initially demonstrated using the homolog AtPIP2;2 [START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF]. Replacement of this histidine by lysine in AtPIP2;2 reduced water conduction and mimicked a closed channel [START_REF] Tournaire-Roux | Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins[END_REF]. The relevant histidine at position 199 in AtPIP2;1 (Fig. 2B) was replaced by lysine (H199K) by site directed mutagenesis. As expected, the sensitivity of H199K mutant expressing yeast to grow on H 2 O 2 was similar to vector transformed controls (Fig. 2C), although a similar protein level was detected from this mutant in Western analysis (Fig. 2A).

Sensitivity of yeast to H 2 O 2 is altered by pore mutations that alter selectivity

Residues in the constriction region of the pores can affect the selectivity properties of aquaporins [28,[START_REF] Tajkhorshid | Control of the selectivity of the aquaporin water channel family by global orientational tuning[END_REF]. Alignments and homology models indicate that the constriction region of all PIP-type aquaporins from Arabidopsis is identical. In AtPIP2;1, the constriction (ar/R) region is composed of the four residues Phe 87, His 216, Thr 225 and Arg 231. In contrast, the "selectivity filter" residues in NIP-type major intrinsic proteins differ, which leads to a larger pore diameter [28]. A mutant, AtPIP2;1 NIP1;2 , in which the ar/R residues in AtPIP2;1 were exchanged to the corresponding residues in AtNIP1;2 (F87W, H216V, T225A) also conducted H 2 O 2 , as was deduced from its ability to confer increased sensitivity to H 2 O 2 when expressed in yeast (Fig. 3C). The ar/R constriction region in this mutant is wider, as can be easily deduced from homology models of this mutant (Suppl. Fig. 1). The residues of the ar/R region of AtPIP2;1 were also exchanged to the corresponding residues of AtNIP5;1, which conducts hydrophobic, non-charged solutes as large as boric acid, but not water, at a high rate [START_REF] Takano | The Arabidopsis major intrinsic protein NIP5;1 is essential for efficient boron uptake and plant development under boron limitation[END_REF]. By introducing the three relevant mutations in AtPIP2;1 (F87A, H216I, T225G), the ar/R constriction region was even wider in this mutant channel (designated AtPIP2;1 NIP5;1 ) (Suppl. Yeast growth is sensitive to high concentrations of toxic boron. AtPIP2;1 expression affected neither the boric acid sensitivity of yeast, nor enhanced urea uptake on selective plates (Fig. 3B;D). The NIP1;2-like aquaporin mutant (AtPIP2;1 NIP1;2 ) did not exhibit novel properties compared to AtPIP2;1. In contrast, the expression of AtPIP2;1 NIP5;1 enhanced its growth sensitivity to boric acid, indicating boron uptake (Fig. 3B). This mutant form of AtPIP2;1 dramatically increased the sensitivity of yeast growth to hydrogen peroxide beyond that of wild type AtPIP2;1 (Fig. 3C). When expressed in a yeast mutant that lacks endogenous urea transporters ( dur3; [START_REF] Liu | AtDUR3 encodes a new type of high-affinity urea/H + symporter in Arabidopsis thaliana[END_REF]) this construct enabled yeast to grow on urea as a sole nitrogen source, indicating urea conduction across the plasma membrane (Fig. 3D). Thus, an AtPIP2;1 aquaporin with selectivity filter as in AtNIP5;1 apparently facilitates the conduction of larger solutes, including hydrogen peroxide. These observations are in agreement with earlier studies [28] that showed that residues in the ar/R region are critical for substrate selectivity. H 2 O 2 conduction by some, but not all aquaporin homologs Representatives of individual sub-groups of the plant aquaporins were tested for their capacity to impair yeast growth on plates containing hydrogen peroxide. While cells expressing either AtPIP1;1 or AtNIP1;1 were similar to controls, the yeast expressing AtNIP1;2 or the tonoplast-intrinsic AtTIP2;3 exhibited an intermediate phenotype (Fig. 4). It is interesting to note that AtNIP1;1 had been found to conduct glycerol across the plasma membrane when expressed in yeast [START_REF] Weig | Functional identification of the glycerol permease activity of Arabidopsis thaliana NLM1 and NLM2 proteins by heterologous expression in Saccharomyces cerevisiae[END_REF]. Despite strong expression of the c-myc tagged AtNIP1;1 in yeast (Fig. 2A), it did not increase the sensitivity to H 2 O 2 (Fig. 4). We conclude that hydrogen peroxide conduction is not supported by NIP1;1, at least when heterologously expressed in yeast. The AtPIP2;4-expressing cells were the most sensitive to H 2 O 2 (Fig. 4). Whether these differences in hydrogen peroxide sensitivity are due to differences in the open probability of the channel, (mis-)targeting of the membrane proteins to other membranes in the heterologous system or due to protein stability, remains unclear at this point. It is, however, notable that AtPIP1;1, AtPIP2;1 and AtPIP2;4 have identical residues at the selectivity filter, which differ in other family members such as AtNIP5;1. It has been repeatedly noted that PIP1-type aquaporins are not active in heterologous systems [START_REF] Chaumont | Regulation of plant aquaporin activity[END_REF]. This may explain why AtPIP1;1 expression did not alter sensitivity to H 2 O 2 (Fig. 4). The tonoplast intrinsic AtTIP2;3, which is known to facilitate ammonia and urea conduction when heterologously expressed in yeast [START_REF] Loqué | Tonoplast aquaporins AtTIP2;1 and AtTIP2;3 facilitate NH3 transport into the vacuole[END_REF] also conducts H 2 O 2 . This homolog is known to facilitate solute fluxes across the plasma membrane of yeast, despite its intracellular localization in planta [START_REF] Loqué | Tonoplast aquaporins AtTIP2;1 and AtTIP2;3 facilitate NH3 transport into the vacuole[END_REF]. Other TIP-type aquaporins, with further different selectivity filter residues in the ar/R region, have been shown to conduct hydrogen peroxide [START_REF] Bienert | Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes[END_REF].

Molecular simulation of conduction through PIP2;1

The plasma membrane localized aquaporin AtPIP2;1 from Arabidopsis is highly similar to other PIP2-type aquaporins, such as the crystallized SoPIP2;1 channel from spinach. The closest Arabidopsis homolog of SoPIP2;1 is AtPIP2;7, but all PIP2-type aquaporins share a very high conservation within the pore and have the identical selectivity filter residues. Open and closed conformations of the SoPIP2;1 structure are available [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. We performed molecular dynamics studies on the open SoPIP2;1 channel to compare H 2 O and H 2 O 2 conduction through plant plasma membrane aquaporins. We initially observed that the closed pores of the tetrameric SoPIP2;1 were highly stable during minimization, equilibration and further simulation, when embedded in a lipid bilayer. In addition, the pores readily filled with a single-file of water during equilibration, but water conduction was impaired by loop D, which closes the pore from the cytosolic side. In contrast, the structures of the lowerresolution open monomers, or homology modeled AtPIP2;1, were less stable during similar simulations and no continuous, stable water line formed, as described in previous simulations [START_REF] Tajkhorshid | Control of the selectivity of the aquaporin water channel family by global orientational tuning[END_REF][START_REF] De Groot | Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF[END_REF]. This was probably mainly due to the lower structural resolution (data not shown).

We therefore constructed open pore monomers based on the high-resolution structure of the closed pore, where the backbone came from the closed pore, but loop D was taken from the open conformation (see methods, Suppl. Fig. 2). Molecular dynamics (MD) simulations on a modified 'open' model of the closed pore structures have already been published [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF]. The 'open' pore tetramer embedded in a POPE lipid bilayer is shown in Fig. 5A. We first tested whether steered molecular dynamics [24] can reproduce the well-established features of H 2 O conduction in aquaporins from other species [START_REF] Tajkhorshid | Control of the selectivity of the aquaporin water channel family by global orientational tuning[END_REF][START_REF] De Groot | Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF[END_REF]. In accordance with previous reports, a stable water line formed in this 'high-resolution open' monomer structure during a MD simulation (Fig. 5B). Furthermore, the dipole orientation of water molecules reversed at the NPA region, similar as in aquaporins from other species [START_REF] Tajkhorshid | Control of the selectivity of the aquaporin water channel family by global orientational tuning[END_REF][START_REF] De Groot | Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF[END_REF]. To increase the probability that a specific water molecule enters a pore, it was positioned close to the external pore vestibule and a constant force was applied to steer the solute into the zdirection (see Fig. 5A) [24]. Since solutes might also pass through the tetramer center, rather than the four channel pores [START_REF] Wang | Exploring gas permeability of cellular membranes and membrane channels with molecular dynamics[END_REF], we positioned a fifth water on top of the tetramer center (Fig. 5A, Suppl. Fig. 3). The trajectories from several independent simulations showed how H 2 O molecules traverse the pores (Fig. 6A). We observed coordinated water conduction, similar to what has been described in earlier simulations on other aquaporins [START_REF] Tajkhorshid | Control of the selectivity of the aquaporin water channel family by global orientational tuning[END_REF][START_REF] De Groot | Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF[END_REF][START_REF] Han | Water transport in AQP0 aquaporin: molecular dynamics studies[END_REF][START_REF] Zhu | Theory and simulation of water permeation in aquaporin-1[END_REF]. After 4 ns, 7 of 11 (64%) water molecules had crossed the pore entirely (Fig. 6A). Individual water molecules showed two preferred residency positions close to the ar/R region and the NPA region (Fig. 6A). Only one single water molecule that had entered the outer channel vestibule did not cross the entire pore during the full simulation time of 16 ns. This molecule moved away from the central pore axis and got stuck in a cavity close to glutamate 168 in transmembrane helix 4. Furthermore, the continuous water line was broken in some cases at the ar/R region, a feature that was also observed when pressure was applied to accelerate the transit of solutes [START_REF] Zhu | Pressure-induced water transport in membrane channels studied by molecular dynamics[END_REF]. In particular cases, the labeled waters did not enter a pore. The trajectories of these waters were excluded from further analysis. The dwell-time estimates derived from the water trajectories indicated that the major energy barriers within the plant aquaporin SoPIP2;1 pore are the ar/R and the NPA region (Fig. 6A,D), which are critical for selective water flux, as had been concluded earlier [START_REF] Tajkhorshid | Control of the selectivity of the aquaporin water channel family by global orientational tuning[END_REF][START_REF] De Groot | Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF[END_REF]. H 2 O 2 conduction in PIP2;1 Similar simulations were done with five H 2 O 2 molecules replacing the waters at identical starting positions. Hydrogen peroxide competed with water for hydrogen bonds, but it was evident from the trajectories that the entrance of H 2 O 2 , as well as the conductance, was slightly less favorable compared to water (Fig. 6B). After 4 ns, only two full conduction events were observed (15%), but all H 2 O 2 molecules finally crossed the pore during the 16 ns simulations. The major residency positions were similar to those of water, but further sites of increased residency close to the internal pore vestibule were detected (Fig. 6B). A closer look at individual simulations showed that the residency at these positions resulted from the exact position of the flexible cytoplasmic loop D that is responsible for closing the pore. The maximal residency time for H 2 O 2 in the ar/R region (indicating a major energy barrier for conduction) is in line with our finding that increasing the pore diameter in this region increases H 2 O 2 conduction/sensitivity (Fig. 3).

Comparison of solute conduction simulations in SoPIP2;1

Similar simulations were also done with urea, a solute that was not conducted by AtPIP2;1, as indicated by our growth assays (Fig. 3D). The trajectories for urea indicated that this solute was also not conducted in simulations. No full conduction event was observed within 16 ns. Only one single urea molecule had traversed most of a pore (Fig. 6C). The trajectories identify the ar/R region as insuperable barrier for urea conduction (Fig. 6C,D). This result is strongly supported by the growth assays in Fig. 3, which indicate that mutations in the ar/R region can convert PIP2;1 into a urea conducting channel. Mutations in the ar/R region result in a wider pore diameter, which agrees with the conduction of urea in these mutants [START_REF] Dynowski | TrpAQP: Computer simulations to determine the selectivity of Aquaporins[END_REF]. The energy profile of each substrate along the z-axis was calculated from the trajectories and further illustrates the differences in the conduction properties (Fig. 6D). The energy barriers for H 2 O 2 along the pore axis was at most positions slightly higher than for water, but the maximal barrier was of similar hight. All three tested substrates have a dipole moment, which was preferentially oriented within the pore. This is well observed at the sites of longer residency, where snapshots of H 2 O 2 and urea in their preferential positions and orientation are given in Fig. 6E,F. Neither hydrogen peroxide nor urea permeated through the tetramer center; the solutes did not enter this central cavity. In contrast, the solutes initially positioned above the tetramer center nearly always entered and permeated a pore. For this reason, we also tested whether smaller simulations with monomers give a similar outcome. Such smaller simulations are advantageous, since they saved 39 % of resource time. The monomer was again embedded into a small patch of lipid bilayer (Suppl. Fig. 4). Interestingly, these simulations yielded essentially the same results (Suppl. Fig. 5). The trajectories of water, H 2 O 2 and urea resembled those from tetramer simulations. This suggests that monomers are sufficiently rigid to computationally simulate conduction in isolated, monomeric pores.

Discussion

The plasma membrane is an important protective barrier against extracellular H 2 O 2 . Yeast strains with impaired ergosterol biosynthesis and, thus, higher membrane permeability to lipophilic substances, were more sensitive to H 2 O 2 [38]. At low millimolar concentrations, hydrogen peroxide induces apoptosis in yeast [START_REF] Madeo | A caspase-related protease regulates apoptosis in yeast[END_REF][START_REF] Jamieson | Oxidative stress responses of the yeast Saccharomyces cerevisiae[END_REF]. Gradients of H 2 O 2 have been reported across the membranes from mammalian cell lines [START_REF] Antunes | Estimation of H 2 O 2 gradients across biomembranes[END_REF], bacteria [START_REF] Seaver | Hydrogen Peroxide Fluxes and Compartmentalization inside Growing Escherichia coli[END_REF] and yeast [38], suggesting that native biological membranes generally prevent the free diffusion of H 2 O 2 . Very recently, tonoplast intrinsic proteins (TIPs) from plants have been shown to specifically conduct H 2 O 2 by different assays, including growth sensitivity on hydrogen peroxide containing plates [START_REF] Bienert | Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes[END_REF]. However, the physiological role of hydrogen peroxide permeability across the tonoplast remains somewhat unclear. We confirmed H 2 O 2 conduction by a tonoplast intrinsic protein (Fig. 4). Tonoplast intrinsic proteins (TIPs) have a relatively weak selectivity and conduct many solutes, including urea, ammonium and water. Our analysis indicates that not only intracellular aquaporins, such as TIPs, but also plasma membrane PIP2-type aquaporins, which were previously believed to be highly selective channels for water, conduct hydrogen peroxide. The Saccharomyces genome encodes four aquaporin genes, two of which are highly homologous to specific water channels. Many laboratory yeast strains, however, contain function-abolishing mutations in these aquaporins [START_REF] Carbrey | Aquaporins in Saccharomyces: Characterization of a second functional water channel protein[END_REF]. Therefore two different laboratory strains were tested for their H 2 O 2 growth sensitivity, but only minor differences were observed and all were unable to grow at > 3 mM H 2 O 2 (data not shown). In both strains, the sensitivity to H 2 O 2 increased several-fold with the heterologous expression of AtPIP2;1 aquaporins. The permeability of the plasma membrane to nonionic substances is generally determined by the membrane fluidity that depends on the content of e.g. sterols [START_REF] Walter | Permeability of small nonelectrolytes through lipid bilayer membranes[END_REF]. Higher plant plasma membranes are known for their high sterol content, which suggests that native plant membranes are relatively impermeable to non-electrolytes [START_REF] Uemura | Cold Acclimation of Arabidopsis thaliana (Effect on Plasma Membrane Lipid Composition and Freeze-Induced Lesions)[END_REF]. In the simulations we used a simple POPE-lipid membrane without sterols, which does not reflect the complex properties of a native plant membrane. This simple membrane is expected to confer only a relatively weak diffusion barrier for solutes. In agreement with this, we occasionally observed full conduction events in simulations with water (three full events from all simulations) and hydrogen peroxide (two full events) across the synthetic lipid membrane.

Here we identified a novel substrate of channels by a combination of functional assays and computer simulations. The combined data suggest that PIP2-type plasma membrane aquaporins are efficient hydrogen peroxide channels (Fig. 6). The aquaporin-mediated conduction of H 2 O 2 may explain previous observations that algal membranes exhibit permeability to H 2 O 2 at high concentrations [20]. The molecular analysis presented here suggests that the ar/R region is critically involved in selectivity towards urea, boron and hydrogen peroxide. Widening the pore allows larger solutes to pass through the channel (Fig. 3B,D), which is in agreement with previous reports [28,[START_REF] Dynowski | TrpAQP: Computer simulations to determine the selectivity of Aquaporins[END_REF]. The simulations and functional data suggest that the ar/R selectivity filter region is the most critical determinant of hydrogen peroxide conduction in aquaporins. Since all plasma membrane PIP2 aquaporins from plants have the identical residues at these positions, it appears reasonable to predict that all eight PIP2-type aquaporins from Arabidopsis conduct hydrogen peroxide. The yeast growth tests further imply that some aquaporins, with a different selectivity filter region, also conduct this solute (Fig. 3, Fig. 4). Interestingly, resource-saving simulations on monomeric SoPIP2;1 yielded an overall result similar to full simulations, although the transit time along the channel seems to be reduced for monomeric simulations. This may be exploited in the future to reduce calculation times and costs. Indeed, simulations on monomers have already been used to probe the conduction properties of aquaporins [45]. Computer simulations on channels also appear to be valuable in predicting novel substrates; this may be used in the future to predict Although the pores were readily filled with a line of waters, the ar/R region was only partially occupied in several independent simulations and the flexible side chain of the arginine (R224) bent briefly into the pore. The flexibility of this arginine had been observed in earlier simulations on other aquaporins [START_REF] Han | Water transport in AQP0 aquaporin: molecular dynamics studies[END_REF][START_REF] Zhu | Theory and simulation of water permeation in aquaporin-1[END_REF], and was therefore fixed in some simulations [START_REF] Zhu | Theory and simulation of water permeation in aquaporin-1[END_REF]. However, the flexibility appears to reflect free arginine rotamers, since this arginine also adopted different conformations within subunits of the AqpZ tetramer [START_REF] Savage | Architecture and selectivity in aquaporins: 2.5 a x-ray structure of aquaporin z[END_REF]. No restrains were applied to any of the side chains during our simulations and the arginine behaved similarly in simulations with water or hydrogen peroxide. When expressed in yeast, a high permeability of aquaporins to hydrogen peroxide results in susceptibility to H 2 O 2 , indicating the putative physiological importance of the hydrogen peroxide conduction by aquaporins. It is known that developmental processes, such as root hair growth and stomatal movements, require NADPH oxidase activity and probably subsequent H 2 O 2 influx to activate Ca 2+ -channels [17]. Whether hydrogen peroxide conduction across plasma membranes is altered in Arabidopsis mutants that lack individual aquaporins remains an unanswered question. However, the large number of plasma membrane aquaporins and their partially overlapping expression pattern and redundant function may require plants in which multiple PIP2s are knocked-out [START_REF] Maurel | Plant aquaporins: novel functions and regulation properties[END_REF]. Arabidopsis mutants with strongly reduced water conduction are not yet available [START_REF] Maurel | Plant aquaporins: novel functions and regulation properties[END_REF]. In line with this, we did not find hydrogen peroxide-related phenotypes with plant lines harboring single T-DNA insertions in PIP2 genes in preliminary experiments (data not shown). It appears likely that plasma membrane aquaporins may be far more than just mediators of water conduction. Recent observations suggest that other small solutes, such as CO 2 , are also channeled by at least some aquaporins [START_REF] Maurel | Plant aquaporins: novel functions and regulation properties[END_REF][START_REF] Kaldenhoff | Functional aquaporin diversity in plants[END_REF]. That excess H 2 O 2 influx kills yeast is supported by the growth tests (Fig. 1) and mutant yeast strains [38]. Aquaporins belong to a multi-gene family in plants and it remains a future challenge to identify the physiological contribution of each individual member to hydrogen peroxide conduction. The ability of pathogens to resist oxidative stress is crucial for their infectiousness and pathogenesis in the host. Aquaporins have also been identified in microbes and pathogens. Pathogens might increase virulence by minimizing the influx of the hydrogen peroxide generated by the plant. Protection against influx of H 2 O 2 may be mediated by pore closure, e.g. by de-phosphorylation, transcriptional down-regulation during infection, or even genomic loss of aquaporins. The AtPIP2;1 NIP5;1 mutant affects yeast sensitivity to boron (B) and H 2 O 2 (C). AtPIP2;1 NIP5;1 rescues growth on urea in absence of an alternative nitrogen source (D). AtPIP2;1 NIP1;2 is a mutant construct with residues as in AtNIP1;2 (F87W, H216V, T225A); the respective residues in the "selectivity filter" of AtNIP1;2 are Trp, Val, Ala and Arg. AtPIP2;1 NIP5;1 has residues F87A, H216I, T225G; the respective residues in the "selectivity filter" of AtNIP5;1 are Ala, Ile, Gly and Arg. The energy profile deduced from the residence time for water (blue), hydrogen peroxide (red), and urea (green) across the SoPIP2;1 pore. The variance along the z-axis of the entrance, selectivity filter (SF), NPA-region and exit is also shown. Open monomers with preferred sites and orientation of H 2 O 2 (E) and urea (F). The residues of the constriction site (ar/R, blue) and the NPA region (yellow) are explicitly shown. Note the flexible cytoplasmic loop D that is involved in closing of the pore (shown in green).

Figure legends

Biochemical

  Journal Immediate Publication. Published on 08 May 2008 as manuscript BJ20080287 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080287 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 08 May 2008 as manuscript BJ20080287 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080287 S t a g e 2 ( a ) P O S T -P R I N T

  Fig. 1). Biochemical Journal Immediate Publication. Published on 08 May 2008 as manuscript BJ20080287 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080287 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 08 May 2008 as manuscript BJ20080287 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080287 S t a g e 2 ( a ) P O S T -P R I N T physiologically important solutes that cannot be easily identified or measured by functional assays.

Fig. 1 .

 1 Figure legendsFig. 1. Growth of Saccharomyces cerevisiae is sensitive to H 2 O 2 and sensitivity is increased by aquaporin expression. (A) Saturated cultures of 23344c cells transformed with pDR195 alone or pDR195-AtPIP2;1 (of same optical density) were spotted in 5 fold dilutions on medium not supplemented with hydrogen peroxide (upper), or supplemented with 0.4 mM H 2 O 2 (middle), or 1.6 mM H 2 O 2 (lower panel). (B) Increased H 2 O 2 levels in Saccharomyces cerevisiae cells expressing the plant plasma membrane aquaporin AtPIP2;1. Yeast yca1 cells transformed with either empty pDR195 or pDR195-AtPIP2;1 were grown to midlog phase and pre-incubated in 2'7'-dichloro-dihydrofluorescein diacetate. Normalized fluorescence (by cell density) detected 30 min after application of 2 mM or 10 mM H 2 O 2 is shown.

Fig. 2 .

 2 Fig. 2. Mutations that affect water conduction also affect sensitivity to H 2 O 2 . (A)Western blot of c-myc tagged AtPIP2;1, the AtPIP2;1 mutant H199K and AtNIP1;1. The monomeric protein band is shown in all cases, Kes1p an oxysterol binding protein from yeast, served as a loading control. (B) Side view of a homology structure of a monomeric AtPIP2;1 pore in ribbon representation. The His199 residue is highlighted by the arrow. (C) Saturated cultures of BY4741 yeast cells transformed with pDR195 alone, pDR195 harboring AtPIP2;1 or the regulatory mutant H199K were spotted in 5-fold serial dilutions. Control plates (no H 2 O 2 added) left upper panel and plates containing the indicated hydrogen peroxide concentrations.

Fig. 3 .

 3 Fig. 3. Pore mutations alter sensitivity to H 2 O 2 and various other substrates (A) Expression of pore mutants in the selectivity filter region (the aromatic/arginine region) does not affect growth on standard media. The yeast strain used in this figure was dur3. (B)The AtPIP2;1 NIP5;1 mutant affects yeast sensitivity to boron (B) and H 2 O 2 (C). AtPIP2;1 NIP5;1 rescues growth on urea in absence of an alternative nitrogen source (D). AtPIP2;1 NIP1;2 is a mutant construct with residues as in AtNIP1;2 (F87W, H216V, T225A); the respective residues in the "selectivity filter" of AtNIP1;2 are Trp, Val, Ala and Arg. AtPIP2;1 NIP5;1 has

Fig. 4 .

 4 Fig. 4. Several plant aquaporins increase H 2 O 2 sensitivity. Spotted dilutions of yeast BY4741 cells transformed with either empty plasmid pDR195 or plant major intrinsic proteins in pDR195 onto media not supplemented with hydrogen peroxide show equal growth (left panel). Spotted 5-fold dilutions at 1 mM, 2 mM and 2.5 mM hydrogen peroxide (right panels).

Fig. 5 .

 5 Fig. 5. Setup of the molecular dynamics simulations on tetrameric open SoPIP2;1. (A) Side view of the tetramer embedded in a lipid bilayer with the individual starting positions of the waters (or H 2 O 2 , urea) explicitly shown (arrows). (B) Snapshot of the water-filled structure of an open pore monomer. Residues of the NPA region (residues 101-103 and 222-224) are explicitly shown in yellow. The external pore exit was defined as C of lysine 144 (orange) and the internal exit as C of proline 33 (orange). Note the coordinated water dipole orientation within the pore.

Fig. 6 .

 6 Fig. 6. Molecular dynamics simulations of H 2 O conduction by SoPIP2;1. Trajectories of water (A), hydrogen peroxide (B) and urea (C) across the open SoPIP2;1 pores from individual simulations (colored lines in each panel). The trajectories were calculated relative to C of valine 206. Note the starting position of solutes at -35 Å. The average residence time for each molecule is shown at the right of each panel (water: blue, hydrogen peroxide: red, urea: green). (D)The energy profile deduced from the residence time for water (blue), hydrogen peroxide (red), and urea (green) across the SoPIP2;1 pore. The variance along the z-axis of the entrance, selectivity filter (SF), NPA-region and exit is also shown. Open monomers with preferred sites and orientation of H 2 O 2 (E) and urea (F). The residues of the constriction site (ar/R, blue) and the NPA region (yellow) are explicitly shown. Note the flexible cytoplasmic loop D that is involved in closing of the pore (shown in green).

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 08 May 2008 as manuscript BJ20080287 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080287 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 08 May 2008 as manuscript BJ20080287 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080287 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 08 May 2008 as manuscript BJ20080287 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080287 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements: We thank Junpei Takano for plasmids and discussions and Vytas Bankaitis for providing reagents. This work was supported in part by a grant of the Landesstiftung Baden Württemberg to U.L. Simulations were carried out at the HPC-center of the University of Tübingen, the Höchstleistungsrechenzentrum of the University of Stuttgart (HLRS) and the Scientific Supercomputing Center (SSC) Karlsruhe, Germany.