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1 Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA
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(Received 4 October 2004;
accepted after revision 27 October 2004;
first published online 28 October 2004)
Corresponding author J. W. Johnson: Department of Neuroscience, 446 Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260, USA. Email: johnson{at}bns.pitt.edu
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Introduction |
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Functional NMDA receptors are believed generally to be heterotetramers of NR1 and NR2 subunits. There are four NR2 gene products, NR2A–NR2D. Expression of NR2 subunits follows distinct developmental and regional patterns of expression. For example, in rat cortex, NR2B subunits are present throughout development and in adult animals, while NR2A subunits are found postnatally. NR2D subunits appear prenatally, their expression peaks near postnatal day 7, and then expression decreases to adult levels (Monyer et al. 1994). NR2 subunit expression can be neurone-specific within brain structures; in the hippocampus, NR2A and 2B are the predominant subunits expressed in pyramidal cells while NR2C and 2D are the predominant subunits expressed in interneurones (Monyer et al. 1994). Different NR2 subunits even have distinct subcellular distributions, such as in cerebellar Golgi cells, where NR2A subunits are expressed both synaptically and extrasynaptically, while NR2D subunits are expressed predominantly at extrasynaptic sites (Brickley et al. 2003).
This tight temporal and spatial control of NR2 subunit expression probably reflects the physiological importance of the NR2 subunit-dependence of NMDA receptor properties (Dingledine et al. 1999). Here, we focus on NMDA receptors composed of NR1 and NR2D subunits (NR1/2D receptors). Although not as extensively studied as NR1/2A and NR1/2B receptors, NR1/2D receptors are involved in many CNS processes, including stress pathways (Miyamoto et al. 2002), epileptogenesis (Bengzon et al. 1999) and synaptic plasticity (Okabe et al. 1998; Bengzon et al. 1999; Hrabetova et al. 2000). The basic pharmacological and biophysical properties of NR1/2D receptors have been characterized in expression systems and in carefully chosen native preparations. NR1/2D receptors have been shown to exhibit unique gating properties, including an extremely slow deactivation rate and an unequal probability in transitions between the main and subconductance states (Monyer et al. 1994; Wyllie et al. 1996, 1998; Vicini et al. 1998; Misra et al. 2000). In addition, the channel properties of NR1/2D (along with NR1/2C) receptors are distinct from the channel properties of NR1/2A or NR1/2B receptors. These properties include single-channel conductance, kinetics (Stern et al. 1992; Momiyama et al. 1996; Wyllie et al. 1996) and sensitivity to external Mg2+ (Mg2+o) block (Monyer et al. 1994; Kuner & Schoepfer, 1996).
Voltage-dependent channel block by Mg2+o (Mayer et al. 1984; Nowak et al. 1984; Ascher & Nowak, 1988) is an NMDA receptor property of fundamental physiological importance. In addition to its functional implications, block by Mg2+o provides a means to explore the structure and gating of NMDA receptors (see Johnson & Qian, 2002). The majority of work on Mg2+o block has been performed on NR1/2A or NR1/2B receptors using expression systems, or on native receptors which are most likely to contain NR1, NR2A and NR2B subunits. The studies that have addressed Mg2+o interaction with NR1/2D and NR1/2C receptors revealed that Mg2+o inhibits these receptors much less effectively than NR1/2A or NR1/2B receptors (e.g. Monyer et al. 1994; Kuner & Schoepfer, 1996). However, single-channel measurements of Mg2+o interaction with NR1/2D or NR1/2C receptors have not been reported. Such measurements are particularly challenging because of the brief open duration of NR1/2D and NR1/2C receptors (Stern et al. 1992; Wyllie et al. 1996, 1998), which is further shortened by Mg2+o. Consequently, basic properties of Mg2+o block of NR1/2D and NR1/2C receptors, including the magnitude and voltage-dependence of the rate of Mg2+o entry into and exit from their channels, were not known.
The goals of this study were 2-fold. The first goal was to perform an integrated whole-cell and single-channel study of Mg2+o block of NR1/2D receptors. While satisfying this goal we also characterized in a mammalian expression system the single-channel properties of NR1/NR2D receptors. The second goal was to explore the origin of the differences in Mg2+o inhibition between cortical receptors (composed of NR1, NR2A and NR2B) and NR1/2D receptors.
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Methods |
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Human embryonic kidney (HEK) 293T cells (ATCC, Manassas, VA, USA) were used for whole-cell experiments and HEK293 cells (ATCC) were used for outside-out patch recordings. These choices were made because 293T cells provided larger whole-cell currents, whereas the success rate for pulling outside-out patches was much higher with HEK293 cells. The cells were cultured at 37°C in 5% CO2–95% air. The culture medium for 293T cells was Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Life Technologies, Carlsbad, CA, USA) with 5% fetal bovine serum (FBS) and 2 mM glutamine. The culture medium for HEK293 cells was DMEM with 10% FBS, 2 mM glutamine and 1 mM sodium pyruvate. The cells were maintained in 100-mm culture dishes and split twice a week. For experiments, the cells were plated onto glass coverslips pretreated with poly D-lysine (0.1 mg ml–1) and rat-tail collagen (0.1 mg ml–1, BD Biosciences, San Jose, CA, USA) in 35-mm culture dishes at 1–4 x 105 cells per dish.
Transfection
HEK293 or 293T cells were transiently transfected with cDNAs for rat NMDA receptor subunits 18–24 h after plating. cDNA for NMDA receptor subunits NR1–1a, NR2A and NR2D (Buller & Monaghan, 1997) were subcloned into mammalian expression vector pcDM8. cDNA of enhanced Green Fluorescent Protein (eGFP) (gift from Dr Mark Fleck, Albany Medical College, NY, USA) was cotransfected as a marker of successfully transfected cells. 293T cells were transfected using LipofectAMINE/PLUS reagents (Invitrogen). Briefly, transfection was performed by adding to each dish 1 ml serum-free medium containing 1 µg total DNA, 5 µl LipofectAMINE and 4 µl PLUS. The ratio of cDNA used was 1 eGFP: 3 NR1: 6 NR2 (A or D). DL-2-amino-5-amino-5-phosphono-valeric acid (DL-APV, (200 µM) was added to prevent NMDA receptor mediated-excitotoxicity. The LipofectAMINE reagents have been reported to weaken the plasma membrane, presumably by incorporating into it, and consequently to be unsuitable for excised patch experiments (Groot-Kormelink et al. 2002). We therefore used a calcium phosphate precipitation procedure to transfect HEK293 cells. The amount of cDNA used per dish was 0.7 µg for eGFP, 0.7 µg for NR1–1a and 1.4 µg for NR2D. Precipitates were washed off with fresh culture medium that contained 200 µMDL-APV, 7–9 h after addition of DNA.
Solutions
Solutions were prepared daily from frozen stock and delivered using an in-house fabricated seven-barrel fast perfusion system (Qian et al. 2002). Currents were activated by 10 or 30 µM NMDA + 30 µM glycine. Mg2+ concentrations from 1 µM to 10 mM were added to external solutions. We did not adjust for changes in osmolality that resulted from adding Mg2+. The external solution contained (mM): NaCl 140, CaCl21, KCl 2.8 and Hepes 10. The internal solutions contained (mM): CsCl 125, EGTA 10 and Hepes 10 for whole-cell experiments; CsF 115, CsCl 10, EGTA 10 and Hepes 10 for outside-out patch recordings. The pH of solutions was adjusted to between 7.1 and 7.2 using HCl or the basic form of the major charge carrier. Sucrose and N-methyl-D-glucamine were used to adjust the osmolality of external and internal solutions, respectively. The junction potentials between the pipette and bath solution were 5 mV in chloride-based internal solution and 9 mV for fluoride-based internal solution. All holding potentials were corrected for junction potentials. Ultrapure salts were used when available. All chemicals were from Sigma Chemical Co. (St Louis, MO, USA), except as indicated in the text.
Whole-cell recordings and analysis
Electrophysiological experiments were performed 20–72 h after transfection,. Whole-cell recordings were performed as previously described (Qian et al. 2002). Briefly, currents were recorded at room temperature with an Axopatch 200A or 200B amplifier (Axon Instruments, Foster City, CA, USA), low-pass filtered at 10 kHz, digitized at 44 kHz with a Neuro-Corder and stored on video tape for off-line analysis. Series resistance was compensated 60–80%. NMDA-activated currents in the absence (Icontrol) and presence (IMg) of multiple concentrations of Mg2+o were measured from –115 mV to –15 mV at 10 mV increments. The IC50 of Mg2+o at each voltage was estimated by fitting IMg/Icontrol at various Mg2+o concentrations using eqn (1):
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| (1) |
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Outside-out patch recordings were performed at room temperature according to standard methods (Hamill et al. 1981). Pipettes (resistance, 5–8 M
) were pulled from borosilicate standard-walled glass with filaments (Warner Instrument Corp., Portland, OR, USA). Pipettes were coated with Sylgard and fire-polished. Single-channel currents were recorded using an Axopatch200A or 200B patch-clamp amplifier, low-pass filtered at 10 kHz, digitized at 44 kHz with a Neuro-Corder and stored on video tape for later analysis. Data were collected at voltages from – 105 mV to –45 mV. At each voltage, single-channel currents in each patch were collected in 50–240 s segments, with the first segment being a control measurement in 0 Mg2+o, followed by one to three additional segments in different Mg2+o concetrations. Data from at least three patches were used at each voltage.
For analysis, each data segment recorded was played back, filtered at 2.5 kHz (–3 dB, eight-pole, low-pass bessel filter) and digitally sampled at 25 kHz using pCLAMP 8 Clampex software (Axon Instruments). The effective filter frequency was 2.43 kHz due to cascaded filters. NR1/2D receptor activation is characterized by relatively short open-duration (dominant mean open time, 0.67 ms) and frequent occupancy of subconductance states (see Fig. 2A). To permit accurate estimation of brief duration events and thereby minimize potential errors from missed events, we used the DC analysis programs (http://www.ucl.ac.uk/Pharmacology/dc/html), which make use of time-course fitting techniques (Colquhoun & Sigworth, 1995). Dwell-time histograms were plotted on square root versus log time scales (Sigworth & Sine, 1987). A chosen time resolution was applied to dwell-time distributions and durations shorter than the value of the time resolution were deleted from both open and closed-duration distributions. The resulting dwell-time histograms were fitted by the maximum likelihood method (Colquhoun & Sigworth, 1995) and the value of the imposed time resolution was subtracted from the time constants of the fit. The time resolution value was 50 µs in most patches (range, 45–85 µs), comparable to studies from other labs (for example Wyllie et al. 1996).
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o) with increasing [Mg2+]o was used to estimate Mg2+o blocking rate (see Results). We did not further characterize the other exponential components. Because open periods (duration of openings regardless of current amplitude) were measured, each open event may contain transitions among openings of different amplitude levels. We did not analyse data from main and subconductance levels separately because of the difficulty of determining conductance level of brief events. To accurately determine the subconductance level of an opening the events must be of long enough duration (at least twice the filter rise time) to reach full amplitude. In NR1/2D receptors, many events were too short to meet this criterion.
Closed-duration histograms in the absence of Mg2+o were adequately fitted by the sum of three or four exponentials (Fig. 2B and C). In both the absence and presence of Mg2+o, closed-duration histograms included the duration of all closures, regardless of the conductance level from which the closure began. In the presence of Mg2+o, in most experiments an additional closed-duration component was observed (time constant, b). This component was interpreted to represent blocking events by Mg2+o because its characteristics were typical of block: it was induced by the presence of Mg2+o and the value of b was insensitive to [Mg2+]o (Fig. 4). The mean amplitude of b (30%) is significantly larger than the mean amplitude of the neighbouring, shorter, time constants (13.6%; P= 0.004) or the mean amplitude of the neighbouring, longer, time constants (12.4%; P < 0.001). Occasionally, when the value of b was very close to the briefest closed-duration component observed in the absence of Mg2+o, the closed-duration histogram was fitted by the same number of exponentials in the absence and presence of Mg2+o. In those cases the value of b still could be estimated with reasonable accuracy because the time constant of the confounding brief closed-duration component was necessarily close to b, and because the amplitude of the b component (mean of 33.7%) was relatively large (e.g. significantly larger than the neighbouring, longer time constant (17.3%; P= 0.013)).
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o because missed brief closings cause neighbouring channel openings to be adjoined during data analysis. The same reasoning applies to overestimation of the mean duration of blocked state due to missed short openings. Because both open- and closed-duration histograms contained multiple components, correction for missed events was not practical (Colquhoun & Sigworth, 1995). To minimize errors introduced by missed events, we optimized our time resolution using time-course fitting (see above) and used only Mg2+o concentrations in which o was substantially longer than the imposed time resolution values. The shortest o measured was 0.228 ms, which was 4.6-fold longer than the time resolution in that patch. The shortest b measured was 0.2 ms (3.3-fold longer than the time resolution). b values were not dependent on [Mg2+]o (Fig. 4B), suggesting that missed openings were not frequent enough to appreciably affect our estimates of b. The observation that single channel-derived dissociation constant (KD) values (see later) agreed well with IC50 values measured in whole-cell experiments over a 60 mV range (Fig. 6) also lends support to the accuracy of our measurements.
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oversus[Mg2+]o (Fig. 3C).
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| (2) |
b. Because 1/b should equal the sum of all rates for leaving the open-blocked state, this estimate assumes that closing rate(s) of blocked channel is low relative to k–,app (Antonov & Johnson, 1996). The accuracy of this assumption is difficult to evaluate because we have no way to estimate the closing rate(s) of blocked channels. If the assumption is incorrect, then k–,app would be slower, and KD lower, than estimated here. Data are expressed as mean ±S.E.M.
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Results |
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We first characterized Mg2+o inhibition of whole-cell NR1/2D responses and compared Mg2+o inhibition of NR1/2A and NR1/2D responses. Figure 1A shows current traces recorded at –85 mV (left) and –35 mV (right) from 293T cells transfected with NR1/2A (top) or NR1/2D (bottom) receptors. During a continuous application of agonist, a single concentration of Mg2+o was applied to inhibit currents. Mg2+o inhibited the currents rapidly in both receptor subtypes. At either voltage, NR1/2A receptor-mediated currents were inhibited much more effectively by [Mg2+]o than NR1/2D receptor-mediated currents. Mg2+o concentration–inhibition curves were constructed to estimate the IC50 of Mg2+o at –85 and –35 mV (Fig. 1B). At either voltage, the concentration–inhibition curve for NR1/2D receptors was right-shifted relative to the curves for NR1/2A receptors. In Fig. 1B, the IC50 values for Mg2+o at –85 mV were 11.8 µM for NR1/2A and 60.0 µM for NR1/2D; at –35 mV, the values were 388 µM for NR1/2A and 2.22 mM for NR1/2D. These results are consistent with previous reports (Monyer et al. 1994; Kuner & Schoepfer, 1996; Momiyama et al. 1996) that Mg2+o inhibited NR1/2A receptor-mediated currents much more effectively than NR1/2D receptor-mediated currents.
Using the procedures shown in Fig. 1B, we measured the Mg2+o IC50 for both NR1/2A and NR1/2D receptor-mediated whole-cell currents from –115 mV to –15 mV at 10 mV intervals (Fig. 1C and Table 1). Mg2+o IC50 values are voltage-dependent in both receptor types, consistent with Mg2+o blocking both channel types by occupying a site within the voltage field. At each voltage tested, Mg2+o IC50 was at least 4-fold higher in NR1/2D than in NR1/2A receptors. Hill coefficient values showed no apparent voltage-dependence; when averaged over all voltages Hill coefficients for NR1/2A responses (0.87 ± 0.04) and for NR1/2D responses (0.86 ± 0.01) were nearly identical and suggested that block by Mg2+ is not cooperative.
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Mg2+o block of NR1/2D receptors at the single-channel level
Mg2+o IC50 measurements, although informative regarding the physiological extent of Mg2+o inhibition, do not reveal the underlying blocking kinetics, as IC50 values are related to the ratio of Mg2+o unblocking and blocking rates. We next characterized Mg2+o block at the single-channel level.
We first characterized the single-channel properties of NR1/2D receptors in the absence of Mg2+o. These control data are particularly important because, to our knowledge, measurements of the single-channel characteristics of NR1/2D receptors in a mammalian expression system have not been published. Single-channel traces at –105 mV (Fig. 2A) show that NR1/2D receptor channel openings are brief and open to both main and subconductance levels. The duration of the largest open duration component (Fig. 2B, left), the only component that was evident in all patches, was voltage-independent (Fig. 2C, left). The duration of the three closed duration components (Fig. 2B, right) that were evident in all patches also were voltage-independent (Fig. 2C, right). Thus, we detected no inherent voltage-dependence of gating of NR1/NR2D receptors. Amplitude histograms (Fig. 2D) were used to plot current–voltage (i–V) curves for the main and subconductance states (Fig. 2E), which exhibited voltage-independent conductances of 37.4 and 19.5 pS, respectively, and extrapolated reversal potentials near 0 mV. Except for the voltage-independence of dwell times of NR1/2D receptors, which has not previously been examined, the NR1/2D receptor characteristics shown in Fig. 2 are consistent with previous measurements in the oocyte expression system (Wyllie et al. 1996, 1998).
Addition of Mg2+o caused the brief NR1/2D receptor channel openings to appear ‘flickery’ (Fig. 3A), consistent with a channel blocker of intermediate kinetics (Hille, 2001). Channel flicker during Mg2+o block of NR1/2D receptors is less obvious than during Mg2+o block of NR1/2A or NR1/2B receptors because of the briefer open durations of NR1/2D receptors.
Mg2+o-induced changes in dwell-time distributions were used to examine the kinetics of Mg2+o block. Mg2+o blocking rates (see Methods) were estimated from open-duration histograms (Fig. 3B). In the control condition (0 Mg2+o) in Fig. 3B, the open-duration histogram was fitted by three exponential components. In the presence of each of the three concentrations of Mg2+o shown, open-duration histograms still were well fitted by three exponential components. The time constant of the main component, o, declined as [Mg2+]o increased. We did not further characterize the other two exponential components in different Mg2+o concentrations. As expected for a open channel blocker (Neher & Steinbach, 1978), the inverse of o depends linearly on [Mg2+]o (Fig. 3C). k+,app was estimated from the slope of the regression line through the data points.
Closed-duration histograms were used to estimate Mg2+o unblocking rate, k–,app (Fig. 4). In the example shown in Fig. 4A, the closed-duration distributions in control condition could be adequately fitted by four exponential components. In the presence of Mg2+o, an additional exponential component was needed to adequately fit the closed-duration histograms. The time constant of this additional component is b (see Methods). k–,app was estimated by averaging the inverse of b in 10, 30 and 50 µM Mg2+o. In contrast to k+,app,k–,app is not correlated with [Mg2+]o at any voltages (P= 0.27 at –85 mV; P-values range from 0.27 to 0.96).
Mechanistic differences between Mg2+o block of cortical and NR1/2D receptors
Measurements of Mg2+ok+,app and k–,app allowed us to probe the underlying mechanism of Mg2+o block of NR1/2D receptors. Both Mg2+ok+,app and k–,app are voltage-dependent. From –45 to –75 mV, k–,app (Fig. 5A and Table 1) decreased with hyperpolarization, consistent with a channel blocker that unblocks predominantly to the extracellular side of the membrane. From –75 to –105 mV, k–,app appeared to increase with hyperpolarization in NR1/2D receptors (not statistically significant; ANOVA, P= 0.07). This observation suggests that, at voltages more hyperpolarized than –75 mV, Mg2+o unblocks from NR1/2D receptors predominantly by permeating the channel Mg2+ok+,app decreased with depolarization (Fig. 5B and Table 1), as expected for an extracellular channel blocker.
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Therefore, Mg2+o inhibits NR1/2D receptors more weakly than cortical (NR1, NR2A and NR2B subunit-containing) NMDA receptors both because block is slower and unblock is faster for NR1/2D receptors. The predominant difference that underlies the NR2 subunit-dependence of inhibition is faster Mg2+o unblock from NR1/2D receptors.
Effect of Mg2+o block on equilibria between kinetic states of NR1/2D receptors
We examined the relationship between Mg2+o IC50 values measured from whole-cell experiments (see Fig. 1) and microscopic Mg2+oKD values calculated from single-channel experiments using the relationship KD=k–,app/k+,app. At each voltage at which IC50, k–,app and k+,app were measured, Mg2+o IC50 and KD values are comparable (Fig. 6). The similarity between the two data sets at all voltages supports the accuracy of our single-channel measurements. In addition, the equality of IC50 and KD suggests that, as was previously reported for cortical receptors (Qian et al. 2002), Mg2+o block does not affect the equilibria between kinetic states of NR1/2D receptors.
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Discussion |
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Comparison with previous studies
The molecular mechanism of the NR2 subunit-dependence of inhibition of whole-cell NMDA currents by Mg2+o has been studied in Xenopus laevis oocytes (Kuner & Schoepfer, 1996). In general, the data presented here and the findings of Kuner & Schoepfer (1996) are similar: both studies reported voltage-dependent inhibition by Mg2+o that is stronger in NR1/2A than NR1/2D receptors. However, there are some notable differences between the studies. In our study, Mg2+o inhibition was substantially weaker in NR1/2D than in NR1/2A receptors at all voltages tested (Fig. 1C) while in the experiments of Kuner & Schoepfer (1996), Mg2+o IC50 values tended to converge at depolarized voltages. The absolute values of Mg2+o IC50 were also lower in their studies. For example, IC50 of NR1/2D receptors at –70 mV is estimated to be about 55 µM by Kuner & Schoepfer (1996) and 145 µM in this study.
We do not know the reasons for these differences. The accuracy of our whole-cell measurements is supported by the very similar IC50 values and voltage-dependence of Mg2+o inhibition measured in two different mammalian systems, 293T cells and rat cortical neurones. One possibility is that differences in results of this study and that of Kuner & Schoepfer (1996) are due to differences in permeant ion concentrations. Previous studies (Antonov & Johnson, 1999; Zhu & Auerbach, 2001a,b; Qian et al. 2002) have shown that permeant ion concentrations shape both the affinity and voltage-dependence of Mg2+o block. The internal ion concentration in oocytes are likely to be significantly different from the ion concentrations used in whole-cell recordings. In addition, the kind of permeant intracellular ions present may influence Mg2+o block. Zhu & Auerbach (2001b) showed that internal K+ can accelerate Mg2+o unblocking rate, while this effect was not observed with Cs+ as the main internal cation (Antonov & Johnson, 1999). It is also possible that the difference in experimental preparation is a source of variability in results. We surveyed published Mg2+o IC50 values for NR1/2A and NR1/2D receptors, and found surprising variation in values measured in the oocyte expression system. In the relevant publications we found, IC50 values varied from as much as 15-fold lower than those reported here (Burnashev et al. 1992; Kawajiri & Dingledine, 1993; Sakurada et al. 1993; Kuner & Schoepfer, 1996; Kupper et al. 1996; Wagner & Leonard, 1996; Williams et al. 1998; Liu et al. 2001b) to as much as 15-fold higher (Wagner & Leonard, 1996; Liu et al. 2001a; Wyllie et al. 1996). Previous measurements in HEK293 cells are far more limited; two publications gave values within a factor of 1.6 of those reported here (Wollmuth et al. 1998; Buck et al. 2000). Note that we could find no whole-cell HEK293 measurements of the Mg2+o IC50 of NR1/2D. Thus, there may be an unrecognized source of variability in measurements in the oocyte expression system of Mg2+o IC50 that complicates comparison of our results with those of previous publications. In contrast, previous measurements of other biophysical properties of NR1/2D receptors in oocytes, such as single-channel conductance and dwell-times (Wyllie et al. 1996, 1998), are consistent with those reported here (Fig. 2). A similar conclusion has been drawn for NR1/2A receptors (Stern et al. 1994).
Properties of Mg2+o block in NR1/2D receptors
We examined the properties of Mg2+o block in NR1/2D receptors using the dual approaches of whole-cell and single-channel recordings. Whole-cell recording of NR1/2D receptor currents revealed the voltage dependence of IC50, but provided no kinetic information (Fig. 1). Single-channel recordings (Fig. 3A) suggested that Mg2+o caused flickers in channel openings, a characteristic typical of blockers of intermediate kinetics (Hille, 2001). Measurements of k–,app and k+, app (Figs 3 and 4) confirmed that Mg2+o block in NR1/2D receptors, as in cortical receptors, is of intermediate kinetics.
Channel blockers typically interact with channel gating transitions. Blockers that have the extreme effect of preventing channel closure are often called ‘sequential blockers’. Channel blockers termed ‘trapping blockers’ allow the channel to close and trap the blocker. Many trapping blockers also affect channel gating (see Johnson & Qian, 2002). A method for estimating the effect of a blocker on the equilibrium between receptor states is to compare IC50 values from whole-cell experiments and KD values from single-channel experiments. If the equilibrium between each pair of states is the same whether or not blocker is bound, then IC50=KD (Johnson & Qian, 2002). Sequential blockers, on the other hand, prolong channel openings by holding the channel in the open state while blocker is bound, increasing the IC50 value of the sequential blocker (Johnson & Qian, 2002). IC50 to KD ratios as high as 60 have been measured for blockers that inhibit channel closure (Sobolevsky, 2003). We show here that there is excellent agreement between the values of IC50 and KD for NR1/2D receptors at all voltages tested (Fig. 6), as is the case for cortical receptors (Qian et al. 2002). This observation suggests that channel occupation by Mg2+o does not affect the equilibrium between states of NMDA receptors, which would be simply achieved if block by Mg2+o does not affect receptor kinetics. However, equality of IC50 and KD values would also be observed if Mg2+o block has compensatory effects on multiple equilibrium or kinetic constants, for example by slowing channel opening and closing equally. The idea that Mg2+o block does not affect NMDA receptor gating kinetics is also supported by Sobolevsky & Yelshansky (2000). However, recent studies of NMDA receptors in cortical neurones have suggested that Mg2+o block does affect the kinetics of NMDA receptor state transitions (Vargas-Caballero & Robinson, 2003, 2004; Kampa et al. 2004). Resolution of these conflicting conclusions will require further research.
Differences between Mg2+o inhibition of cortical and NR1/2D receptors
Mg2+o inhibition is weaker in NR1/2D than in NR1/2A or NR1/2B receptors. The mechanistic basis of this subunit-dependent difference was addressed by measuring Mg2+o blocking and unblocking rate constants in single-channel experiments. Our results show that the rates of Mg2+o block of NR1/2D receptors are moderately slower than the rates of block of cortical receptors over a wide range of voltages (Fig. 5C). Mg2+o unblocks much more rapidly from NR1/2D receptors than from cortical receptors. Consequently, the weaker Mg2+o inhibition of NR1/2D receptors is primarily the result of faster unblocking rates.
The Mg2+ok–,app of NR1/2D receptors decreases with hyperpolarization from –45 to –75 mV, suggesting that in this voltage range Mg2+o unblocks predominantly to the external solution. However, k–,app then goes through a minimum value and increases with further hyperpolarization (Fig. 5A). This observation indicates that Mg2+o unblocks predominantly to the internal solution (permeates) at voltages more hyperpolarized than –75 mV. Previous studies have suggested that Mg2+o can permeate native NMDA receptors (Mayer & Westbrook, 1987; Stout et al. 1996). A model of Mg2+o block based on single-channel measurements indicated that, when permeant ions are at physiological concentrations, the predominant direction of Mg2+o unblock from cortical receptors changes from outward to inward at about –105 mV (Antonov & Johnson, 1999). Using this model, the ratio of k–,app of NR1/2D and cortical receptors (Fig. 5C) can be reproduced by simply setting the NR1/2D receptor Mg2+o permeation rate to be about five times faster than the corresponding rate for cortical receptors (not shown). No change in the voltage-dependence of any rate constant is needed. This explanation surely is an oversimplification, given the multiple external and internal regions of NMDA receptors that have been reported to influence the NR2 subunit-dependence of inhibition by Mg2+o (Kuner & Schoepfer, 1996). However, our data are consistent with the idea that Mg2+o inhibits NR1/2D receptors less effectively than cortical receptors in part because of faster Mg2+ permeation of NR1/2D receptors. This conclusion would suggest that one of the regions identified by Kuner & Schoepfer (1996) to be responsible for NR2 subunit-dependence of inhibition by Mg2+o contributes to the energy barrier between the Mg2+o binding site and the internal solution.
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) and NR1/2D (
) receptor currents at the indicated voltages. C, Mg2+o IC50 values measured from –115 to –15 mV plotted for NR1/2A (
) are plotted as a function of voltage. Data for cortical receptors were estimated by linear interpolation of data from