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. 2004 Apr 8;557(Pt 3):795–808. doi: 10.1113/jphysiol.2003.059212

Regulation of single NMDA receptor channel activity by alpha-actinin and calmodulin in rat hippocampal granule cells

Beth K Rycroft 1, Alasdair J Gibb 1
PMCID: PMC1665152  PMID: 15073274

Abstract

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 (Popen), whereas calmodulin binding reduced single channel mean open time, resulting in an overall reduction in Popen. The physiological implications of these findings are discussed.

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 Popen. Mean shut time was unchanged in the presence of calmodulin alone or alpha-actinin plus calmodulin, resulting in overall 29 and 49% decreases in Popen, 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

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 MΩ. 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 Popen (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). Popen 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

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.

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.

Figure 1

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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.

Single channel conductance in the presence of alpha-actinin and calmodulin

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.

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.

Figure 2

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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.

Table 1.

Single channel current amplitude from control, 2.5 μm alpha-actinin-, 800 nm calmodulin- and 2.5 μm alpha-actinin + 800 nm calmodulin-treated patches at −60 mV

Gaussian components
1st (pA) 2nd (pA) 3rd (pA)
Control (n = 6) 3.53 ± 0.16 2.73 ± 0.14 1.64 ± 0.18
 84 ± 2.1%  15 ± 2.2%  1.4 ± 0.5%
Alpha-actinin (n = 5) 3.32 ± 0.1 2.34 ± 0.03 1.51 ± 0.07
 82 ± 5.5%  14 ± 4.8%  4.4 ± 1.6%
Calmodulin (n = 7) 3.34 ± 0.13 2.46 ± 0.10 1.55 ± 0.09
 83 ± 2.8%  15 ± 2.3%  2.1 ± 0.5%
Alpha-actinin + calmodulin (n = 6) 3.58 ± 0.09 2.67 ± 0.05 1.8 ± 0.1
 86 ± 1.3%  13 ± 1.4% 1.1 ± 0.4%
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Shown is the mean ± s.e.m. for the mean of each Gaussian component detected in each amplitude distribution and below the mean ± s.e.m of the relative area of each component (n = number of patches).

Mean channel amplitudes for the first and second amplitude components were plotted against holding potential to determine the channel slope conductance (Fig. 3). The main conductance level was 57.2 ± 2.7 pS for control (n = 6), 51.5 ± 3.2 pS for alpha-actinin-treated patches, 53.6 ± 2.3 pS for calmodulin-treated patches (n = 7) and 55.5 ± 1.5 pS for alpha-actinin + calmodulin-treated patches (n = 6). The associated subconductance level had slope conductances of 44.7 ± 3.1, 37.6 ± 5.8, 43.3 ± 2.3 and 48.0 ± 1.5 pS, respectively. Analysis of covariance indicated that the presence of alpha-actinin, calmodulin or alpha-actinin + calmodulin did not significantly affect NMDA receptor single channel conductance when compared to control. However, the subconductance but not the main conductance level of single channel openings in the presence of alpha-actinin alone was significantly reduced when compared to control.

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.

Figure 3

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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).

NMDA channel shut time is reduced in the presence of alpha-actinin but not calmodulin

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).

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.

Figure 4

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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.

Table 2.

Distribution of shut times and open times from control, 2.5 μm alpha-actinin-, 800 nm calmodulin- and 2.5 μm alpha-actinin + 800 nm calmodulin-treated patches at −60 mV

τ1 (μs) τ2 (ms) τ3 (ms) τ4 (ms) τ5 (ms) Mean (ms)
Control (n = 6)
 Shut times 157 ± 60 1.03 ± 0.24 12.7 ± 2.8 316 ± 156 897 ± 290 211 ± 53
 26 ± 5%  26 ± 2%  13 ± 3%  12 ± 2%  24 ± 3%
 Open times 139 ± 32 2.07 ± 0.7 6.26 ± 1.0 — — 4.14 ± 0.6
 17 ± 4%  24 ± 6%  59 ± 7% — —
Alpha-actinin (n = 5)
 Shut times 129 ± 33 0.91 ± 0.15 11.1 ± 2.2 94.8 ± 6.7 162 ± 13 57.7 ± 13
 14 ± 4%  24 ± 6%  14 ± 2%  29 ± 7%  19 ± 9%
 Open times 118 ± 41 1.69 ± 0.2 3.12 ± 0.4 — — 2.08 ± 0.2
 16 ± 7%  39 ± 12%  45 ± 9% — —
Calmodulin (n = 7)
 Shut times 237 ± 65 0.99 ± 0.07 13.5 ± 4.2 184 ± 66 1037 ± 413 291 ± 89
 24 ± 3%  23 ± 5%  12 ± 3%  14 ± 3%  27 ± 5%
 Open times 89 ± 24 1.63 ± 0.6 4.61 ± 0.5 — — 2.93 ± 0.4
 31 ± 4%  9 ± 4%  60 ± 5% — —
Alpha-actinin + calmodulin (n = 6)
 Shut times 227 ± 36 1.06 ± 0.16 21.0 ± 6.9 280 ± 91 772 ± 258 321 ± 103
 17 ± 4%  25 ± 5%  12 ± 2%  10 ± 4%  36 ± 3%
 Open times 133 ± 34 1.35 ± 0.3 3.77 ± 0.4 — — 1.84 ± 0.4
 21 ± 3%  45 ± 13%  34 ± 12% — —
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Shown is the mean ± s.e.m. for the time constant of each exponential component detected in the shut and open time distributions and below the mean ±s.e.m. of the relative area of each component (n = number of patches).

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.

Figure 5

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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).

Upon examination of individual shut time components, the reduction in mean shut time by alpha-actinin was found to be due to a shortening of the fifth and longest time constant. Distributions of shut time components from patches in the presence of calmodulin and alpha-actinin + calmodulin, however, exhibited remarkable similarity to control shut time components. The weighted mean shut time of the first four exponential components, which can used to approximate the mean shut time within an activation (Gibb & Colquhoun, 1992; Wyllie et al. 1998), was also calculated (Fig. 5B). This was found to be not significantly different (by analysis of covariance) between any of the treated groups over all membrane potentials, indicating that the effect of alpha-actinin is predominately on the longest component of the shut times.

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.

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).

Figure 6

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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.

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.

Figure 7

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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 (graphic file with name tjp0557-0795-fu1.jpg).

The voltage dependence of the channel open time observed in these experiments is likely to be due to channel block by residual magnesium present in the recording solution, as this is removed in the presence of divalent chelators such as EDTA (Gibb & Colquhoun, 1992). Analysis of covariance indicated that the voltage dependence of the mean open time (slope of the line) did not significantly change in the presence of all three internal solutions in comparison to control. Therefore the voltage dependence of the channel open time is consistent between groups, suggesting that the magnesium sensitivity of the channel was unaltered. Mean open time increased e-fold for every 48, 47, 47 and 45 mV depolarization, in control, alpha-actinin-, calmodulin- and alpha-actinin + calmodulin-treated patches, respectively (Fig. 7A).

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 Popen 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). Popen 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 Popen caused by alpha-actinin was significant at all membrane potentials tested (analysis of covariance), as was the reduction in Popen caused by alpha-actinin + calmodulin but not calmodulin alone. However, both calmodulin alone and alpha-actinin + calmodulin significantly reduced Popen in comparison to alpha-actinin alone at all membrane potentials recorded.

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.

Figure 8

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Popen expressed as a percentage of control for alpha-actinin (n = 5) (▪), calmodulin (n = 7) (□), alpha-actinin + calmodulin (n = 6) (graphic file with name tjp0557-0795-fu2.jpg) and a low concentration (12 nm) of calmodulin (n = 7) (▪).

Discussion

In this study alpha-actinin was found to reduce NMDA receptor single channel mean open time by 45% and decrease mean shut time by 83%, presumably due to binding at the C0 region of the NR1 C-terminus (Wyszynski et al. 1997; Krupp et al. 1999). This resulted in an overall 77% increase in channel Popen. At a concentration of calmodulin predicted to fully occupy both C1 and C0 regions of the NR1 C-terminus (Ehlers et al. 1996), mean open time was reduced by 29% and mean shut time was unaffected at all potentials recorded. Furthermore, a combination of alpha-actinin plus calmodulin, predicted to result in maximal binding of calmodulin to the C1 region and alpha-actinin to the C0 region, was found to reduce mean open time by 51%, with no effect on channel shut time.

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 Popen 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 Popen. 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).

Figure 9. Scheme to describe NMDA receptor modulation by alpha-actinin (1) and calmodulin (•) during rest (low [Ca2+]in) and neuronal activation (high [Ca2+]in).

Figure 9

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Although the effective free concentration of alpha-actinin in the cell is unknown it exhibits a higher affinity for the NR1 subunit C0 region than calmodulin (Ehlers et al. 1996; Krupp et al. 1999). In addition, although alpha-actinin in some isoforms is calcium sensitive, a calcium concentration of 100 nm was found to be insufficient to antagonize the binding of alpha-actinin-2 to the NR1 subunit in vitro (Wyszynski et al. 1997). Alpha-actinin is therefore likely to be bound to the NMDA receptor at rest (depending on the relative concentrations of calmodulin and alpha-actinin in the cell), tethering the NMDA receptor to the cytoskeleton (Wyszynski et al. 1997, 1998; Allison et al. 1998; Krupp et al. 1999).

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 Popen 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 Popen, 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 Popen, 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 Popen.

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

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 Popen. 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 Popen. 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.

Acknowledgments

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

References

  1. Allison DW, Gelfand VI, Spector I, Craig AM. Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurones. J Neurosci. 1998;18:2423–2436. doi: 10.1523/JNEUROSCI.18-07-02423.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anson LC, Schoepfer R, Colquhoun D, Wyllie DJ. Single-channel analysis of an NMDA receptor possessing a mutation in the region of the glutamate binding site. J Physiol. 2000;527:225–237. doi: 10.1111/j.1469-7793.2000.00225.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banke TG, Traynelis SF. Activation of NR1/NR2B NMDA receptors. Nat Neurosci. 2003;6:144–152. doi: 10.1038/nn1000. [DOI] [PubMed] [Google Scholar]
  4. Bear MF, Malenka RC. Synaptic plasticity: LTP and LTD. Curr Opin Neurobiol. 1994;4:389–399. doi: 10.1016/0959-4388(94)90101-5. [DOI] [PubMed] [Google Scholar]
  5. Bliss TVP, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature. 1993;361:31–39. doi: 10.1038/361031a0. [DOI] [PubMed] [Google Scholar]
  6. Bliss TVP, Collingridge GL. Memories of NMDA receptor and LTP. Trends Neurosci. 1995;28:54–56. [PubMed] [Google Scholar]
  7. Carroll RC, Zukin RS. NMDA-receptor trafficking and targeting: implications for synaptic transmission and plasticity. Trends Neurosci. 2002;25:571–577. doi: 10.1016/s0166-2236(02)02272-5. [DOI] [PubMed] [Google Scholar]
  8. Chen L, Huang LY. PKC decreases magnesium block of the NMDA receptor channels as a mechanism of modulation. Nature. 1992;356:521–523. doi: 10.1038/356521a0. [DOI] [PubMed] [Google Scholar]
  9. Clapham DE. Calcium signalling. Cell. 1995;80:259–268. doi: 10.1016/0092-8674(95)90408-5. [DOI] [PubMed] [Google Scholar]
  10. Colquhoun D, Sigworth FJ. Fitting and statistical analysis of single-channel records. In: Sakmann B, Neher E, editors. Single-Channel Recording. 2. New York: Plenum Press; 1995. pp. 483–587. [Google Scholar]
  11. Edwards FA, Konnerth A, Sakmann B, Takahashi T. A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflugers Arch. 1989;414:600–612. doi: 10.1007/BF00580998. [DOI] [PubMed] [Google Scholar]
  12. Ehlers MD, Fung ET, O'Brien RJ, Huganir RL. Splice variant–specific interaction of the NMDA receptor subunit NR1 with neuronal intermediate filaments. J Neurosci. 1998;18:720–730. doi: 10.1523/JNEUROSCI.18-02-00720.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ehlers MD, Zhang S, Bernhardt JP, Huganir RL. Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell. 1996;84:745–755. doi: 10.1016/s0092-8674(00)81052-1. [DOI] [PubMed] [Google Scholar]
  14. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Meth Enzymol. 1988;157:378–417. doi: 10.1016/0076-6879(88)57093-3. [DOI] [PubMed] [Google Scholar]
  15. Gaertner TR, Putkey JA, Waxham MN. Kinetics of calmodulin binding to RC3/neurogranin. Soc Neuro Abstracts. 2002;436:15. [Google Scholar]
  16. Gibb AJ, Colquhoun D. Glutamate activation of a single NMDA receptor-channel produces a cluster of channel openings. Proc R Soc Lond B Biol Sci. 1991;243:39–45. doi: 10.1098/rspb.1991.0007. [DOI] [PubMed] [Google Scholar]
  17. Gibb AJ, Colquhoun D. Activation of N-methyl-D-aspartate receptors by 1-glutamate in cells dissociated from adult rat hippocampus. J Physiol. 1992;456:143–179. doi: 10.1113/jphysiol.1992.sp019331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Haiech J, Klee CB, Demaille JG. Effects of cations on affinity of calmodulin: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes. Biochem. 1981;20:3890–3897. doi: 10.1021/bi00516a035. [DOI] [PubMed] [Google Scholar]
  19. Hisatsune C, Umemori H, Inoue T, Michikawa T, Kohda K, Mikoshiba K, et al. Phosphorylation-dependent regulation of N-methyl-D-aspartate receptors by calmodulin. J Chem Biol. 1997;272:20805–20810. doi: 10.1074/jbc.272.33.20805. [DOI] [PubMed] [Google Scholar]
  20. Holmes WR. Models of calmodulin trapping and CaM kinase II activation in a dendritic spine. J Comput Neurosci. 2000;81:65–85. doi: 10.1023/a:1008969032563. [DOI] [PubMed] [Google Scholar]
  21. Howe JR, Cull-Candy SG, Colquhoun D. Currents through single glutamate receptor channels in outside-out patches from rat cerebellar granule cells. J Physiol. 1991;432:143–202. doi: 10.1113/jphysiol.1991.sp018381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. James P, Vorherr T, Carafoli E. Calmodulin-binding domains: just two faced or multi-faceted. Trends Biol Sci. 1995;20:38–42. doi: 10.1016/s0968-0004(00)88949-5. [DOI] [PubMed] [Google Scholar]
  23. Kakalis LT, Kennedy M, Sikkink R, Rusnak F, Armitage IM. Characterisation of the calcium-binding sites of calcineurin B. FEBS Lett. 1995;362:55–58. doi: 10.1016/0014-5793(95)00207-p. [DOI] [PubMed] [Google Scholar]
  24. Kakiuchi S, Yasuda S, Yamazaki R, Teshima Y, Kanda K, Kakiuchi R, et al. Quantitative determinations of calmodulin in the supernatant and particulate fractions of mammalian tissues. J Biochem (Tokyo) 1982;92:1041–1048. doi: 10.1093/oxfordjournals.jbchem.a134019. [DOI] [PubMed] [Google Scholar]
  25. Kennedy MB. Signal-processing machines at the postsynaptic density. Science. 2000;290:750–754. doi: 10.1126/science.290.5492.750. [DOI] [PubMed] [Google Scholar]
  26. Kim JH, Huganir RL. Organization and regulation of proteins at synapses. Curr Opin Cell Biol. 1999;11:248–254. doi: 10.1016/s0955-0674(99)80033-7. [DOI] [PubMed] [Google Scholar]
  27. Koh DS, Geiger JR, Jonas P, Sakmann B. Ca2+ -permeable AMPA and NMDA receptor channels in basket cells of rat hippocampal dentate gyrus. J Physiol. 1995;485:383–402. doi: 10.1113/jphysiol.1995.sp020737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Krupp JJ, Vissel B, Heinemann SF, Westbrook GL. Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific. Mol Pharmacol. 1996;50:1680–1688. [PubMed] [Google Scholar]
  29. Krupp JJ, Vissel B, Thomas CG, Heinemann SF, Westbrook GL. Interactions of calmodulin and α-actinin with the NR1 subunit modulate calcium-dependent inactivation of NMDA receptors. J Neurosci. 1999;19:1165–1178. doi: 10.1523/JNEUROSCI.19-04-01165.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lan J-Y, Skeberdis VA, Jover T, Grooms SY, Lin Y, Araneda RC, et al. Protein kinase C modulates NMDA receptor trafficking and gating. Nat Neurosci. 2001;4:382–390. doi: 10.1038/86028. [DOI] [PubMed] [Google Scholar]
  31. Legendre P, Rosenmund C, Westbrook GL. Inactivation of NMDA channels in cultured hippocampal neurons by intracellular calcium. J Neurosci. 1993;13:674–684. doi: 10.1523/JNEUROSCI.13-02-00674.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Leonard AS, Bayer KU, Merrill MA, Lim IA, Shea MA, Schulman H, et al. Regulation of calcium/calmodulin-dependent protein kinase II docking to N-methyl-D-aspartate receptors by calcium/calmodulin and alpha-actinin. J Biol Chem. 2002;277:48441–48448. doi: 10.1074/jbc.M205164200. [DOI] [PubMed] [Google Scholar]
  33. Leonard AS, Hell JW. Cyclic AMP-dependent protein kinase and protein kinase C phosphorylate N-methyl-D-aspartate receptors at different sites. J Biol Chem. 1997;272:12107–12115. doi: 10.1074/jbc.272.18.12107. [DOI] [PubMed] [Google Scholar]
  34. Lester RA, Jahr CE. NMDA channel behavior depends on agonist affinity. J Neurosci. 1992;12:635–643. doi: 10.1523/JNEUROSCI.12-02-00635.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Liebermann DN, Mody I. Regulation of NMDA channel function by endogenous Ca2+-dependent phosphatase. Nature. 1994;369:235–239. doi: 10.1038/369235a0. [DOI] [PubMed] [Google Scholar]
  36. Lin JW, Wyszynski M, Madavan R, Sealock R, Kim JU, Sheng M. Yotiao, a novel protein of neuromuscular junction and brain that interacts with specific splice variants of NMDA receptor subunit NR1. J Neurosci. 1998;18:2017–2027. doi: 10.1523/JNEUROSCI.18-06-02017.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lisman JE. A mechanism for the Hebb and the anti-Hebb processes underlying learning and memory. Proc Natl Acad Sci U S A. 1989;86:9574–9578. doi: 10.1073/pnas.86.23.9574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lisman JE. Three Ca2+ levels affect plasticity differently: the LTP zone, the LTD zone and no man's land. J Physiol. 2001;532:285. doi: 10.1111/j.1469-7793.2001.0285f.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lu WY, Xiong ZG, Lei S, Orser BA, Dudek E, Browning MD, et al. G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors. Nat Neurosci. 1999;2:331–338. doi: 10.1038/7243. [DOI] [PubMed] [Google Scholar]
  40. Matsuda S, Hirai H. The clustering of NMDA receptor NR1 subunit is regulated by the interaction between the C-terminal exon cassettes and the cytoskeleton. Neurosci Res. 1999;34:157–163. doi: 10.1016/s0168-0102(99)00046-2. [DOI] [PubMed] [Google Scholar]
  41. McManus OB, Blatz AL, Magleby KL. Sampling, log binning, fitting, and plotting durations of open and shut intervals from single channels and the effects of noise. Pflugers Arch. 1987;410:530–553. doi: 10.1007/BF00586537. [DOI] [PubMed] [Google Scholar]
  42. Medina I, Leinekugel X, Ben-Ari Y. Calcium-dependent inactivation of the monosynaptic NMDA EPSCs in rat hippocampal neurons in culture. Eur J Neurosci. 1999;11:2422–2430. doi: 10.1046/j.1460-9568.1999.00664.x. [DOI] [PubMed] [Google Scholar]
  43. Mosior M, Epand RM. Characterisation of the calcium-binding site that regulates association of protein kinase C with phospholipid bilayers. J Biol Chem. 1994;19:13798–13805. [PubMed] [Google Scholar]
  44. Piña-Crespo JC, Gibb AJ. Subtypes of NMDA receptors in new-born rat hippocampal granule cells. J Physiol. 2002;541:41–64. doi: 10.1113/jphysiol.2001.014001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rafiki A, Gozlan H, Ben-Ari Y, Khrestchatisky M, Medina I. The calcium-dependent transient inactivation of recombinant NMDA receptor-channels does not involve the high affinity calmodulin binding site of the NR1 subunit. Neurosci Lett. 1997;223:137–139. doi: 10.1016/s0304-3940(97)13413-9. [DOI] [PubMed] [Google Scholar]
  46. Rosenmund C, Feltz A, Westbrook GL. Calcium-dependent inactivation of synaptic NMDA receptors in hippocampal neurons. J Neurophysiol. 1995;73:427–430. doi: 10.1152/jn.1995.73.1.427. [DOI] [PubMed] [Google Scholar]
  47. Rosenmund C, Westbrook GL. Calcium-induced actin depolymerisation reduces NMDA channel activity. Neuron. 1993;10:805–814. doi: 10.1016/0896-6273(93)90197-y. [DOI] [PubMed] [Google Scholar]
  48. Rycroft BK, Gibb AJ. Direct effects of calmodulin on NMDA receptor single channel gating in rat hippocampal granule cells. J Neurosci. 2002;22:8860–8868. doi: 10.1523/JNEUROSCI.22-20-08860.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sattler R, Xiong Z, Lu WY, MacDonald JF, Tymianski M. Distinct roles of synaptic and extrasynaptic NMDA receptors in excitotoxicity. J Neurosci. 2000;20:22–33. doi: 10.1523/JNEUROSCI.20-01-00022.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sigworth FJ, Sine SM. Data transformations from improved display and fitting of single-channel dwell time histograms. Biophys J. 1987;52:1047–1054. doi: 10.1016/S0006-3495(87)83298-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Slemmon JR, Feng B, Erhardt JA. Small proteins that modulate calmodulin-dependent signal transduction: effects of PEP-19, neuromodulin, and neurogranin on enzyme activation and cellular homeostasis. Mol Neurobiol. 2000;22:99–113. doi: 10.1385/MN:22:1-3:099. [DOI] [PubMed] [Google Scholar]
  52. Tingley WG, Ehlers MD, Kameyama K, Doherty C, Ptak JB, Riley CT, et al. Characterisation of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies. J Biol Chem. 1997;272:5157–5166. doi: 10.1074/jbc.272.8.5157. [DOI] [PubMed] [Google Scholar]
  53. Tong G, Jahr CE. Regulation of glycine-insensitive desensitisation of the NMDA receptor in outside-out patches. J Neurophysiol. 1994;71:754–761. doi: 10.1152/jn.1994.72.2.754. [DOI] [PubMed] [Google Scholar]
  54. Tong G, Shepherd D, Jahr CE. Synaptic desensitization of NMDA receptors by calcineurin. Science. 1995;267:1510–1512. doi: 10.1126/science.7878472. [DOI] [PubMed] [Google Scholar]
  55. Umemiya M, Chen N, Raymond LA, Murphy TH. A calcium-dependent feedback mechanism participates in shaping single NMDA miniature EPSCs. J Neurosci. 2001;21:1–9. doi: 10.1523/JNEUROSCI.21-01-00001.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wagner DA, Leonard JP. Effect of protein kinase C activation on the Mg2+-sensitivity of cloned NMDA receptors. Neuropharmacol. 1996;35:29–36. doi: 10.1016/0028-3908(95)00177-8. [DOI] [PubMed] [Google Scholar]
  57. Wechsler A, Teichberg VI. Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin. EMBO J. 1998;17:3931–3939. doi: 10.1093/emboj/17.14.3931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Weiss DS, Magleby KL. Gating scheme for single GABA-activated Cl-channels determined from stability plots, dwell-time distributions, and adjacent-interval durations. J Neurosci. 1989;9:1314–1324. doi: 10.1523/JNEUROSCI.09-04-01314.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wyllie DJ, Behe P, Colquhoun D. Single-channel activations and concentration jumps: comparison of recombinant NR1a/NR2A and NR1a/NR2D NMDA receptors. J Physiol. 1998;510:1–18. doi: 10.1111/j.1469-7793.1998.001bz.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wyszynski M, Kharazia V, Shanghvi R, Rao A, Beggs AH, Craig AM, et al. Differential regional expression and ultra-structural localisation of α-actinin-2 a putative NMDA receptor-anchoring protein, in rat brain. J Neurosci. 1998;18:1383–1392. doi: 10.1523/JNEUROSCI.18-04-01383.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wyszynski M, Lin J, Rao A, Nigh E, Beggs AH, Craig AM, et al. Competitive binding of the α-actinin-2 and calmodulin to the NMDA receptor. Nature. 1997;385:439–442. doi: 10.1038/385439a0. [DOI] [PubMed] [Google Scholar]
  62. Xiong ZG, Raouf R, Lu WY, Wang LY, Orser BA, Dudek EM, et al. Regulation of N-methyl-D-aspartate receptor function by consitutively active protein kinase C. Mol Pharmacol. 1998;54:1055–1063. [PubMed] [Google Scholar]
  63. Zar JH. Comparing simple linear regression equations. In: Zar JH, editor. Biostatistical Analysis. 4. New Jersey: Prentice Hall; 1999. pp. 360–376. [Google Scholar]
  64. Zhang S, Ehlers MD, Bernhardt JP, Su C-T, Huganir RL. Calmodulin mediates calcium-dependent inactivation of N-methyl-D-aspartate receptors. Neuron. 1998;21:443–453. doi: 10.1016/s0896-6273(00)80553-x. [DOI] [PubMed] [Google Scholar]

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