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Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK

	Abstract

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The NMDA receptor is modulated by changes in the intracellular calcium concentration, through activation of various intracellular calcium-dependent proteins. We have investigated regulation of single NMDA receptor channel activity by the calcium-sensing proteins alpha-actinin and calmodulin. Both of these proteins bind to the NMDA receptor NR1 subunit C-terminus at the C0 region where they compete for occupation of the C0 site and contribute to calcium-dependent inactivation of NMDA receptor-mediated whole-cell currents. Calmodulin has also been shown to bind to the neighbouring C1 region where it has been shown to reduce single channel open time. To investigate regulation of single NMDA channel activity by alpha-actinin and calmodulin, we selected concentrations of these two proteins that would result in maximal binding to the C0 region and/or the C1 region in the case of calmodulin. Alpha-actinin binding was found to predominantly decrease single channel shut time, resulting in an increased open probability (P_open), whereas calmodulin binding reduced single channel mean open time, resulting in an overall reduction in P_open. The physiological implications of these findings are discussed.

(Received 8 December 2003; accepted after revision 5 April 2004; first published online 8 April 2004)
Corresponding author B. K. Rycroft: Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. Email: b.rycroft{at}ucl.ac.uk

	Introduction

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NMDA receptor activity is influenced by changes in intracellular calcium concentration through activation of various intracellular calcium-dependent proteins including: alpha-actinin (Wyszynski et al. 1997; Zhang et al. 1998; Krupp et al. 1999), calmodulin (Ehlers et al. 1996; Hisatsune et al. 1997; Rafiki et al. 1997; Zhang et al. 1998; Krupp et al. 1999), calcineurin (Liebermann & Mody, 1994; Tong & Jahr, 1994; Tong et al. 1995) and protein kinase C (PKC) (Chen & Huang, 1992; Wagner & Leonard, 1996; Xiong et al. 1998; Lu et al. 1999; Lan et al. 2001). This provides a feedback mechanism, linking a rise in intracellular calcium concentration, in part due to flux through the NMDA receptor channel (Legendre et al. 1993; Krupp et al. 1996), to a change in receptor activity. These regulatory second messenger pathways may serve to fine-tune NMDA receptor-dependent synaptic plasticity (Bliss & Collingridge, 1993, 1995; Bear & Malenka, 1994), enabling synapses to adapt to past activity (Lisman, 1989, 2001; Kim & Huganir, 1999; Kennedy, 2000). The complexity of these cellular pathways is reflected in their integrative nature, illustrated by the competitive relationship for binding to the NMDA receptor between alpha-actinin and calmodulin (Wyszynski et al. 1997), PKC and calmodulin (Hisatsune et al. 1997) and calcium–calmodulin kinase II (CaMKII) and alpha-actinin (Leonard et al. 2002).

Alpha-actinin binds to the NMDA receptor NR1 subunit C-terminus at the C0 region cross-linking receptors to the actin cytoskeleton (Wyszynski et al. 1997). Calmodulin also binds to the NMDA receptor at the C0 site (Ehlers et al. 1996; Zhang et al. 1998) where it competes with alpha-actinin. Displacement of alpha-actinin from, and calmodulin occupation of, the C0 region is implicated in calcium-dependent inactivation of NMDA receptor-mediated whole-cell currents (Zhang et al. 1998; Krupp et al. 1999). In addition, calmodulin binds to the neighbouring C1 region (Ehlers et al. 1996) where it inhibits single NMDA channel activity (Ehlers et al. 1996; Rycroft & Gibb, 2002).

Here we have investigated regulation of single NMDA channel activity by alpha-actinin and calmodulin. Active calmodulin at 800 nM (expected to saturate both the C1 and C0 site), alpha-actinin at 2.5 µM (expected to saturate the C0 region) and a mixture of both proteins that should result in saturation of the C1 region by calmodulin and of the C0 region by alpha-actinin were applied to the intracellular membrane of outside-out patches from hippocampal granule cells. Control experiments were made with 800 nM calcium in the pipette solution.

At all membrane potentials tested, alpha-actinin or a combination of alpha-actinin and calmodulin reduced mean open time by 45 and 51%, respectively, whereas calmodulin alone reduced mean open time by 29%. This suggests that both alpha-actinin and calmodulin reduce single channel open time, albeit to a lesser extent in the case of calmodulin. However, mean shut time was also reduced by 83% in the presence of alpha-actinin alone, resulting in an overall 77% increase in P_open. Mean shut time was unchanged in the presence of calmodulin alone or alpha-actinin plus calmodulin, resulting in overall 29 and 49% decreases in P_open, respectively. These results suggest that dissociation of alpha-actinin and association of calmodulin with the NMDA receptor reduces single channel activity and that this may underlie calcium-dependent inactivation of whole-cell and synaptic currents.

	Methods

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Twelve-day-old Sprague-Dawley rats were killed humanely by decapitation and 300 µm hippocampal slices made in an ice-cold (< 4°C) oxygenated slicing solution (composition (mM): sucrose, 250; KCl, 2.5; CaCl₂, 1.0; MgCl₂, 4.0; NaH₂PO₄, 1.25; NaHCO₃, 26; glucose, 25; pH 7.4) using a vibroslicer (Vibroslice 752, Campden Instruments, UK). Slices were maintained for 1–8 h at room temperature in an incubation chamber in Krebs solution containing (mM): NaCl, 125; KCl, 2.5; CaCl₂, 1.0; MgCl₂, 4.0; NaH₂PO₄, 1.25; NaHCO₃, 24; glucose, 25 (pH 7.4). Slices were viewed on the stage of an upright microscope (Zeiss Axioscope FS) using Normaski differential interference contrast optics (Edwards et al. 1989) and dentate gyrus granule cells were identified by their location, size and morphology (Koh et al. 1995). All experiments performed in this study conformed with UK Home Office regulation guidelines.

For single-channel recording, slices were bathed in Mg²⁺-free Krebs solution containing (mM): NaCl, 125; KCl, 2.5; CaCl₂, 1.0; NaH₂PO₄, 1.25; NaHCO₃, 24; glucose, 25 (pH 7.4) continuously gassed with a mixture of O₂ (95%) and CO₂ (5%). Control outside-out patch recordings were made with patch pipettes filled with a low-chloride (10 mM) pipette solution containing (mM): NaCl, 10; EGTA, 10; Hepes, 10; sodium gluconate acid, 140; adjusted to pH 7.3 with NaOH (Gibb & Colquhoun, 1991) with a buffered free calcium concentration (calibrated with a calcium electrode) of 800 nM. Total calcium, 9.87 mM, was calculated using the program ‘ALEX’ by Michael Vivaudou which is based on that described by Fabiato (1988).

Alpha-actinin purified from rabbit skeletal muscle, containing isoforms 2 and 3, is calcium insensitive and has an affinity of 48 nM for the C0 region of the NMDA receptor NR1 subunit C-terminus (Krupp et al. 1999). The concentration of alpha-actinin present in these experiments (2.5 µM) would therefore result in 98% occupation of the C0 region. Calmodulin has four calcium binding sites where occupancy of two or more binding sites is needed to give active calmodulin (James et al. 1995). The concentration of active calmodulin (calmodulin with 2 or more binding sites occupied) was calculated using the equilibrium constants for calcium binding given by Haiech et al. (1981) of K₁= 67 nM, K₂= 170 nM, K₃= 600 nM, K₄= 900 nM. A concentration of 800 nM active calmodulin was thus obtained at a total pipette concentration of 80 µM calmodulin and a free calcium concentration of 800 nM. The affinity of calmodulin for fusion peptides of the NR1 subunit C-terminal C1 and C0 regions was estimated by Ehlers et al. (1996) to be 4 nM and 87 nM, respectively, suggesting 800 nM active calmodulin would result in 99% occupancy of the NR1 subunit high affinity C1 region and 90% occupancy of the low affinity C0 region. When both 2.5 µM alpha-actinin and 800 nM active calmodulin are present, occupation of the C1 region by calmodulin would be 99% whereas (assuming simple competitive antagonism) occupation of the C0 region by alpha-actinin would be 84%.

Outside-out patch-clamp single channel recordings were made with patch pipettes pulled from thick-walled aluminosilicate glass capillaries containing an internal filament (SM150F-7.5, outer diameter 1.5 mm, inner diameter 0.80 mm, Clark Electromedical, Reading, UK) coated with Sylgard 184 resin (Dow Corning, USA) and fire polished on a microforge (Narishige MF-83) to a final resistance of 20–30 M. Single channel currents were recorded using an Axopatch 200A patch-clamp amplifier (Axon Instruments) and stored on digital audiotape (BioLogic DTR 1202). Before recording was attempted, the patch noise level was checked and an RMS noise level below 300 fA at a bandwidth of 5 kHz was considered acceptable. Patches showing spontaneous channel activity in the absence of agonists were discarded. Each outside-out patch was exposed to a constant high concentration of 10 mM glycine, and 0.1–10 µM NMDA (Tocris) for approximately 10 min, at different membrane potentials changing in 10 mV steps between –30 and –80 mV at room temperature (20–24°C).

Data acquisition and analysis

Single channel currents were replayed from tape, amplified and filtered at 2 kHz (8 pole Bessel) and digitized at 20 kHz using an analog-to-digital converter (CED 1401plus, Cambridge Electronic Design, UK). Each digitized record was analysed using ‘SCAN’, an interactive computer program (that can be requested at http://www.ucl.ac.uk/Pharmacology/dc.html) that fits the time course of each event based on the step response of the recording system (Colquhoun & Sigworth, 1995). Display and analysis of single channel data distributions was done using ‘EKDIST’ (Colquhoun & Sigworth, 1995). Before analysis, a fixed resolution for open times and closed times that gave a false event rate less than or equal to 10^–12 events per second was imposed (Colquhoun & Sigworth, 1995). This was 110 ms for open and closed times at –60 mV for the patches analysed in this study. Before a patch was accepted for detailed analysis, the long-term stability of the data records was checked by making stability plots of channel amplitudes, open times, shut times and P_open (Weiss & Magleby, 1989). Once the stability of the record had been confirmed, amplitude distributions were made containing individual channel amplitudes of openings longer than 2.0 filter rise-times (332 µs). Distributions of channel amplitudes were best fitted with the sum of two or three Gaussian components, as appropriate, with the standard deviation constrained to be the same for each component. The relative area occupied by each Gaussian component therefore represents the relative frequency of openings to each particular amplitude level. Distributions of closed times and open times were displayed using a logarithmic transformation of the x-axis (McManus et al. 1987; Sigworth & Sine, 1987) and a square root transformation of the y-axis (Sigworth & Sine, 1987). Distributions were fitted using the maximum likelihood method with probability density functions that were a mixture of three exponential components for open times and five exponential components for closed times (Colquhoun & Sigworth, 1995). P_open was calculated from mean open time and mean shut time (mean open time/(mean open time + mean shut time)) for each patch.

Data are expressed as mean ±S.E.M. For statistical comparisons, either a randomization test was used that does not involve making any assumptions about the shape of the distribution of the observations (that can be requested at http://www.ucl.ac.uk/Pharmacology/dcpr95.html) or for correlated data sets, analysis of covariance was used (Zar, 1999). Statistical significance was set at P < 0.05 unless otherwise indicated.

	Results

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Single NMDA channel recordings

Application of 0.1–10 mM NMDA and 10 mM glycine to outside-out patches from hippocampal dentate gyrus granule cells resulted in single channel activity characteristic of NMDA receptor activation. For the majority of control, calmodulin- and alpha-actinin + calmodulin-treated patches, the NMDA concentration was adjusted in the range 0.1–10 µM; NMDA was applied to give a level of channel activity sufficient for detailed analysis. However, alpha-actinin-treated patches were all recorded in the presence of 100 nM NMDA. Figure 1 shows examples of channel traces from control, alpha-actinin-, calmodulin- and alpha-actinin + calmodulin-treated patches. Qualitatively, channel behaviour is similar in each recording solution with channel openings occurring in similar bursts with at least two conductance levels observed in all groups at holding potentials between –30 and –80 mV. Although it is noticeable in Fig. 1B that the baseline noise level is higher than in panels A, C or D, this was not consistently observed with alpha-actinin-treated patches and illustrates the variation in baseline noise observed between recordings.

View larger version (17K): [in this window] [in a new window]   Figure 1.  Examples of NMDA receptor-channel openings from control, 2.5 µM alpha-actinin-, 800 nM calmodulin- and 2.5 µM alpha-actinin + 800 nM calmodulin-treated patches Downward deflections in baseline indicate single channel openings in the presence of 0.1–10 µM NMDA and 10 µM glycine, recorded from outside-out patches taken from dentate gyrus hippocampal granule cells. Patches were recorded at holding potentials between –30 and –80 mV. Currents were low-pass filtered at 2 kHz (–3 dB, 8 pole Bessel filter). Each trace is 162 ms long.

Distributions of single channel amplitude were best fitted with the sum of three (or sometimes two) Gaussian components for all groups (Fig. 2A–D). A large main amplitude and high probability component, a second smaller amplitude or sublevel component of lower probability, and a third sublevel of smallest amplitude and lowest probability were observed. Mean amplitude and mean relative area at –60 mV of the first, second and third Gaussian components are presented in Table 1. Analysis of variance indicated there were no significant differences between the channel amplitudes in each recording condition at –60 mV. In addition, the 2nd: 1st Gaussian component area ratio did not differ significantly in the presence of alpha-actinin (0.19 ± 0.08, n= 5), calmodulin (0.19 ± 0.04, n= 7) or alpha-actinin + calmodulin (0.15 ± 0.02, n= 6) when compared to control (0.18 ± 0.03, n= 6) at –60 mV, indicating that the relative frequency of main and sublevel openings was not affected by calmodulin or alpha-actinin.

View larger version (14K): [in this window] [in a new window]   Figure 2.  Amplitude histograms from control (A), 2.5 µM alpha-actinin-treated (B), 800 nM calmodulin-treated (C), and 2.5 µM alpha-actinin + 800 nM calmodulin-treated (D) patches Distributions of channel amplitudes were fitted with the sum of three Gaussian components for amplitudes of openings longer than two filter rise-times (332 µs). Mean amplitude, standard deviation and relative area for each Gaussian component are inset. Membrane potential –60 mV.

View larger version (18K): [in this window] [in a new window]   Figure 3.  Relationship between holding potential and channel amplitude for control, 2.5 µM alpha-actinin-, 800 nM calmodulin- and 2.5 µM alpha-actinin + 800 nM calmodulin-treated patches Plots show current–voltage relationships for single channel currents activated by 0.1–10 µM NMDA and 10 µM glycine. The following values for main and subconductance states were estimated from the slope of the line fitted by linear regression for all patches, 60 and 47 pS for control (n= 6) (•), 54 and 37 pS for 2.5 µM alpha-actinin (n= 5) ( ), 52 and 42 pS for 800 nM calmodulin (n= 7) () and 56 and 48 pS for 2.5 µM alpha-actinin + 800 nM calmodulin (n= 7) ( ). Alpha-actinin alone significantly reduced the subconductance but not main conductance level in comparison to control (analysis of covariance F < 0.05).

Shut time distributions were best fitted with the sum of five exponential components (Fig. 4A–D); mean data collected at –60 mV are presented in Table 2. Neither calmodulin, nor the combination of alpha-actinin plus calmodulin significantly affected channel shut times in comparison to control. However mean shut time was significantly reduced (analysis of covariance) in the presence of alpha-actinin alone and this was consistent at all membrane potentials recorded (Fig. 5A).

View larger version (20K): [in this window] [in a new window]   Figure 4.  Distribution of shut times for control (A), 2.5 µM alpha-actinin-treated (B), 800 nM calmodulin-treated (C), and 2.5 µM alpha actinin + 800 nM calmodulin-treated (D) patches In these distributions shut time intervals ranging from 0.11 to 3541 ms were best fitted with the sum of five exponential components, time constants and associated areas are inset. Predicted mean shut times (and observed and predicted number of observations) were 103 ms (1022 and 1213) in control; 97 ms (1825 and 1978) in 2.5 µM alpha-actinin-treated patches; 129 ms (880 and 963) in 800 nM calmodulin-treated patches and 157 ms (733 and 812) in 2.5 µM alpha-actinin + 800 nM calmodulin-treated patches.

View larger version (21K): [in this window] [in a new window]   Figure 5.  Comparison of overall mean shut time (A) and mean shut time (B) for the first four shut time components for control, 2.5 µM alpha-actinin-, 800 nM calmodulin- and 2.5 µM alpha-actinin + 800 nM calmodulin-treated patches Each point represents the mean shut time of all patches (A) or weighted mean shut time of the first four shut time components (B), at each membrane potential, calculated from the exponential components fitted to the shut time distributions. The data were fitted by linear regression with a continuous line for control (•) (n= 6), long dashed line for 2.5 µM alpha-actinin ( ) (n= 5), short dashed lines for 800 nM calmodulin () (n= 7) and dashed–dotted line for 2.5 µM alpha-actinin + 800 nM calmodulin ( ) (n= 7). Alpha-actinin significantly reduced mean shut time in comparison to control, calmodulin- and alpha-actinin + calmodulin-treated patches over all membrane potentials recorded (analysis of covariance F < 0.05).

NMDA channel open time is reduced in the presence of alpha-actinin and calmodulin

Open time distributions were best fitted with a mixture of three exponential components; mean data recorded at –60 mV are presented in Table 2. It is apparent from the distributions shown in Fig. 6A–D that application of all three internal solutions shifted the distribution of open times to the left, giving overall shorter open times when compared to control. The reduction in mean open time by all three internal solutions was consistent at all membrane potentials studied in comparison to control (analysis of covariance) (Fig. 7A). In addition, the reduction in mean open time by calmodulin alone was significantly less than that caused by alpha-actinin or alpha-actinin + calmodulin.

View larger version (16K): [in this window] [in a new window]   Figure 6.  Comparison of distributions for all individual open times in control (A), 2.5 µM alpha-actinin-treated patches (B), 800 nM calmodulin-treated patches (C), and 2.5 µM alpha-actinin + 800 nM calmodulin-treated patches (D) In these distributions open time intervals ranging from 0.11 to 40 ms were fitted with the sum of three exponential components (time constants and associated areas are inset). Predicted mean open time (and observed and predicted number of observations) were 4.15 ms (1021 and 1143) for control, 2.58 ms (1332 and 1592) for 2.5 µM alpha-actinin-treated patches, 2.78 ms (879 and 1097) for 800 nM calmodulin-treated patches and 1.48 ms (732 and 1245) for 2.5 µM alpha-actinin + 800 nM calmodulin-treated patches.

View larger version (19K): [in this window] [in a new window]   Figure 7.  Comparison of the effects of 2.5 µM alpha-actinin, 800 nM calmodulin and 2.5 µM alpha-actinin + 800 nM calmodulin on mean open time A, mean open time for control, 2.5 µM alpha-actinin-treated patches, 800 nM calmodulin-treated patches and 2.5 µM alpha-actinin + 800 nM calmodulin-treated patches. Each point represents the mean open time of all patches, at that membrane potential, calculated from the three exponential components fitted to the open time distributions. The data were fitted using linear regression with a continuous line for control (•) (n= 6), long dashed line for 2.5 µM alpha-actinin ( ) (n= 5), short dashed lines for 800 nM calmodulin () (n= 7) and dashed–dotted line for 2.5 µM alpha-actinin + 800 nM calmodulin ( ) (n= 7). 2.5 µM alpha-actinin, 800 nM calmodulin and 2.5 µM alpha-actinin + 800 nM calmodulin significantly reduced mean open time in comparison to control. 2.5 µM alpha-actinin and 2.5 µM alpha-actinin + 800 nM calmodulin significantly reduced mean open time in comparison to 800 nM calmodulin alone over all membrane potentials recorded (analysis of covariance F < 0.05). B, reduction in mean open time, expressed as a percentage of control, for 2.5 µM alpha-actinin, 800 nM calmodulin and 2.5 µM alpha-actinin + 800 nM calmodulin. Percentage open time in the presence of a calmodulin concentration of 12 nM(n= 7) is also included for comparison. Mean open time was reduced by 45% in the presence of 2.5 µM alpha-actinin ( ), 29% in the presence of 800 nM calmodulin (), 51% in the presence of 2.5 µM alpha-actinin + 800 nM calmodulin ( ) and 50% in the presence of 12 nM calmodulin ( ).

The percentage reduction in mean open time by alpha-actinin, calmodulin and alpha-actinin + calmodulin was also compared at each membrane potential to the reduction in mean open time observed in the presence of a lower concentration of calmodulin (12 nM) predicted to occupy predominantly the C1 region (Fig. 7B). The effect of alpha-actinin and alpha-actinin + calmodulin on mean open time was identical to that observed in the presence of 12 nM calmodulin. However, the high concentration of calmodulin (800 nM) used in this study, predicted to occupy both C1 an C0 binding sites, had significantly less effect on mean open time when compared with the low concentration of calmodulin (12 nM).

Alpha-actinin caused an increase in channel P_open indicating that, compared to the effect on channel open times, the observed reduction in mean shut time is the more dominant effect on NMDA channel kinetics (Fig. 8). P_open was reduced in the presence of calmodulin alone and alpha-actinin + calmodulin in comparison to control, a result of their ability to shorten mean open time without significantly reducing channel shut time. The increase in P_open caused by alpha-actinin was significant at all membrane potentials tested (analysis of covariance), as was the reduction in P_open caused by alpha-actinin + calmodulin but not calmodulin alone. However, both calmodulin alone and alpha-actinin + calmodulin significantly reduced P_open in comparison to alpha-actinin alone at all membrane potentials recorded.

View larger version (11K): [in this window] [in a new window]   Figure 8.  Comparison of the effects of 2.5 µM alpha-actinin, 800 nM calmodulin and 2.5 µM alpha-actinin + 800 nM calmodulin on Popen Popen expressed as a percentage of control for alpha-actinin (n= 5) ( ), calmodulin (n= 7) (), alpha-actinin + calmodulin (n= 6) ( ) and a low concentration (12 nM) of calmodulin (n= 7) ().

	Discussion

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Intriguingly, both alpha-actinin and alpha-actinin + calmodulin reduced mean open time to the same extent as that observed in the presence of 12 nM calmodulin (Rycroft & Gibb, 2002), a low concentration predicted to predominantly occupy the high affinity C1 region. However, at the high concentration of calmodulin used in this study (800 nM), predicted to maximally bind to both the C1 and C0 regions, mean open time was reduced to a significantly lesser extent than with 12 nM calmodulin. Of the three pipette solutions used in this study the effects of alpha-actinin + calmodulin exhibited a remarkable similarity to 12 nM calmodulin, reducing mean open time whilst leaving mean shut time unaffected. These results and their physiological implications are discussed below.

The major effect of alpha-actinin on NMDA receptor single channel characteristics is a reduction in mean shut time, due to a reduction in the time constant of the slowest component of the shut time distribution. This could be caused by an increase in agonist association rate (caused by alpha-actinin binding), which could be investigated by measuring the EC₅₀ for NMDA, in association with kinetic measurements, or an increase in the density of receptors in the patch in the presence of alpha-actinin, which would be indicated by an increase in the peak macroscopic current. The slowest component probably represents the closed receptor in the unbound state, as, at least for recombinant NR1/NR2A receptors (Anson et al. 2000), this time constant is dependent upon agonist concentration. However, the slowest shut time component could also reflect a desensitized state of the receptor and therefore the effect of alpha-actinin binding could alternatively be to shorten the lifetime of this desensitized state. These two possibilities cannot be distinguished from these data.

In view of the agonist concentration dependence of the slowest exponential components of the NMDA receptor shut time distributions (Anson et al. 2000), care was taken to ensure these effects of alpha-actinin were not due to the range of agonist concentrations used in this study (100 nM–10 µM). All alpha-actinin-treated patches were recorded in the presence of 100 nM NMDA in comparison to a range of 100 nM to 10 µM for the control, calmodulin- and alpha-actinin + calmodulin-treated patches. The reduction of mean shut time by alpha-actinin compared to the control recordings cannot therefore be attributed to the agonist concentrations used in these recordings.

There are limitations to the interpretation of mean shut time and changes in the time constant of the fifth shut time component. For instance, it is not possible to guarantee that the size of the membrane patch (and hence number of receptors) is consistent between recordings, although if care is taken to ensure that pipette resistance and shape is consistent this should follow. In addition, any explanation of the effect of alpha-actinin also needs to take into account why the presence of alpha-actinin + calmodulin does not also have the same effect on shut time as alpha-actinin alone.

Despite these considerations, perhaps the most likely explanation for the effect of alpha-actinin on shut time is an increase in the number of receptors on the surface of the outside-out patch. For example a decrease in NMDA channel shut time in the presence of PKC (Chen & Huang, 1992; Xiong et al. 1998) has been shown to result from an increase in NMDA receptor trafficking to the cell surface (Lan et al. 2001). Although dynamic regulation of NMDA channel density is unlikely in an outside-out patch, in whole-cell configuration during formation of an outside-out patch, the cell will be flooded with alpha-actinin from the patch pipette. Depending on the rate of NMDA receptor trafficking (Carroll & Zukin, 2002) and the length of time taken to form an outside-out patch, these conditions could result in a change of NMDA receptor density at the cell surface.

An increase in cell membrane receptor density in the presence of alpha-actinin, and equally, a reduction in the absence of alpha-actinin, is consistent with the role of alpha-actinin as a cross-linking protein, binding the NMDA receptor to the cytoskeleton. Actin depolymerization reduces channel activity (Rosenmund & Westbrook, 1993) and NMDA receptor clustering at the cell surface is disrupted when C-terminal segments containing the NR1 subunit C0 region are coexpressed with recombinant receptors, presumably due to displacement of alpha-actinin from functional receptors (Matsuda & Hirai, 1999). In addition, disruption of actin filaments releases NMDA receptor clusters away from their postsynaptic sites, selectively reducing the activity of synaptically activated NMDA receptors (Allison et al. 1998; Sattler et al. 2000). Since the NMDA receptor is tethered to the actin cytoskeleton by alpha-actinin it might therefore be expected that alpha-actinin dissociation will also reduce the activity of synaptically activated receptors.

The calmodulin-mediated reduction of channel open time observed in this study suggests that the synaptic current could be shortened when calmodulin is bound to both C1 and C0 sites, in addition to just the C1 region as predicted from the results of Rycroft & Gibb (2002). However, one of the most surprising results of this study was that a high concentration of calmodulin (800 nM) had significantly less effect on open time when compared to a low concentration of calmodulin (12 nM). From this result it is tempting to speculate that calmodulin binding at C1 and C0 exhibits negative co-operativity.

Does alpha-actinin- and calmodulin-mediated modulation of single channel kinetics underlie calcium-dependent inactivation of whole-cell and synaptic currents?

Alpha-actinin dissociation and calmodulin binding to the C0 region of NMDA receptors have been shown to mediate calcium-dependent inactivation of whole-cell currents (Zhang et al. 1998; Krupp et al. 1999). Furthermore, calmodulin-mediated inhibition is predicted to shorten the duration and amplitude of synaptic currents (Rycroft & Gibb, 2002) and therefore may underlie calcium-dependent inactivation of synaptic currents (Rosenmund et al. 1995; Medina et al. 1999; Umemiya et al. 2001). Changes in channel shut times may be responsible for calcium-dependent inactivation of whole-cell currents (shut time will increase and P_open will decrease following calcium- or calmodulin-dependent dissociation of alpha-actinin from C0), whereas an alpha-actinin- or calmodulin-induced reduction in mean open time may underlie calcium-dependent inactivation of synaptic currents.

By examining single channel kinetics, macroscopic effects of alpha-actinin and calmodulin on the synaptic current can be predicted by calculating the ensemble current from supercluster alignment (Wyllie et al. 1998). However, because recordings were made at several membrane potentials in this study in order to investigate any voltage dependence of the actions of alpha-actinin and calmodulin, the individual recordings are too short to conduct a reliable analysis of the NMDA receptor supercluster length and P_open. In addition, NMDA and glycine were used as agonists, rather than the endogenous neurotransmitter glutamate. Whilst channel open times are similar in the presence of glutamate or NMDA (Howe et al. 1991; Piña-Crespo & Gibb, 2002; Banke & Traynelis, 2003), the duration of macroscopic relaxations are shorter in the presence of NMDA compared to glutamate (Lester & Jahr, 1992), implying that the duration of activations or superclusters will be shorter in NMDA (as demonstrated by Piña-Crespo & Gibb, 2002). These differences can be attributed to different agonist properties such as receptor affinity (Lester & Jahr, 1992) and the length of closed times within the activation (Banke & Traynelis, 2003).

A scheme to predict modulation of NMDA channel open time by alpha-actinin and calmodulin under physiological conditions

Based on the results described here, a scheme for NMDA receptor modulation by alpha-actinin and calmodulin is presented in Fig. 9. It is assumed that the resting calcium concentration of the cell is less than 100 nM (Clapham, 1995), and the intracellular calmodulin concentration is approximately 10 µM (Kakiuchi et al. 1982; Holmes, 2000), although the free calmodulin concentration is likely to be lower due to buffering by neurogranin (Slemmon et al. 2000; Gaertner et al. 2002).

View larger version (16K): [in this window] [in a new window]   Figure 9.  Scheme to describe NMDA receptor modulation by alpha-actinin ( ) and calmodulin (•) during rest (low [Ca2+]in) and neuronal activation (high [Ca2+]in)

Due to the high affinity of calmodulin for the C1 region (Ehlers et al. 1996) it is likely that dephosphorylated NMDA receptors are tonically inhibited by calmodulin. However, due to the many modulatory and cytoskeletal proteins currently known to associate with the C1 region including PKC and PKA (Leonard & Hell, 1997; Tingley et al. 1997), brain spectrin (Wechsler & Teichberg, 1998), NF-L (Ehlers et al. 1998) and yotiao (Lin et al. 1998), and specifically those known to compete with calmodulin at this site such as PKC (Hisatsune et al. 1997) and brain spectrin (Wechsler & Teichberg, 1998), occasions will arise when calmodulin is not bound to the C1 region. At a low calcium concentration, the calcium-dependent phosphatase calcineurin will be inactive, given its relatively low affinity for calcium (15 µM; Kakalis et al. 1995), whereas PKC, with a higher affinity for calcium (700 nM; Mosior & Epand, 1994), will more likely be active. This would suggest that the receptor is likely to be phosphorylated under resting conditions.

Calmodulin binding to the C0 region is likely to occur when the intracellular calcium concentration is above resting levels. For example at a resting calcium concentration of 100 nM (Clapham, 1995), calmodulin may occupy the C0 region by about 50%, given a dissociation constant of 87 nM (Ehlers et al. 1996). This would be reduced by the competitive presence of alpha-actinin, as the latter has a higher affinity for the C0 region with an equilibrium constant of 48 nM (Krupp et al. 1999).

The sequence of events in a cell may therefore be as follows (Fig. 9): at a low calcium concentration (states (1) and (2)) the receptor has a high P_open because alpha-actinin is bound, thereby shortening mean shut time. Calmodulin is not bound to the C1 region because the receptor is phosphorylated, in this example, or otherwise occupied by a protein that competes with calmodulin for this site. State (3) will occur at low-to-intermediate levels of intracellular calcium following phosphatase activation, dephosphorylation of the receptor and dissociation of alpha-actinin. Thus in the absence of alpha-actinin, calmodulin and phosphorylation, the receptor will have a long mean shut time and long mean open time and therefore moderate P_open, as in our control experiments. In this scheme states (2) to (3), dissociation of alpha-actinin resulting in an increase in channel shut time and a reduction of P_open, resemble the cellular events underlying calcium-dependent inactivation. As the calcium concentration continues to rise during neuronal excitation and calmodulin binds in states (4) and (5), the channel also has a short mean open time, long mean shut time and therefore low P_open.

In the process of deriving the scheme in Fig. 9, it became apparent that data obtained in the presence of alpha-actinin and calmodulin, presumably bound to the C0 and C1 region, respectively, were not consistent. Channel kinetics exhibited a remarkable similarity to that observed in the presence of a low concentration of calmodulin (12 nM), predicted to predominantly occupy the C1 region. A possible reason for this outcome is that alpha-actinin and calmodulin cannot be bound to the C0 and C1 regions, respectively, at the same time. If binding of alpha-actinin and calmodulin were mutually exclusive, the effects we observed in the presence of alpha-actinin and calmodulin may actually be the mixed effects of calmodulin bound at the C1 region or alpha-actinin bound at C0, for which the net effect is similar to 12 nM calmodulin; a low concentration that will occupy mainly C1. This would explain the apparent lack of calmodulin-mediated effects, via the C1 site, in calcium-dependent inactivation of whole-cell currents (Rafiki et al. 1997; Zhang et al. 1998; Krupp et al. 1999). As suggested in Fig. 9, if the receptor is bound to alpha-actinin at rest it may not be able to bind calmodulin at the C1 region. Therefore effects of calmodulin at C1 would not be evident until alpha-actinin has dissociated from C0.

	Conclusions

Top Abstract Introduction Methods Results Discussion Conclusions References

 
We have investigated regulation of single NMDA channel activity by alpha-actinin and calmodulin. We conclude that under resting cellular conditions whilst bound to alpha-actinin, the NMDA receptor will have a short mean shut time and a short mean open time and therefore higher P_open. When the intracellular calcium concentration increases during neuronal excitation, alpha-actinin will dissociate and calmodulin bind to the receptor, causing an increase in mean shut time, a decrease in mean open time and an overall reduction in P_open. These changes in channel shut time caused by alpha-actinin and in open time caused by calmodulin may underlie calcium-dependent inactivation of NMDA receptor-mediated whole-cell currents (Zhang et al. 1998; Krupp et al. 1999) and synaptic currents (Rosenmund et al. 1995; Medina et al. 1999; Umemiya et al. 2001), and therefore contribute to the dynamic regulation of NMDA receptor activity during excitatory synaptic transmission in the brain.

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	Acknowledgements

 
This work was supported by the Medical Research Council and the Wellcome Trust. We are grateful to David Colquhoun for providing software.

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