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First published online September 23, 2005; 10.1104/pp.105.066670 Plant Physiology 139:1015-1029 (2005) © 2005 American Society of Plant Biologists
NH4+ Currents across the Peribacteroid Membrane of Soybean. Macroscopic and Microscopic Properties, Inhibition by Mg2+, and Temperature Dependence Indicate a SubpicoSiemens Channel Finely Regulated by Divalent Cations1Molecular Plant Physiology, Division of Allergy and Immunobiology, Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria (G.O.); and School of Agriculture and Wine, University of Adelaide, Glen Osmond 5042, South Australia, Australia (S.D.T.)
The control of ammonium (NH4+) transport is critical in preventing futile cycles of NH4+/ammonia transport. An unusual nonselective cation channel with subpicoSiemens single-channel conductance permeable to NH4+ had previously been identified in the peribacteroid membrane (PBM) of symbiosomes from soybean (Glycine max) nodules. Here, we investigate the proposed channel mechanism and its control by luminal magnesium. Currents carried by NH4+ were measured in inside-out PBM patches by patch clamp. NH4+ transport corresponding to the physiological direction of net transfer showed time-dependent activation and associated single-channel-like events. These could not be resolved to discrete conductances but had the same selectivity as the total current. The voltage dependence of the steady-state current was affected by temperature consistent with the rate constant of channel opening being reduced with decreased temperature. This resulted in steady-state currents that were more temperature sensitive at voltages where the current was only partially activated. When fully activated, the current reflected more the ion conduction through open channels and had an activation energy of 28.2 kJ mol–1 (Q10 = 1.51, 8°C–24°C). Increased Mg2+ on the symbiosome lumen side blocked the current (ID50 = 351 µM, with 60 mM NH4+). Complete inhibition with 2 mM Mg2+ was relieved with a small increase in NH4+ on the lumen side of the membrane (shift of 60–70 mM). With Mg2+ the selectivity of the transport for divalent cations increased. From these features, we propose a divalent-dependent feedback regulation of the PBM-nonselective cation channel that could maintain a constant NH4+ gradient across the membrane.
The transport of ammonium (NH4+)/ammonia (NH3) has important roles in nitrogen assimilation in plants, algae, and bacteria, and for pH balance in animals. The control of NH4+ transport is critical to prevent futile cycles that may dissipate pH gradients, waste cell energy, and could account for NH4+ toxicity (Britto et al., 2001a  ). Recently, the structure of the AmtB NH4+ transporter from Escherichia coli has indicated that the protein functions as a channel transporting NH3 after the NH4+ is deprotonated in the periplasmic vestibule of the channel at a site that effectively reduces its pKa, and then returning to equilibrium as NH4+ through protonation of NH3 in the cytoplasmic vestibule (Khademi et al., 2004). This transport would not be electrogenic and also does not require conformational changes during the transport cycle as indicated from the structural study. The human rhesus glycoproteins that share conserved sequences with the Amt family show electroneutral NH4+/H+ exchange characteristics (Ludewig, 2004) and NH3 stimulated NH4+ transport (Bakouh et al., 2004) when expressed in Xenopus laevis oocytes. Aquaporins have also been implicated in NH3 transport (Niemietz and Tyerman, 2000; Jahn et al., 2004; Loqué et al., 2005) that would function as a low affinity version of the AmtB mechanism. Increased NH3 permeability facilitated by aquaporins would increase the rate of passive NH3 flux into an endomembrane-bound acidic compartment provided there is an appropriate gradient (e.g. if cytoplasmic [NH4+] >10 mM; see Britto et al., 2001b), and NH4+ would be trapped in the acid compartment (Loqué et al., 2005). However, NH4+ could move passively back from the acid compartment (Britto et al., 2001b), say through a nonselective cation channel (NSCC), causing a futile cycling of nitrogen and dissipation of the pH gradient. It is thus essential that tight control should occur over the fluxes of both NH4+ and NH3 across membranes.
In legumes, a membrane that has a high density of NH4+/NH3 transport that surrounds an acidic compartment is the peribacteroid membrane (PBM; Day et al., 2001
The PBM-NSCC is novel among NSCC in plants (Demidchik et al., 2002
Another novel feature of the PBM-NSCC is its very low apparent single-channel conductance and very high density in the membrane (Tyerman et al., 1995
In summary, the properties of the PBM-NSCC that stand out as being unusual for an ion channel-mediated transport in plant membranes are (1) inward rectification caused by Mg2+ on the cytosolic face; (2) the very low open-channel conductance (0.11 pS); (3) saturation of the conductance with increasing NH4+ concentration; and (4) stationary noise spectra (power proportional to inverse of the frequency) that is typical for carriers, or ion channels with high cooperativity or a high number of kinetic states (Tyerman et al., 1995
In view of the emerging novel mechanisms for NH4+ transport (Khademi et al., 2004
Channel-Like Activity Can Sometimes Be Observed Associated with the NH4+ Currents
After formation of a "giga seal" at the symbiosome membrane in the cell-attached configuration, currents were recorded by clamping the voltage to different positive and negative voltages resulting in a voltage-dependent current with strong rectification properties as reported previously (Tyerman et al., 1995 In approximately 90% of successful inside-out patches (n = 57), only a time-dependent activation of the inward current without any visible single-channel events was observed, although the noise on the currents was variable (Fig. 1A, trace 1). In about 10% of recordings the current had single-channel-like events superimposed (Fig. 1A, trace 2). These events did not have the characteristics of another type of channel superimposed on a rising baseline current; rather, the magnitude of the current deviations increased in proportion to the magnitude of the baseline current. For six different patches displaying this behavior and using data from repetitive pulses to the same voltage, we were able to calculate the maximum fluctuation as a function of time from the start of the current activation. Plotting this as a function of the average current revealed a linear trend. The mean slope was 0.48 (SEM = 0.13), with a mean intercept of –6.5 pA (SEM = 5.27). In other words, the time dependence of the increase in magnitude of the channel fluctuations paralleled the time dependence of the baseline current.
The occurrence of these deviations in the time-dependent current were unpredictable, and so far did not depend on any pretreatments of the symbiosomes nor on any special experimental conditions. In some instances the two types of behavior (smooth and channel like) were recorded on the same symbiosome patch under the same conditions (Fig. 1, A–C). In some current traces, channel-like closing events could be noticed followed by a slow "opening" showing almost the same time-dependent activation as the macroscopic current (Fig. 1B). It seems to be very likely that channel events and the macroscopic current are closely linked as reported by Figure 1C.
A fully activated, time-dependent current at –180 mV was subject to a depolarizing voltage ramp (–180–100 mV) reflecting the voltage dependence of the activated transporters. Two limiting current amplitudes were observed, and channel-like closing and opening events between these two currents were recorded. No difference in voltage dependence of the current voltage (IV) curve or reversal potential (Erev) was observed between the current amplitudes in Figure 1D. There appeared to be a continuum between smooth to more noisy, to more channel-like activity. The extremes of these behaviors are illustrated in Figure 1. For the channel-like extreme (Fig. 1D), it was not possible to obtain a consistent single-channel conductance when channel events were clear (Fig. 2). By subtracting the baseline time-dependent current, we then attempted to analyze the transitions using a technique that examines the transition rates between current levels (transition amplitude analysis [TRAMP]; Tyerman et al., 1992
Magnesium Ions Block the Current on the Symbiosome Lumen Face A Mg2+ concentration of 1 mM in the bath (corresponding to the symbiosome lumen face of the PBM) blocked about 75% of the time-dependent current (Fig. 3A) when 60 mM NH4+ was in the bath solution. The effective concentration at which 50% of the NH4+ current was inhibited (ID50) was 351 µM (Fig. 3B). The dose response could be well fitted by a dose-response curve where the Hill slope was equal to 1. There was no significant difference between the fitted dose-response curves and hence for the ID50, within the range of –180 to –100 mV for which current activation was clear. Thus, it appears that the voltage dependency of Mg2+ block is absent or very weak and not detectable.
The NH4+ current could be completely inhibited by 2 mM Mg2+ when the bath contained 60 mM NH4+; in fact, a small decaying inward current became apparent (Fig. 4B). When the NH4+ concentration was only slightly increased to 70 mM, the time-dependent activation of the NH4+ currents reappeared (Fig. 4C) and were similar to those observed with 1 mM Mg2+ at 60 mM NH4+(Fig. 4A). The reduction in the Mg2+ inhibition by increasing the NH4+ concentration may indicate a competition between these two ions for the same binding site, or interactions between two separate binding sites. To investigate a possible interaction between NH4+ and Mg2+, inward currents were recorded at various NH4+ concentrations in the absence or the presence of 2 mM MgCl2, and the relative conductance was calculated from the resulting IV curves. The results of Figure 5 showed a saturation of the NH4+ current with bath NH4+ concentration. With no Mg2+ present, a Km of 26.4 mM was determined by nonlinear regression. With 2 mM Mg2+, no inward currents are observed until 70 mM NH4+ and further increases in NH4+ concentration indicated that saturation occurred at higher NH4+ concentrations (Fig. 5). With Mg2+ present, none of the commonly known interaction types (competitive, uncompetitive, or noncompetitive interactions) were recognized (data not shown).
Figure 6 shows the effect of Mg2+ on the Erev of the NH4+ current that were obtained by tail current analysis. Without Mg2+ present in the bath, the Erev shifted to more positive potentials when the NH4+ concentration of the bath was increased (Fig. 6A), indicating a flux of cations (NH4+) from the symbiosome lumen (bath) to the cytoplasm (pipette). The shift in Erev followed closely the predicted Nernst equilibrium potential for monovalent ions with the assumption that the permeabilities of K+ and NH4+ were equal (Tyerman et al., 1995 ), giving a slope of the regression line that was not significantly different to 58 mV per decade NH4+. This contrasts to data obtained under similar solution regimes for L. japonicus PBM where the observed membrane potentials were offset in the negative direction (below the Nernst equilibrium line; Roberts and Tyerman, 2002). Upon addition of Mg2+ (2 mM) to the bath solution (lumen face of the PBM), the Erev of the time-dependent current became more negative, suggesting an additional permeability of a cation whose electrochemical gradient is directed into the symbiosome lumen (bath). Note that an increase in anion permeability under the conditions of the experiment would shift the Erev more positive because the Cl– concentration in the pipette is higher than in the bath. Previous experiments had also established that the PBM-NSCC showed no appreciable anion selectivity (Tyerman et al., 1995; Whitehead et al., 1998; Roberts and Tyerman, 2002). The high deviation from the predicted Nernstian Erev is reflected by the regression line with a slope of 95 mV (decade NH4+)–1. When maintaining the NH4+ concentration at 60 mM and increasing the Mg2+ concentration, the Erev shifted to more negative voltages (Fig. 6B).
This shift in Erev of about 10 to 15 mV relative to the predicted Nernst potential (see Fig. 6A) for univalent cations is not enough to substantially effect the conductances that we measured for the Mg2+ data in Figure 5. Also, inspection of Figure 4B shows that there is absolutely no time-dependent activation of inward current (tails also have reversed) at any membrane potential (up to –180 mV) at the threshold point of 60 mM NH4+ with 2 mM Mg2+ present. Also note that at 150 mM NH4+ there was no difference between the Erev with and without 2 mM Mg2+ (Fig. 6A), yet there was a reduction in conductance of about 50% (Fig. 5). Thus, the shift in Erev with Mg2+ present in the bath cannot explain the apparent threshold effect illustrated by the data in Figure 5.
We examined the noise associated with the macroscopic time-dependent currents to attempt to clarify the variable behavior of currents (e.g. Fig. 1), and to examine whether or not Mg2+ caused additional noise due to open-channel blockade. Some patches were analyzed where differences in noise of the time-dependent current were observed over the duration of the experiment, and where the same voltage pulse protocol was used (40 current traces record; Fig. 7A). The spectral densities of smooth and noisy current traces of the same recording batch were analyzed separately to detect any difference in the Mg2+ block on these current types. In total, each of at least 6x40 current traces on three different PBM patches that were recorded under various conditions of NH4+ and Mg2+ concentrations (100 mM NH4+, 2 mM Mg2+; 150 mM NH4+, 2 mM Mg2+; 150 NH4+, 1 mM Mg2+; as shown in Fig. 5) were analyzed. A typical example is shown in Figure 7, B and C. No major Mg2+ effect on the frequency dependence of the spectral densities was noticed when current traces with small fluctuations were analyzed (Fig. 7B), indicating that the Mg2+-induced inhibition of the current does not cause any obvious channel flickering. Nevertheless, a decrease in the averaged spectral density especially at lower frequencies can be observed in these (Fig. 7C) as well as in current traces with initially larger fluctuations present (Fig. 7C, insert). Note that all data were obtained from the same patch and the same recording (40 current traces). In the other two experiments, similar results were observed: Mg2+ did not affect the frequency dependence, but only reduced the amplitude of the spectra in both smooth and more noisy time-dependent currents (data not shown).
Block by Mg2+ of the time-dependent NH4+ currents occurred irrespective of the presence or absence of the channel-like activity within the time-dependent current. No effect could be determined on the magnitude of the channel-like steps occasionally observed. Nonstationary noise analysis was performed on the time-dependent currents to determine the effect on the single-channel amplitude. No significant difference was observed ± Mg2+ at 100 mM and 150 mM NH4+, but this may be due to the statistical power of the analysis because very high standard errors of the single channel's estimates were obtained.
The temperature sensitivity of the NH4+ currents may differentiate between different molecular mechanisms of transport across the PBM, since channel- and carrier-mediated transport show different temperature dependence (Table I).
To ensure that a good estimate of the temperature dependence of open-channel conductance was made, several precautions were taken in the experiments. We have observed some variability in currents over the duration of experiments on a single patch, apart from the noise described above. Often, the conductance increased as though more transporters were recruited into becoming active. To check that this did not occur during temperature experiments, we compared conductance before and after the temperature variations. On one occasion the conductance increased, and this correlated with a high Q10 and activation energy (EA) being measured. It was also known from other studies that the activation rates of channels can be extremely temperature sensitive (Acerbo and Nobile, 1994
Another complication arises because we cannot observe single-channel events and therefore cannot get a direct measure of open-channel current. Therefore, time-dependent currents must be activated at voltages where the channel open probability (Popen) is maximal and preferably near 1. To reveal the voltage dependency of current activation, the corresponding IV curves for initial current (Ii) and final current (If) were plotted (Fig. 8C). A temperature dependence of the current at all voltage steps was observed where initial current and final steady-state current, as well as the time-dependent component (If – Ii), showed no obvious change of their voltage dependence at 8.9°C or 22.7°C.
These current recordings were used to examine the temperature dependence as a function of voltage in more detail. The activation of the current can be described as a Boltzmann function with a V50 of approximately –60 mV and an effective gating charge of near 1 (Whitehead et al., 1998
The EA at different voltages was obtained from Arrhenius plots of the time-dependent current amplitudes measured at the same voltage. At voltages near V50, the EA was much higher (41 kJ mol–1 at –80 mV in Fig. 9B) than at –180 mV (18 kJ mol–1 in Fig. 9B). In total, an average EA of 28.2 kJ mol–1 ± 2.87 (SEM, n = 7) was measured for the currents when they were maximally activated. The range in EA for currents that were fully activated was from 18.3 to 39.8 kJ mol–1. The average Q10 was 1.51 ± 0.06 (SEM, n = 7). At a membrane voltage of –100 mV, the average EA was 48.7 kJ mol–1 ± 8.6 (SEM, n = 4).
We also analyzed the effect of temperature on activation and deactivation kinetics of the time-dependent currents. As reported by Whitehead et al. (1998)
Previous studies have assigned the NH4+ current to a channel mechanism (PBM-NSCC) despite the absence of channel current transitions in patch-clamp recordings so far reported and the equivocal stationary noise characteristics as displayed in this study (Fig. 7). The assignment is based on the passive nature of the currents that are uncoupled from other ion movements and the ability to model the variance during current activation to simple gating (Tyerman et al., 1995 ). Also, blockade by divalent cations, polyamines (Whitehead et al., 2001), and verapamil (Whitehead et al., 1998) is consistent with channel-mediated transport. In this study, we have demonstrated that single-channel-like events are associated with the NH4+ current. These events appear to lie at one extreme of a variable noise level on the time-dependent currents. The events we observe are closely associated with the time-dependent current, both in terms of their activation and their selectivity, and we propose they represent a form of cooperative gating between variable numbers of the subpicoSiemens channel conductance. We have applied nonstationary noise analysis and have confirmed that the single-channel amplitudes lie in the range of 5 to 50 fA depending on voltage, giving a conductance of between 0.11 and 0.22 pS as reported by Tyerman et al. (1995). From the size of the current steps observed in some patches (e.g. Fig. 1C), this would suggest that up to 1,000 single channels may activate and deactivate simultaneously under some circumstances. Similar behavior has been observed for anion channels in Chara plasma membrane, although with larger unit conductance (McCulloch et al., 1997). In Chara, the anion current could be modeled as unit channels forming a larger cluster that allowed cooperative gating. The specific conditions that cause this behavior are so far unknown.
The temperature dependence of the currents is more consistent with a channel mechanism. An EA of 28 kJ mol–1 when the current is fully activated lies within the range of values obtained for other ion channels; although slightly on the higher side of the range, it is certainly less than the subpicoSiemens voltage-dependent H+ channels (DeCoursey and Cherny, 1998
The limiting NH4+ conductance in water gives an EA of 12.3 kJ mol–1 (data from appendix 6.2 in Robinson and Stokes, 1968 The higher EAs recorded at intermediate voltages where the currents are about half activated also indicates that the transitions that determine Popen have some steps with a high EA. The kinetics of current activation were highly temperature dependent (Q10 = 2.7) in contrast to channel deactivation (Q10 = 1), implying that the rate constant(s) for channel opening is more temperature dependent than channel closing. If this is the case, then the Popen would decline with decrease in temperature, and this should be indicated in our conductance data at voltages where Popen is near 0.5. We observed a negative shift in the V50 of the Boltzman curves fitted to relative conductance as a function of voltage of 2.3 mV per degree reduction in temperature without any change in the shape of the curves. Assuming that the Q10s of the channel opening and closing rate constants are the same as the current activation and deactivation rate constants, we can calculate that at a voltage where Popen was initially 0.5, Popen would decline to 0.3 for a 10°C reduction in temperature. This corresponds very closely to the change in relative conductance at V = –60 mV (0.5–0.3) that we observe from the Boltzman fits for a 10°C reduction in temperature. Note that at a voltage where the current is near maximally activated (–180 mV), the reduction in relative conductance is only from 0.98 to 0.96 (from Boltzman fits). This is explained by the rate constant for channel opening being much larger at this voltage than the rate constant for channel closing (greater than 10-fold), so a reduction in the rate constant of channel opening caused by lower temperature has less of an effect on Popen at more negative voltages.
There are only a limited number of studies on the temperature dependence of plant ion channels (see Table I), with one whole-cell study giving rather high values for well-established classes of plant ion channels (Colombo and Cerana, 1993
In terms of the biology of nitrogen fixation, for the energized PBM where hyperpolarization of the symbiosome membrane is proposed to occur via H+-ATPase activity (Udvardi and Day, 1989
Activation of PBM-NSCC is highly temperature dependent (EA = 64 kJ mol–1), indicating that some conformational changes must occur in the channel protein during opening. This has implications for the proposed model of a simple divalent blocking and unblocking mechanism of PBM-NSCC gating proposed by Whitehead et al. (1998)
It is interesting to note that this mechanism of channel gating is similar to that proposed for the SV channel in Beta vulgaris, where there is a site within the channel that allows low affinity blockade by divalent cations (Ca2+ and Mg2+) that also permeate the channel, and a Ca2+-selective site outside of the voltage field that stabilizes the closed state (Pottosin et al., 2004
Despite the external site for divalent blockade of PBM-NSCC, there were unusual interactions between the permeating ion and Mg2+. The interaction cannot be described by a simple competitive inhibition or other known forms of interaction with the permeating ion. The most interesting observation with respect to NH4+ permeation is that at a concentration of Mg2+ that totally blocks the channel for a specific NH4+ concentration, it only requires a relatively small increment in NH4+ concentration to relieve this complete blockade. Thus, if the Mg2+ concentration was set relatively constant within the symbiosome lumen, there would exist a threshold concentration of NH4+ below which there would be no transport to the cytoplasm. As N2 fixation by the bacteroids built up the lumen concentration of NH4+, the threshold concentration would be overcome and transport to the cytoplasm would proceed. Complicating this is the substantial increase in NH4+:K+ selectivity of the PBM-NSCC when lumen NH4+ declines (Whitehead et al., 1998
Another interesting observation regarding blockade by Mg2+ was the change in selectivity of the channel. With increasing Mg2+ concentration, as the channel currents became reduced, they also became more selective for an ion with an electrochemical gradient from the pipette solution to the bath. Under the conditions of our experiment, this is likely to be an increased Ca2+ permeability. We believe we can discount the alternative, an increase in K+ permeability, because all studies to date have shown the reverse is normally the case, i.e. at lower NH4+ concentrations, the relative permeability of K+: NH4+ decreases (Tyerman et al., 1995 The permeation of divalents through the PBM-NSCC may determine the concentrations of divalents in the symbiosome lumen, which in turn will determine the set point for NH4+ transport. This may work like a negative feedback mechanism whereby progressive blockade by higher symbiosome lumen concentrations of Ca2+ or Mg2+ causes the channel to conduct more of these divalents out of the symbiosome lumen to the cell cytoplasm, thus relieving the block. The small volume of the symbiosome lumen would mean that this could be quite a sensitive control system.
The degree of control evident over PBM-NSCC suggests that other NH4+ permeable NSCCs in different membranes could have similar sophisticated control over activity, and this may be very important to prevent futile cycling of NH4+ (Britto et al., 2001a
The cycling of NH3 back into the symbiosome lumen from the cytoplasm depends on the cytoplasmic concentration of NH4+ and the NH3 permeability of the PBM. The PBM has a relatively low NH3 permeability despite being channel mediated and it is reduced by ATP preincubation (Niemietz and Tyerman, 2000
The molecular identity of the PBM-NSCC has been assigned to GmSAT1, which is a novel protein situated on the PBM that has an N-terminal helix-loop-helix motif similar to transcription factors and a hydrophobic transmembrane domain (Kaiser et al., 1998
The NH4+ current in the PBM of soybean nodules has been positively associated with a channel-mediated transport mechanism from its temperature dependence, despite its unusual characteristics of cooperative gating, stationary noise characteristics, and very low single-channel conductance. Magnesium ions change the channel conformation, and to open the channel a high energy step is required. With partial Mg2+ block the transport of divalent cations becomes possible, and this may be involved in setting the threshold for NH4+ transport through the channel by virtue of the novel interaction between divalent concentration and NH4+ concentration in the symbiosome lumen.
Plant Material
Nodules were collected just before the experiments from 5- to 7-week-old soybean plants (Glycine max L. cv Stephens or Forest) that were grown in a naturally illuminated greenhouse with additional illumination to maintain 16-h-light/8-h-dark cycles. Plants were inoculated with Bradyrhizobium japonicum U.S. Department of Agriculture 110 and grown in pots containing a mixture of perlite and vermiculite. To isolate the symbiosomes, three to five nodules were cut with a razor blade or gently crushed in standard bath medium with 5 mM dithiothreitol added, at 4°C in a petri dish. After 5 to 10 min on ice, a small volume was transferred into the perfusion chamber filled with standard bath medium. Symbiosomes were allowed to settle at the bottom of a glass coverslip for another 5 min. The chamber was then perfused with approximately 5 to 10 mL of fresh standard bath medium, and the symbiosomes that remained adherent to the glass coverslip formed high G
In all experiments, a standard pipette medium was used containing (in mM) 80 mannitol, 150 KCl, 10 CaCl2, 5 HEPES adjusted to pH 7.0 with KOH. The standard bath solution contained (in mM) 170 mannitol, 100 K+Glu, 2 MgCl2, 10 EGTA, 2.3 CaCl2 (giving a free Ca2+ concentration of 84.8 nM), 5 HEPES adjusted to pH 7.0 with Tris. Other bath media with various NH4+ concentrations were made on the basis of a 10 mM HEPES/Tris buffer, pH 7.0, with 1 mM EGTA. Osmolalities of all solution were adjusted with mannitol to approximately 400 mOsmol. The free Ca2+ concentration (approximately 100 nM) was calculated in respect to the Mg2+ concentration, pH, and ionic strength of the solutions using the GEOCHEM program (Parker et al., 1987
Patch pipettes were pulled from borosilicate glass capillaries (GC150-10, Clark Electronic Instruments) to an o.d. of approximately 0.5 to 1 µm, and tips were coated with Sylgard (Dow Corning). In general, the pipettes were fire polished and back filled with standard pipette medium. Best sealing rates were obtained with pipettes and showed a resistance of 15 M The currents were measured using an Axopatch 200A (Axon Instruments) or a List EPC-7 amplifier (List Electronics). The currents were filtered at 1 or 2 kHz and digitized at frequencies of 2 and 5 kHz, respectively, using a Strobes (Strobes Engineering) or DigiData 1200B (Axon Instruments) analog to digital converter running under pClamp8 data acquisition and voltage pulse protocol software. Current data were analyzed by use of Clampfit (versions 8 and 9), MathCad, SigmaPlot, or Graphpad Prism softwares. A pulse protocol was applied to excised membrane patches starting from a holding potential of 0 mV (0.1 s) and stepping the voltage for 2 s to values between 0 and –180 mV at 20 mV intervals, and finally clamping the voltage at 50 mV for 0.4 s. The time-depended currents were obtained by subtracting the instantaneous current component at the beginning of the negative voltage step from the total current (mean current of last 200 ms). To obtain the Erev of the respective currents, the patched membrane was hyperpolarized for 2 s (–160 mV) to activate all channels and then stepped to more positive values ranging between –140 and 100 mV in 20 mV intervals. The resulting tail currents were analyzed according to standard electrophysiological protocols, namely, the potential at which a "zero" tail current was detected was taken as the Erev.
Stationary noise analysis examines the fluctuations in current as a function of time while the average current is constant in time. This occurs at a fixed voltage and requires that all time-dependent changes in current have ceased. Nonstationary noise analysis examines the kinetics of current fluctuations during current activation or deactivation, generally after a change in voltage. For the stationary noise analysis carried out in this paper 40 current traces were recorded after at voltages of –120, –140, –160, and –180 mV. The current was filtered at a cut-off frequency of 2 kHz (Bessel-type filter) and digitized at 5 kHz. Noise spectra were generated from every one of these current traces (1 s, 4,250 data points) after manual removal of the direct current component and using a Poisson periodogram (factor
To observe the temperature dependence of the currents, a thermistor-based (RS Components, nos. 151–136) thermometer was inserted into the perfusion chamber as close as possible to the patch pipette. After recording the currents at room temperature, prechilled bath medium was perfused through the chamber until a stable temperature was recorded. The bath medium was then allowed to warm up to room temperature again while currents at –180 mV steps as well as the corresponding temperature were recorded. Temperature changes in the chamber were slow and did not change more than 1°C during the recording of one set of currents. The EA was calculated from the slope of a regression line of an Arrhenius diagram (ln [-I] versus T–1), and Q10 values, the relative changes in a parameter for a 10°C change in temperature, were obtained from a linear regression line of I-T diagrams with T ranging from 8°C to 18°C, or Q10 was calculated for other temperature ranges by:
We are very grateful for the excellent technical assistance provided by Wendy Sullivan. Received June 9, 2005; returned for revision July 28, 2005; accepted July 30, 2005.
1 This work was supported by an Australian Research Council Discovery Grant and by an Australian Research Council International Research Exchange grant.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.066670. * Corresponding author; e-mail steve.tyerman{at}adelaide.edu.au; fax 618–83037116.
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ABSTRACT
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mep2
seals when they were gently touched by a patch pipette. 
= 6, pClamp9 software). 
i
MR, Andjus PR (2000) Temperature sensitivity of the tonoplast Ca2+-activated K+ channel in Chara: the influence of reversing the sign of membrane potential. J Membr Biol 178: 215–224