Neuron,
Vol. 8, 181-187, January, 1992, Copyright
0 1992 by Cell Press
Slow VoltageDependent Changes in Channel Open-State Probability Underlie Hysteresis of NMDA Responses in Mg*+-Free Solutions Linda M. Nowak and Jerry M. Wright* Department of Pharmacology College of Veterinary Medicine Cornell University Ithaca, New York 14853
Summary Many single-channel studies rely on the assumption that the channels are functioning under steady-state conditions. In examining the basis for nonlinear whole-cell current-voltage curves in Mgz+-free solutions we discovered that N-methyl-o-aspartate (NMDA) channels in excised patches reversibly shifted their open-state prob ability (P,,) in a voltagedependent way, exhibiting approximately 3-to 4-fold greater POat positive potentials than at rest. Changes in P, were mainly attributable to shifts in frequency of channel opening. P,,changed remarkably slowly (2-15 min), explaining the hysteresis of whole cell current-voltage curves obtained in nonequilibrium conditions. The slow increase in P. provides a mechanism by which NMDA channels can substantially increase Ca*+ influx in cells depolarized for prolonged periods of time and may play a role in excitotoxicity. Introduction The N-methyl-o-aspartate (NMDA) receptor is directly coupled to a nonselective cation channel permeated by small monovalent cations (Na+, K+, and Cs’) (Nowak et al., 1984; Ascher et al., 1988) and Ca*+ (MacDermott et al., 1986; Mayer and Westbrook, 1987; Jahr and Stevens, 1987; Ascher and Nowak, 1988; lino et al., 1990). In physiological solutions the voltagedependent blockade of NMDA channels by extracellular Mg2+ results in a marked decrease of whole-cell current at negative voltages (Nowak et al., 1984; Mayer et al., 1984; Mayer and Westbrook, 1985; Jahr and Stevens, 1990). By contrast, the relatively small degree of voltage dependence observed for the whole-cell NMDA current-voltage (I-V) curves in nominally Mg2+-free solutions has received little attention. The degreeof nonlinearity in whole-cell NMDA I-Vcurves that can be observed in published data varies from report to report, and this rectification of NMDA I-V curves is generally not specifically mentioned. The nonlinearity of the I-V curves may be partially explained by the recording conditions in the experiments. For example, elevated extracellular Ca*+ concentration decreases NMDA single-channel currents at negative potentials (Ascher and Nowak, 1988), and thus extracellular Cati can contribute to the rectifica*Present address: The National Alcoholism, 12501 Washington 20852.
Institute on Alcohol Avenue, Rockville,
Abuse and Maryland
tion of NMDA responses in cells in some cases (lino et al., 1990). In addition, the presence of residual Mg*+ may also be responsible for NMDA responses being somewhat smaller at negative potentials in othercases (Mayer and Westbrook, 1985). However, preliminary reports on single NMDA channels in excised patches (Strecker and Jackson, 1990; J. M. W. and L. M. N., unpublished data) and NMDA receptor-mediated postsynaptic currents in cells (Konnerth, et al., 1990) suggested an intrinsic voltage dependence of NMDA channel open-state probability (Pd. For the experiments in this report, the voltage dependence of NMDA responses was investigated in conventional whole-cell recordings and in single-channel recordings in nominally Mg2+-free solutions, and the time course of the changes in P, that underlies the voltage dependence was examined. Results NMDA whole-cell I-V curves shown in Figure 1 illustrate data obtained from two neurons using experimental protocols that differ only in the intervals over which measurements were made following voltage changes. They also represent the two conditions investigated by single-channel recordings in this study, namely the rectification of macroscopic NMDA I-V curves in Mg2+-free solutions recorded in steady-state conditions and an apparent hysteresis in the NMDA I-V curves observed when responses were measured prior to reaching equilibrium, The difference between the curve shown in Figure IAand FigurelBisthat inFigure1Athemeasurements were taken 2-4 min after each membrane potential change, while the curves in Figure IB were taken 3050 s after the membrane potential change. In the protocol allowing the 2-4 min waiting period, the I-V curve obtained between -80 to +60 mV was similar to the one obtained from +60 to -80 mV. Thus, the NMDA I-V curve plotted in Figure IA is the average of the two sets of measurements. The data obtained at shorter intervals (25-50 s) produced different I-V curves in each direction (in three of three cells), with a nearly linear I-V curve observed as the potential was stepped from -80 mV to +60 mV and a nonlinear I-V curve from +60 to -80 mV (Figure IB). For the nonlinear curve, taken after the cell had spent several minutes at depolarized potentials, larger NMDA currents were obtained at all potentials except at -80 mV where thetwo curves converged (Figure 18). The short interval protocol used to demonstrate the hysteresis shown in Figure IB was adopted following singlechannel studiesexaminingthetimecourseofthevoltage dependence of the shift in P, (see below). In summary, whole-cell I-V curves taken under steady-state conditions (Figure 1A) indicate the NMDA current is clearly smaller at negative potentials than at positive
A
400-r
Figure 1. Whole-Cell NMDA I-V Curves Exhibit Outward Rectification and a Hysteresis
pA
0 from
In both examples NMDA I-V curves were constructed in the following sequence: measuring whole-cell currents in the absence of NMDA (control I-V curves), repeating the procedure in the continuous presence of 20 PM NMDA plus 1 PM glytine to obtain a combined I-V curve, and subtracting the appropriate control curves from the total I-V curves to obtain the NMDA I-V curves displayed. Membrane potential waschanged in 20 mV increments between -80 and f60 mV and back again from f60 to -80 mV, thus obtaining two sets of control and NMDA I-Vcurves from each ceil. neuron shows rectification at negative potentials. The ratio of inward to obtained from measurements at each membrane potential made 2-4 min intervals at negative voltages. The range of values were within the symbol The higher values obtained at +40 and f60 mV were measured during the
+60
-- -200 -- -300
(A) The average I-V curve obtained from a small multipolar outward current at -60 and +60 mV is 1:1.7. The curve was following each 20 mV change in potential, with the longest except at +40 and +60 mV where it is shown by error bars. return from +68 toward -80 mV. (B) In a second larger multipolar neuron, two different I-V curves were obtained: the first during potential changes from -80 to +I% potential held mV (open squares) and the second during potential changes from +60 to -80 mV (closed circles), with each membrane for only 30-68 s before the next voltage step.
potentials.
In contrast,
ing equilibrium depending was
-80
tials,
and
age
steps.
The
(Figure upon
mV or f60 the
basis
length for
the
I-V
curves
taken
IB)
may
be linear
whether mV,
the the
of the nonlinearity
time time
initial spent interval
before
reach-
or nonlinear
holding at these between
in steady-state
voltage potenvoltwhole-
I-V curves was investigated by examining P, of NMDA channels in outside-out patches at different potentials. Three parameters of single-channel activity were measured, including channel amplitude (in PA), open time (G, in ms), and frequency of opening (events.min-I), to determine the influence of each on the rectification. In multichannel patches and whole cells the total open-state probability is the product of the number (N) of channels and the P, for each channel. When P, is likely to be considerably less than 0.5, it is generally not feasible to determine either the value of N or P, unambiguously (Sachs et al., 1982). Thus, our data are presented in terms of the product NP,. Results from a patch containing occasional openings to a fourth level (i.e., N 2 4) are shown in Figure 2 for dataobtained between -1OOand +50 mV. The raw data traces in Figure 2A illustrate that greater channel activity at +40 mV than at -60 mV very likely contributes to the increase in current observed at positive potentials. As reported previously (Now!ak et al., 1984; Ascher et al., 1988; Ascher and Nowak, 1988; CullCandy and Usowicz, 1987; Jahr and Stevens, 1987) single NMDA channel current amplitude is a linear function of membrane potential with a larger absolute current measured at -60 mV compared with +40 mV (Figure 2B). There is also a small contribution from mean channel G with z0 having slightly longer durations at positive potentials. The difference in z,, with cell
respect to potential (rO = 7.2 ms at +40 mV; z, = 4.3 ms at -60 mV) was more extreme for the patch in Figure 2 than was typically observed, but it serves to indicate that changes in rO, as well as frequency of opening, may contribute to the larger average currentsobserved at positive potentials (parameters compared in Figure 3). Thus, increases in frequency of opening and z, both contribute to the larger average current through NMDA channels at +40 mV compared with -60 mV, with the average current versus voltage curve from this patch (Figure 2C) resembling the nonlinear steady-statewhole-cell I-Vcurve shown in Figure IA. These data indicate that the nonlinear I-V curves seen in whole-cell recordings at equilibrium can arise from single-channel behavior. The relative contributions of z,, and frequency of opening to NP, at different potentials in 12 patches are shown in Figure 3. Data were normalized to the values obtained at -60 mVfor each patch before combining them. As shown in Figure 3A, there was a small increase in z0 above -60 mV and a larger decrease in z,, at more negative potentials. Below -60 mV the influence of -iO on the overall NP, is apparent. From about -30 mV to more positive potentials, the increase in event frequency made the larger contribution to the increase in NP, (Figure 3B), since r0 did not change in this voltage range. Data obtained from an additional 5 patches were consistent with this picture, but were not included due to lack of data at -60 mV, which was used for normalizing values within patches. In Figure 3C, NP, values seem to increase steadily from -100 to +60 mV, although in some patches NP, appeared to approach a plateau at positive potentials. Previous studies have suggested NMDA channel P, is low (Heuttner and Bean, 1988; Traynelis and Cull-
NMDA 183
Channel
Open
Probability
Varies
with
Voltage
A +40 m V
02-D 01-b C--W
-BO m V
B Channel
C
Amplitude
Patch Current
(PA) 6.0
4.0
Potential -100
-60
t --
(mV)
Potential
-60
I
I
I
20
40
60
(mV) 20
40
60
---4.0 -0.6
t
-r-6.0
Figure 2. NMDA Channels in Outside-Out
Patches Exhibit
Voltage-Dependent
Behavior
(A) Singlechannel records in IO u M NMDA plus 1 p M glycine are shown at +40 and -60 m V after reaching steady-state open probability at each voltage. Inspection of the record indicated a voltage-dependent change in the frequency of opening. Upper trace: 1860 events.min-’ at +40 mV; bottom trace: 1260 events.min-’ at -60 mV. (B) The singlechannel current amplitude versus voltage (l-v) plot was linear with a slope conductance of 55 pS. (C) Average current through the patch (I in PA), obtained by dividing the total charge through the patch per min by 60 s, is plotted as a function of membrane potential to give the average patch I-V curve shown.
Candy, 1990); however, it should depend upon agonist concentration. W e attempted to estimate P, and the effect of NMDA concentration on P, by assuming that N equaled the number of open levels seen in patches. At -60 mV, estimated channel P, values were 0.024 f 0.004 in 10 PM NMDA (n = 3), 0.042 f 0.019 in 20 ~.LMNMDA (n = 4), and 0.077 in 40 PM NMDA (n = 1). None of these 8 patches were made with ATP-containing pipettes. Dose dependence of P, was examined in 1 patch made with an ATP-containing pipette where it was 0.007 in 2 PM NMDA, 0.010 in 4 PM NMDA, and 0.050 in 10 PM NMDA. These P, values are similar to those reported by Traynelis and Cull-Candy (1990), but higher than those reported by Huettner and Bean (1988) by another method. During the course of these studies we were concerned that a decrease in channel activity might signify either a slow desensitization or a “rundown” effect (MacDonald et al., 1989), rather than a true voltage dependence in NP,. To accomodate these possibilities, recordings were initiated at -60 mV, proceeding afterward to more positive voltages at which NP, increases. Also, the patches were allowed to stabilize
for between 2 and 10 min after initial application of NMDA before sampling. In several experiments in which data acquisition was repeated at -60 mV, it was obvious that if a patch had been held at one extreme potential or another (e.g., +40 or -100 mV), the NMDA channels continued to retain the NP, of the previous potential for relatively long periods of time before returning to previous activity levels at -60 mV. Preliminary experiments were conducted in patches (n = 5) to investigate the time dependence of the shift in NP,overaIimitedvoltagerange. Inthepatchshown in Figure 4, a series of 12 changes in holding potential were performed in the range of 0 to -80 mV. It was observed that holding the patch at 0 mV for 2 min increased NP, at -60 mV by 5 0 % and that NP, did not return to control levels for 4-5 min following repolarization. For the data presented in Figure 4,, patch potential was held at -30 mV, -60 mV, and -80 mV in recordings lasting between IO and 25 min at each potential. Complete reversal of theeffect of the previous holding voltage on the NP, was achieved at all three potentials. Figure4showsstripchart recordsof NMDAchannel
A
Relative
20
.
2l O-1
B
,
,
[
,
,
,
,
,
,
-100
-80
-60
-40
-20
0
20
40
60
Relative 4
Frequency
1
I I 1
O-J
,
,
,
,
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,
,
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,
-100
-80
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-20
0
20
40
60
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I
-100
I
-80
NPo
I
-60
I
-40
1
-20
Potential
I
8
I
0
20
40
I
60
(mV)
Figure 3. Changes in ~,and Frequency of Events Occur at Different Potentials and Contribute to the Voltage Dependence of NP, (A) 70 increased by 18% between -60 mV and positive potentials. Average values (* SD) obtained were 5.4 + 0.8 ms (n = 12) at -60 mV; 6.0 * 1.1 ms (n = 7) at -40 mV; and 6.6 f 1.4 ms (n = 11) at positive potentials. 5, did not exhibit marked voltage dependence above -60 mV, but it was considerably shorter at -100 mV (2.2 ms) than at -60 mV. The curve was hand drawn. (8) The frequency of events increased in a voltagedependent manner above -60 mV, although this was not generally obvious until -20 mV. In individual patches the increase in event frequency appeared to reach a plateau at positive potentials. Below -80 mV the frequency of opening increased again in some patches,consistentwith thepresenceof residual Mg2+.Thecurve was hand drawn. (C) NP,, the product of mean z, and event frequency, appears to increase almost steadily from -100 to l-60 mV. In individual patches the increase in NP, appeared to reach a plateau at positivepotentials.Thecurvewasdrawnasacompositeofthecurves in (A) and (B). Measurements (n = 45) for (A), (B), and (0 were taken in 12 patches. The error is shown as the standard deviation and in some instances is within the symbol. Exceptions are at -90 mV, -70 mV, and -30 mV, where symbols represent single determinations. ro, frequency of opening, and the NP, values obtained at each potential were normalized to the values obtained at -60 mV (open squares) for each patch before they were combined. High variability at positive potentials was partially due to our initial lack of recognition that 10 min or more may be required for NMDA channel activity to reach equilibrium at these poten-
activity at equilibrium (Figure 4A) for -30 and -80 mV and also for the 100 s interval following the changes from -30 to -80 mV and from -80 to -30 mV (Figure 4B). At equilibrium, NP, was nearly 2 times greater at -30 mV than at -80 mV. However, in the first minute after changing the potential from -30 to -80 mV, NP, at -80 mV was nearly twice the equilibrium value. Conversely, in the first minute after changing the potential from its equilibrated state at -80 mV, to -30 mV, NP, was one-half its -30 mV equilibrium value. In all instances z, appeared to change rapidly, reaching equilibrium values within 1 min. By contrast, the component of NP, associated with the frequency of events shifted slowly. The effect of changing NP, values on the average current through the patch is shown in Figure 4C at equilibrium for -30 mV, -60 mV, and -80 mV and following voltage perturbations. At equilibrium the average patch current rectifies at negative potentials and resembles the whole-cell recording in Figure IA and the average patch I-V in Figure 2C. The time dependence of the change in NP, following a voltage step is intriguing. For channels recorded in the first minute following a voltage perturbation from an earlier holding potential, NP, remains at or close to the value observed at the previous potential. This indicated that the use of a ramp voltage stimulus would produce linear whole-cell I-V curves, whereas the steady-state I-V curve would be nonlinear as illustrated in Figure I. The time course of the shift in NP, was measured in the patch shown in Figure 4 for overlapping 60 s intervals beginning at the onset of each voltage change. The difference in NP, between -30 and -60 mV was not large (0.034 at -30 mV versus 0.025 at -60 mV), thus making this region difficult to study using single-channel data. In contrast, for voltage shifts between -80 and -30 mV, the time course could be followed adequately as shown in Figure 4D. In Figure 4Dl the patch reached its equilibrium NP, at -80 mV within 2-3 min after stepping from -30 mV. In contrast, it took 12-14 min for this patch to regain its -30 mvequilibrium NP,valueafterhaving beenat-80mV (Figure 4D2). The slow time course of the P, change observed going from -80 to -30 mV is consistent with the nearly linear I-V curves obtained in whole-cell recordings between -80 and +60 mV illustrated in Figure 18. The more rapidly occurring shift in NP, seen in the single-channel data from -30 to -80 mV was also consistent with observations in whole-cell recordings in this voltage range, giving rise to the rectification seen in whole-cell I-Vcurves as shown in Figure IB and the convergence of the -80 mV current values. tials, the intrinsic problem of normalizing data from patches containing an unknown N, and the smaller number of patches represented (n = 3 for most points) at positive potentials. NMDA (2-40 PM) was coapplied with 1 pM glycine. Three of the 12 patches were made with ATP-containing pipettes. Resting membrane potentials were between -56 and -87 mV.
NMDA Channel 185
Al
Open Probability
equilibrium
Varies with Voltage
at -30
A2
mv
NPo = 0.034
@-SO
after -8OmV
NPo = 0.016
C4 04 04
BI
equilibrium
at -80
B2 (~~40after-30mV
mV
NPo = 0.017
NPo = 0.029
-80
y...‘+
C-b + if
04
./’
I equilibrium
./’
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-80
0 after
-30
-120
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15 s
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after -3OmV
o
80
it E
60
8 tJ a 20,
a-30
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40 207,
0
2
4
Elapsed
6
8
Time
Figure 4. Time- and Voltage-Dependent pletely Reversible
10
12
0
(min) Changes
2
4
6
Elapsed in NMDA
Channel
Open-State
8
Time
Probability
10
12
14
(min)
Observed
in Excised Patches Were Com-
NMDA (2 uM NMDA plus 1 uM glycine) channel data recorded from 1 patch using an ATP-containing (4 m M Mg-ATP) pipette were collected in the following sequence: (Al) events recorded in the 12-13 min interval at -30 mV (equilibrium), (B2) events immediately after changing the potential to -80 mV, (Bl) events recorded in the 15-16 min interval at -80 mV (equilibrium), and (A2) events immediately after changing the potential to -30 mV. (A)At -3OmVequilibrium NP,was0.034. (Al) NP,,changed toa newequilibriumofO.O17at-80mV(A2). Fourteen minutesafter returning the patch to -30 mV from the -80 mV condition displayed and analyzed here, NP, was 0.033 (data not shown). (B) In the first minute followingvoltagetransitions NP, remained close to thevalueof the previous equilibrium (in Bl at-30 mV, stepped from -88 mV, NP,, was initially 0.016; in 82 at -80 mV, stepped from -30 mV, NP, was initially 0.029). (C)Average I-V data from the patch are shown in two conditions, at equilibrium (open squares), and for the first minute after a voltage transition (e.g., from -80 mV equilibrium [closed squares] to -30 mV [open squares], from -30 mV equilibrium [closed squares] to -80 mV [half closed squares]). Dashed lines with arrows indicate the sequence of voltage- and time-dependent changes. (D) The time course for the reestablishment of equilibrium NP, values is shown after transitions from -30 to -80 mV (Dl) and from -80 to -30 mV (D2). The equilibrium NP, at -80 mV was 0.017; at -30 mV it was 0.035, determined from 3 min segments of recordings to minimize the variability introduced by the normal bursting behavior of the channels. Data points in the graphs were obtained by counting the number of events and measuring r0 for adjacent and/or overlapping 68 s intervals in each record. Symbols are placed 38 s into the interval. Normal variability of NP, at equilibrium can be seen in Dl between the 4th and 12th min.
Otherfactorsthat mayinfluenceoverall POwerealso examined briefly with respect to the voltage dependence, but the time course of the NP, change was not typically examined. Including ATP in the patch pipette may have decreased the rate of rundown of NMDA channel
activity
(MacDonald
et al., 1989),
but
its pres-
ence did not alter the voltage dependence of P, (n = 5). Extracellulardithiothreitol(1 mM) increased overall P, compared with untreated patches (n = 3), as expected from whole-cell responses (Aizenman and Lipton, 1989). Although both 70 and frequency of events increased in 1 mM dithiothreitol, the voltage dependence of NP, was similar in the dithiothreitol-treated patches and untreated patches. Again, an increase in
event frequency made the greater contribution to the increase in P, at positive potentials. Having observed the slow changes in P, in excised membrane patches exposed to simple saline solutions, with and without ATP present, it appears that the phenomenon is closely associated with NMDA receptor channel proteins. Discussion
While not nearly so dramatic as the voltage dependence seen in the presence of extracellular Mg2+, there is nonetheless an obvious sensitivity of NMDA responses to membrane voltage in the nominally Mg2+-
NWKWl 186
free solutions. The slowness of the voltage-dependent change in NMDA channel P, was exploited to demonstrate a hysteresis in whole-cell NMDA I-Vcurves (Figure IB). Thus, the observation that NMDA currents had not reached asteady-statevalueat most potentials within 2 min, coupled with the observation that voltage-dependent changes of currents in the control I-V curves had reached equilibrium within 20 s, led ustouse25-60sintervalsforvoltagechangesin Figure IB. Such slow changes in P, have important implications for interpretation of NMDA channel kinetics studies and for determination of the voltage sensitivityof association and dissociation rates of slow NMDA channel blockers like MK-801 (MacDonald and Nowak, 1990). The voltage dependence of P, is most remarkable because its slow time dependence gives the impression of a system with memory. Because much of the change in NP, occurs between -60 and f40 mV and its time course is slow, this phenomenon cannot be explained by a rapid block of channels by residual Mg 2+. In contrast, at -90 and -100 mV we did obtain evidence for the presence of residual Mg2+ in some recordings: openings were grouped in bursts containing brief closures (rc = 1.1 ms), and z,, was decreased. The apparent number of events also increased as predicted if the current through open channels was being interrupted by Mg2+. Together, these results suggest that a block by residual Mg2+ in the nominally Mg2+-free solution is a possible explanation for an outward rectification at large negative potentials. This result was not readily apparent in all patches. In our experiments the nominally”Mg*+-free” extracellular solutions typically contained I-2 PM Mg2+, as measured by atomic absorption spectroscopy (Wright et al., 1991). Further study is needed to elucidate the mechanism(s) underlying the voltage-dependent changes in NMDA channel P,. The increase in P, with depolarization occurs at considerably lower rates than would be anticipated for a rearrangement of dipoles in the protein(s), or the association/dissociation of ions, which might influencechannel gating, unless the process is linked to a particular state of the channel. For example, if the process underlyingchanging P,occurs in association with channel opening, it would be slow because NMDA channels open infrequently. In addition, if an NMDA channel must open in order to reset its activity level, it would take longer if the membrane is depolarized following a long period of hyperpolarization because the channel opening rates at negative potentials are lower. The process would be faster for hyperpolarization after a prolonged period at positive potentials because the channel activity would be equilibrated to a higher level. Alternatively, the slow increase in P, with depolarization may arise from a decrease in desensitization (Clark et al., 1990). Due to the relatively high permeability of NMDA channels to Ca2+(MacDermott et al., 1986; Mayer and Westbrook, 1987) and the link between Ca2+ entry and
excitotoxicity (Choi, 1987; Meldrum and Garthwaite, 1990), tight control of NMDA receptor channel activity appears vital for normal brain function. The slow increase in NP, was observed clearly at -30 mV, and the influence of holding patches at 0 mV was observed as an increase in NMDA channel activity that persisted for 4-5 min upon repolarization to -60 mV (n = 2 patches). Increasing NMDA channel activity in this potential range is important since glutamatergic synaptic activity tends to depolarize cells to near 0 mVand the increase in P, is likely to foster hyperexcitability in cells that are not voltage clamped. Thus, in contrast to voltage-gated Caz+channels, which exhibit voltagedependent and Ca2+-dependent inactivation with depolarization, NMDA channel activity is likely to increase following prolonged depolarization. The slow rise in NMDA channel open probability following sustained depolarization would result in a much higher degree of Ca*+ loading than would occur if NP, did not increase by between 2-and 3-fold near 0 mV, compared with NP, at resting membrane potential. Thus, persistent depolarization of cells in vivo by NMDA agonists leading to the increase in NMDA channel activity through the slow voltage-dependent increase in P, is likely to contribute to neurodegeneration. In several reports it appears that NMDA-induced excitotoxicity requires IO-15 min exposures to NMDA in the presence of extracellular Ca2+ to obtain a reliably high percentage of cell death in many preparations (Meldrum and Garthwaite, 1990); however, Hartley and Choi (1989) have observed that a majority of mouse cortical neurons in culture were killed by 3-5 min exposures to high concentrations of NMDA. It is difficult to compare the time scale of the changes in P, observed in our experiments with the time course of excitotoxic damage because low doses of NMDA (2-5 GM) were used for the P, studies. Thus, additional experiments directed at testing the effect of agonist concentration on the rate of change of P,, as well as other factors that influence overall P,, will be necessary in isolated patches and in intact cells in order to determine whether changes in P, play a role in regulating the physiological state of neurons in vivo.
General Methods Mouse forebrain neurons were prepared from 15 to I7-day old fetuses as described previously (Ascher et al., 1988). Recordings were performed at 20°C-25°C on neurons in culture for 4-43 days. Whole-cell and single-channel NMDA current responses were recorded in the presence of 1 u M glycine (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988) with a List EPC-7 patch-clamp amplifier using borosilicate glass patch pipettes (3-7MD; WPI TW150) containing the following: 140 m M CsCI, 10 m M K-EGTA/l m M CaCI,, and 10 m M K-HEPES (pH 7.2). NaNO, (75 mM) or 70 m M Cs-acetate was sometimes substituted for the equivalent amount of CsCl in intracellular solutions without any effect on NMDA channel 7, or conductance (Ascher et al., 1988; Wright et al., 1991). Mg-ATP (2-4 mM)was included in the pipette solutions for some experiments. Extracellular (bath) solutions contained the following: 150 m M NaCI, 2.8 m M KCI, 1.0 m M
NMDA 187
Channel
Open Probability
Varies with Voltage
CaCb, 10 m M Na-HEPES (pH 7.2) with 300 nM tetrodotoxin and 1 J&I glycine. NMDA (Cambridge Research Biochemicals), dithiothreitol (Sigma), and glycine (Sigma) were dissolved in the bath solution and applied by rapid local perfusion directed at the patch pipette through a large bore Pasteur pipette. Total bath solution was continually exchanged by slow perfusion of the 35 m m culturedish. Recordings were initially filtered (4 kHz; apole Bessel), digitized (44 kHz; Medical Systems, PCM-IB), and stored on cassettes by video (Sony Beta SL HF450) for later analysis. Single-Channel Methods Singlechannel data were reconverted to analog form, filtered at 2 kHz (Bessel), sampled at 10 kHz (Axolab, Axon Instruments), and analyzed on personal computers (AST Premium 286) using the CAP software (R. C. Electronics; Goleta, CA) and additional software developed in the laboratory (Wright et al., 1991). Channel ~~ was determined from histograms generated from events lists obtained from single channels recorded in patches where the channel openings to a second level represented less than 10% of openings in the list. In a few patches with many openings to a second level, open times were estimated from events lists obtained by the method of Jackson (1985). Open durations were fitted by the maximum likelihood method from unbinned data as described by Colquhoun and Sigworth (1983). Amplitude histograms were constructed as probability density functions of open NMDA channels using all of the points sampled in each segment of recording that was analyzed. Single-channel current amplitude was obtained by fitting a Gaussian function to the peak representing single-level openings. Additional Guassian functions were fitted to the amplitude peaks representing openings to second, third, and fourth levels as necessary to obtain the total charge per min (in pC.mirt-‘) by integrating the area under all of the Gaussian functions. The average current was determined by measuring total charge per minute from amplitude probability density functions standardized to represent 60 s of recordingand dividingthechargepermin by60sasdescribed previously(Wrightetal., 1991).This measureoftheaverage patch current determined at different potentials was used in constructing the I-V curves in Figure 2C and Figure 4C.
analysisof single-channel records. In Single-Channel E. Neher and B. Sakmann, eds. (New York: Plenum Corp.), pp. 191-263.
Recording, Publishing
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P. (1987). Glycine potentiates mouse brain neurons. Nature
the 325,
Kleckner, N. W., and Dingledine, R. (1988). Requirement for glytine in activation of NMDA-receptors expressed in Xenopus oocytes. Science 241,835-837. Konnerth, A., Keller, B. U., Ballanyi, K., and Yaari, Y. (1990). Voltage sensitivity of NMDA-receptor mediated postsynaptic currents. Exp. Brain Res. 87, 209-212. MacDermott, A. B., Mayer, M. L., Westbrook, G. L., Smith, S. J., and Barker, J. L. (1986). NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones. Nature 327, 519-522. MacDonald, J. F., and Nowak, L. M. (1990). Mechanisms of blockade of excitatory amino acid receptor channels. Trends Pharmacol. Sci. 77, 167-172.
We thank Philippe Ascher for his comments on an earlierversion of the manuscript, Paul A. Kline for writing the computer software, and Valerie S. Engle and Cornelia Poulopoulou for cell cultures. This project was supported by National Institutes of Health grant #NS24l67. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertiseme& in accordance with 18 USC Section 1734 solely to indicate this fact. Received
August
15, 1991; revised
October
30, 1991.
Aizenman, E., Lipton, S. A., and Loring, R. H. (1989). Selective modulation of NMDA responses by reduction and oxidation. Neuron 2, 1257-1263. cations in neurones
Ascher, P., Bregestovski, P., and Nowak, L. (1988). N-methyl-oaspartateactivated channels of mouse central neurones in magnesium-free solutions. j. Physiol. 399, 207-226. Choi, D. W. (1987). Ionic dependenceof J. Neurosci. 7, 369-379.
glutamate
neurotoxicity.
Clark, G. D., Clifford, D. B., and Zorumski, C. F. (1990). Theeffect of agonist concentration, membrane voltage and calcium on N-methyl-o-aspartate receptor desensitization. Neuroscience39, 787-797. Colquhoun,
D., and Sigworth,
F. (1983). Fitting
Mayer, M. L., and Westbrook, C. L. (1985). The action of N-methylo-aspartic acid on mouse spinal neurones in culture. J. Physiol. 367, 65-90. Mayer, M. L., and Westbrook, G. L. (1987). Permeabon and block of N-methyl-o-asparticacid receptor channels bydivalent cations in mouse cultured central neurones. J. Physiol. 394, 501-527. Mayer, M. L., Westbrook, G. L., and Guthrie, P. B. (1984).Voltage dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature 309, 261-263.
References
Ascher, P., and Nowak, L. (1988). The role of divalent the N-methyl-D-aspartate responses of mouse central in culture. J. Physiol. 399, 247-266.
MacDonald, J. F., Mody, I., and Salter, M. W. (1989). Regulation of N-methyl-o-aspartate receptors revealed by intracellular dialysis of murine neurons in culture. J. Physiol. 474, 17-34.
and statistical
Meldrum, B., and Garthwaite, J. (1990). Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11, 379-387. Nowak, L., Bregestovski, P., Ascher, P., Herbet, A., and Prochiantz, A. (198-I). Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307, 462-465. Sachs, F., Neil, J., and Barkakati, N. (1982).The automated analysis of data from single ionic channels. Pfliigers Arch. 395,331-340. Strecker, C. J., and Jackson, M. B. (1990). NMDA-activated channel currents in thin hippocampal slices. Biophys. J. 57, 121. Traynelis, S. F., and Cull-Candy, S. G. (1990). Proton inhibition of N-methyl-oaspartate receptors in cerebellar neurons. Nature 345, 347-350. Wright, J. M., Kline, P. A., and Nowak, L. M. (1991). Multiple effects of tetraethylammonium on N-methyl-o-aspartate recep tar-channels in mouse brain neurons in cell culture. J. Physiol. 439,579~604.
Vol. 8, 181-187, January, 1992, Copyright
0 1992 by Cell Press
Slow VoltageDependent Changes in Channel Open-State Probability Underlie Hysteresis of NMDA Responses in Mg*+-Free Solutions Linda M. Nowak and Jerry M. Wright* Department of Pharmacology College of Veterinary Medicine Cornell University Ithaca, New York 14853
Summary Many single-channel studies rely on the assumption that the channels are functioning under steady-state conditions. In examining the basis for nonlinear whole-cell current-voltage curves in Mgz+-free solutions we discovered that N-methyl-o-aspartate (NMDA) channels in excised patches reversibly shifted their open-state prob ability (P,,) in a voltagedependent way, exhibiting approximately 3-to 4-fold greater POat positive potentials than at rest. Changes in P, were mainly attributable to shifts in frequency of channel opening. P,,changed remarkably slowly (2-15 min), explaining the hysteresis of whole cell current-voltage curves obtained in nonequilibrium conditions. The slow increase in P. provides a mechanism by which NMDA channels can substantially increase Ca*+ influx in cells depolarized for prolonged periods of time and may play a role in excitotoxicity. Introduction The N-methyl-o-aspartate (NMDA) receptor is directly coupled to a nonselective cation channel permeated by small monovalent cations (Na+, K+, and Cs’) (Nowak et al., 1984; Ascher et al., 1988) and Ca*+ (MacDermott et al., 1986; Mayer and Westbrook, 1987; Jahr and Stevens, 1987; Ascher and Nowak, 1988; lino et al., 1990). In physiological solutions the voltagedependent blockade of NMDA channels by extracellular Mg2+ results in a marked decrease of whole-cell current at negative voltages (Nowak et al., 1984; Mayer et al., 1984; Mayer and Westbrook, 1985; Jahr and Stevens, 1990). By contrast, the relatively small degree of voltage dependence observed for the whole-cell NMDA current-voltage (I-V) curves in nominally Mg2+-free solutions has received little attention. The degreeof nonlinearity in whole-cell NMDA I-Vcurves that can be observed in published data varies from report to report, and this rectification of NMDA I-V curves is generally not specifically mentioned. The nonlinearity of the I-V curves may be partially explained by the recording conditions in the experiments. For example, elevated extracellular Ca*+ concentration decreases NMDA single-channel currents at negative potentials (Ascher and Nowak, 1988), and thus extracellular Cati can contribute to the rectifica*Present address: The National Alcoholism, 12501 Washington 20852.
Institute on Alcohol Avenue, Rockville,
Abuse and Maryland
tion of NMDA responses in cells in some cases (lino et al., 1990). In addition, the presence of residual Mg*+ may also be responsible for NMDA responses being somewhat smaller at negative potentials in othercases (Mayer and Westbrook, 1985). However, preliminary reports on single NMDA channels in excised patches (Strecker and Jackson, 1990; J. M. W. and L. M. N., unpublished data) and NMDA receptor-mediated postsynaptic currents in cells (Konnerth, et al., 1990) suggested an intrinsic voltage dependence of NMDA channel open-state probability (Pd. For the experiments in this report, the voltage dependence of NMDA responses was investigated in conventional whole-cell recordings and in single-channel recordings in nominally Mg2+-free solutions, and the time course of the changes in P, that underlies the voltage dependence was examined. Results NMDA whole-cell I-V curves shown in Figure 1 illustrate data obtained from two neurons using experimental protocols that differ only in the intervals over which measurements were made following voltage changes. They also represent the two conditions investigated by single-channel recordings in this study, namely the rectification of macroscopic NMDA I-V curves in Mg2+-free solutions recorded in steady-state conditions and an apparent hysteresis in the NMDA I-V curves observed when responses were measured prior to reaching equilibrium, The difference between the curve shown in Figure IAand FigurelBisthat inFigure1Athemeasurements were taken 2-4 min after each membrane potential change, while the curves in Figure IB were taken 3050 s after the membrane potential change. In the protocol allowing the 2-4 min waiting period, the I-V curve obtained between -80 to +60 mV was similar to the one obtained from +60 to -80 mV. Thus, the NMDA I-V curve plotted in Figure IA is the average of the two sets of measurements. The data obtained at shorter intervals (25-50 s) produced different I-V curves in each direction (in three of three cells), with a nearly linear I-V curve observed as the potential was stepped from -80 mV to +60 mV and a nonlinear I-V curve from +60 to -80 mV (Figure IB). For the nonlinear curve, taken after the cell had spent several minutes at depolarized potentials, larger NMDA currents were obtained at all potentials except at -80 mV where thetwo curves converged (Figure 18). The short interval protocol used to demonstrate the hysteresis shown in Figure IB was adopted following singlechannel studiesexaminingthetimecourseofthevoltage dependence of the shift in P, (see below). In summary, whole-cell I-V curves taken under steady-state conditions (Figure 1A) indicate the NMDA current is clearly smaller at negative potentials than at positive
A
400-r
Figure 1. Whole-Cell NMDA I-V Curves Exhibit Outward Rectification and a Hysteresis
pA
0 from
In both examples NMDA I-V curves were constructed in the following sequence: measuring whole-cell currents in the absence of NMDA (control I-V curves), repeating the procedure in the continuous presence of 20 PM NMDA plus 1 PM glytine to obtain a combined I-V curve, and subtracting the appropriate control curves from the total I-V curves to obtain the NMDA I-V curves displayed. Membrane potential waschanged in 20 mV increments between -80 and f60 mV and back again from f60 to -80 mV, thus obtaining two sets of control and NMDA I-Vcurves from each ceil. neuron shows rectification at negative potentials. The ratio of inward to obtained from measurements at each membrane potential made 2-4 min intervals at negative voltages. The range of values were within the symbol The higher values obtained at +40 and f60 mV were measured during the
+60
-- -200 -- -300
(A) The average I-V curve obtained from a small multipolar outward current at -60 and +60 mV is 1:1.7. The curve was following each 20 mV change in potential, with the longest except at +40 and +60 mV where it is shown by error bars. return from +68 toward -80 mV. (B) In a second larger multipolar neuron, two different I-V curves were obtained: the first during potential changes from -80 to +I% potential held mV (open squares) and the second during potential changes from +60 to -80 mV (closed circles), with each membrane for only 30-68 s before the next voltage step.
potentials.
In contrast,
ing equilibrium depending was
-80
tials,
and
age
steps.
The
(Figure upon
mV or f60 the
basis
length for
the
I-V
curves
taken
IB)
may
be linear
whether mV,
the the
of the nonlinearity
time time
initial spent interval
before
reach-
or nonlinear
holding at these between
in steady-state
voltage potenvoltwhole-
I-V curves was investigated by examining P, of NMDA channels in outside-out patches at different potentials. Three parameters of single-channel activity were measured, including channel amplitude (in PA), open time (G, in ms), and frequency of opening (events.min-I), to determine the influence of each on the rectification. In multichannel patches and whole cells the total open-state probability is the product of the number (N) of channels and the P, for each channel. When P, is likely to be considerably less than 0.5, it is generally not feasible to determine either the value of N or P, unambiguously (Sachs et al., 1982). Thus, our data are presented in terms of the product NP,. Results from a patch containing occasional openings to a fourth level (i.e., N 2 4) are shown in Figure 2 for dataobtained between -1OOand +50 mV. The raw data traces in Figure 2A illustrate that greater channel activity at +40 mV than at -60 mV very likely contributes to the increase in current observed at positive potentials. As reported previously (Now!ak et al., 1984; Ascher et al., 1988; Ascher and Nowak, 1988; CullCandy and Usowicz, 1987; Jahr and Stevens, 1987) single NMDA channel current amplitude is a linear function of membrane potential with a larger absolute current measured at -60 mV compared with +40 mV (Figure 2B). There is also a small contribution from mean channel G with z0 having slightly longer durations at positive potentials. The difference in z,, with cell
respect to potential (rO = 7.2 ms at +40 mV; z, = 4.3 ms at -60 mV) was more extreme for the patch in Figure 2 than was typically observed, but it serves to indicate that changes in rO, as well as frequency of opening, may contribute to the larger average currentsobserved at positive potentials (parameters compared in Figure 3). Thus, increases in frequency of opening and z, both contribute to the larger average current through NMDA channels at +40 mV compared with -60 mV, with the average current versus voltage curve from this patch (Figure 2C) resembling the nonlinear steady-statewhole-cell I-Vcurve shown in Figure IA. These data indicate that the nonlinear I-V curves seen in whole-cell recordings at equilibrium can arise from single-channel behavior. The relative contributions of z,, and frequency of opening to NP, at different potentials in 12 patches are shown in Figure 3. Data were normalized to the values obtained at -60 mVfor each patch before combining them. As shown in Figure 3A, there was a small increase in z0 above -60 mV and a larger decrease in z,, at more negative potentials. Below -60 mV the influence of -iO on the overall NP, is apparent. From about -30 mV to more positive potentials, the increase in event frequency made the larger contribution to the increase in NP, (Figure 3B), since r0 did not change in this voltage range. Data obtained from an additional 5 patches were consistent with this picture, but were not included due to lack of data at -60 mV, which was used for normalizing values within patches. In Figure 3C, NP, values seem to increase steadily from -100 to +60 mV, although in some patches NP, appeared to approach a plateau at positive potentials. Previous studies have suggested NMDA channel P, is low (Heuttner and Bean, 1988; Traynelis and Cull-
NMDA 183
Channel
Open
Probability
Varies
with
Voltage
A +40 m V
02-D 01-b C--W
-BO m V
B Channel
C
Amplitude
Patch Current
(PA) 6.0
4.0
Potential -100
-60
t --
(mV)
Potential
-60
I
I
I
20
40
60
(mV) 20
40
60
---4.0 -0.6
t
-r-6.0
Figure 2. NMDA Channels in Outside-Out
Patches Exhibit
Voltage-Dependent
Behavior
(A) Singlechannel records in IO u M NMDA plus 1 p M glycine are shown at +40 and -60 m V after reaching steady-state open probability at each voltage. Inspection of the record indicated a voltage-dependent change in the frequency of opening. Upper trace: 1860 events.min-’ at +40 mV; bottom trace: 1260 events.min-’ at -60 mV. (B) The singlechannel current amplitude versus voltage (l-v) plot was linear with a slope conductance of 55 pS. (C) Average current through the patch (I in PA), obtained by dividing the total charge through the patch per min by 60 s, is plotted as a function of membrane potential to give the average patch I-V curve shown.
Candy, 1990); however, it should depend upon agonist concentration. W e attempted to estimate P, and the effect of NMDA concentration on P, by assuming that N equaled the number of open levels seen in patches. At -60 mV, estimated channel P, values were 0.024 f 0.004 in 10 PM NMDA (n = 3), 0.042 f 0.019 in 20 ~.LMNMDA (n = 4), and 0.077 in 40 PM NMDA (n = 1). None of these 8 patches were made with ATP-containing pipettes. Dose dependence of P, was examined in 1 patch made with an ATP-containing pipette where it was 0.007 in 2 PM NMDA, 0.010 in 4 PM NMDA, and 0.050 in 10 PM NMDA. These P, values are similar to those reported by Traynelis and Cull-Candy (1990), but higher than those reported by Huettner and Bean (1988) by another method. During the course of these studies we were concerned that a decrease in channel activity might signify either a slow desensitization or a “rundown” effect (MacDonald et al., 1989), rather than a true voltage dependence in NP,. To accomodate these possibilities, recordings were initiated at -60 mV, proceeding afterward to more positive voltages at which NP, increases. Also, the patches were allowed to stabilize
for between 2 and 10 min after initial application of NMDA before sampling. In several experiments in which data acquisition was repeated at -60 mV, it was obvious that if a patch had been held at one extreme potential or another (e.g., +40 or -100 mV), the NMDA channels continued to retain the NP, of the previous potential for relatively long periods of time before returning to previous activity levels at -60 mV. Preliminary experiments were conducted in patches (n = 5) to investigate the time dependence of the shift in NP,overaIimitedvoltagerange. Inthepatchshown in Figure 4, a series of 12 changes in holding potential were performed in the range of 0 to -80 mV. It was observed that holding the patch at 0 mV for 2 min increased NP, at -60 mV by 5 0 % and that NP, did not return to control levels for 4-5 min following repolarization. For the data presented in Figure 4,, patch potential was held at -30 mV, -60 mV, and -80 mV in recordings lasting between IO and 25 min at each potential. Complete reversal of theeffect of the previous holding voltage on the NP, was achieved at all three potentials. Figure4showsstripchart recordsof NMDAchannel
A
Relative
20
.
2l O-1
B
,
,
[
,
,
,
,
,
,
-100
-80
-60
-40
-20
0
20
40
60
Relative 4
Frequency
1
I I 1
O-J
,
,
,
,
‘
,
,
,
,
-100
-80
-60
-40
-20
0
20
40
60
Relative
I
-100
I
-80
NPo
I
-60
I
-40
1
-20
Potential
I
8
I
0
20
40
I
60
(mV)
Figure 3. Changes in ~,and Frequency of Events Occur at Different Potentials and Contribute to the Voltage Dependence of NP, (A) 70 increased by 18% between -60 mV and positive potentials. Average values (* SD) obtained were 5.4 + 0.8 ms (n = 12) at -60 mV; 6.0 * 1.1 ms (n = 7) at -40 mV; and 6.6 f 1.4 ms (n = 11) at positive potentials. 5, did not exhibit marked voltage dependence above -60 mV, but it was considerably shorter at -100 mV (2.2 ms) than at -60 mV. The curve was hand drawn. (8) The frequency of events increased in a voltagedependent manner above -60 mV, although this was not generally obvious until -20 mV. In individual patches the increase in event frequency appeared to reach a plateau at positive potentials. Below -80 mV the frequency of opening increased again in some patches,consistentwith thepresenceof residual Mg2+.Thecurve was hand drawn. (C) NP,, the product of mean z, and event frequency, appears to increase almost steadily from -100 to l-60 mV. In individual patches the increase in NP, appeared to reach a plateau at positivepotentials.Thecurvewasdrawnasacompositeofthecurves in (A) and (B). Measurements (n = 45) for (A), (B), and (0 were taken in 12 patches. The error is shown as the standard deviation and in some instances is within the symbol. Exceptions are at -90 mV, -70 mV, and -30 mV, where symbols represent single determinations. ro, frequency of opening, and the NP, values obtained at each potential were normalized to the values obtained at -60 mV (open squares) for each patch before they were combined. High variability at positive potentials was partially due to our initial lack of recognition that 10 min or more may be required for NMDA channel activity to reach equilibrium at these poten-
activity at equilibrium (Figure 4A) for -30 and -80 mV and also for the 100 s interval following the changes from -30 to -80 mV and from -80 to -30 mV (Figure 4B). At equilibrium, NP, was nearly 2 times greater at -30 mV than at -80 mV. However, in the first minute after changing the potential from -30 to -80 mV, NP, at -80 mV was nearly twice the equilibrium value. Conversely, in the first minute after changing the potential from its equilibrated state at -80 mV, to -30 mV, NP, was one-half its -30 mV equilibrium value. In all instances z, appeared to change rapidly, reaching equilibrium values within 1 min. By contrast, the component of NP, associated with the frequency of events shifted slowly. The effect of changing NP, values on the average current through the patch is shown in Figure 4C at equilibrium for -30 mV, -60 mV, and -80 mV and following voltage perturbations. At equilibrium the average patch current rectifies at negative potentials and resembles the whole-cell recording in Figure IA and the average patch I-V in Figure 2C. The time dependence of the change in NP, following a voltage step is intriguing. For channels recorded in the first minute following a voltage perturbation from an earlier holding potential, NP, remains at or close to the value observed at the previous potential. This indicated that the use of a ramp voltage stimulus would produce linear whole-cell I-V curves, whereas the steady-state I-V curve would be nonlinear as illustrated in Figure I. The time course of the shift in NP, was measured in the patch shown in Figure 4 for overlapping 60 s intervals beginning at the onset of each voltage change. The difference in NP, between -30 and -60 mV was not large (0.034 at -30 mV versus 0.025 at -60 mV), thus making this region difficult to study using single-channel data. In contrast, for voltage shifts between -80 and -30 mV, the time course could be followed adequately as shown in Figure 4D. In Figure 4Dl the patch reached its equilibrium NP, at -80 mV within 2-3 min after stepping from -30 mV. In contrast, it took 12-14 min for this patch to regain its -30 mvequilibrium NP,valueafterhaving beenat-80mV (Figure 4D2). The slow time course of the P, change observed going from -80 to -30 mV is consistent with the nearly linear I-V curves obtained in whole-cell recordings between -80 and +60 mV illustrated in Figure 18. The more rapidly occurring shift in NP, seen in the single-channel data from -30 to -80 mV was also consistent with observations in whole-cell recordings in this voltage range, giving rise to the rectification seen in whole-cell I-Vcurves as shown in Figure IB and the convergence of the -80 mV current values. tials, the intrinsic problem of normalizing data from patches containing an unknown N, and the smaller number of patches represented (n = 3 for most points) at positive potentials. NMDA (2-40 PM) was coapplied with 1 pM glycine. Three of the 12 patches were made with ATP-containing pipettes. Resting membrane potentials were between -56 and -87 mV.
NMDA Channel 185
Al
Open Probability
equilibrium
Varies with Voltage
at -30
A2
mv
NPo = 0.034
@-SO
after -8OmV
NPo = 0.016
C4 04 04
BI
equilibrium
at -80
B2 (~~40after-30mV
mV
NPo = 0.017
NPo = 0.029
-80
y...‘+
C-b + if
04
./’
I equilibrium
./’
l-
4 PA
0 after
-80
0 after
-30
-120
1
15 s
DI
Q-BO
D2
after -3OmV
o
80
it E
60
8 tJ a 20,
a-30
after -8OmV
40 207,
0
2
4
Elapsed
6
8
Time
Figure 4. Time- and Voltage-Dependent pletely Reversible
10
12
0
(min) Changes
2
4
6
Elapsed in NMDA
Channel
Open-State
8
Time
Probability
10
12
14
(min)
Observed
in Excised Patches Were Com-
NMDA (2 uM NMDA plus 1 uM glycine) channel data recorded from 1 patch using an ATP-containing (4 m M Mg-ATP) pipette were collected in the following sequence: (Al) events recorded in the 12-13 min interval at -30 mV (equilibrium), (B2) events immediately after changing the potential to -80 mV, (Bl) events recorded in the 15-16 min interval at -80 mV (equilibrium), and (A2) events immediately after changing the potential to -30 mV. (A)At -3OmVequilibrium NP,was0.034. (Al) NP,,changed toa newequilibriumofO.O17at-80mV(A2). Fourteen minutesafter returning the patch to -30 mV from the -80 mV condition displayed and analyzed here, NP, was 0.033 (data not shown). (B) In the first minute followingvoltagetransitions NP, remained close to thevalueof the previous equilibrium (in Bl at-30 mV, stepped from -88 mV, NP,, was initially 0.016; in 82 at -80 mV, stepped from -30 mV, NP, was initially 0.029). (C)Average I-V data from the patch are shown in two conditions, at equilibrium (open squares), and for the first minute after a voltage transition (e.g., from -80 mV equilibrium [closed squares] to -30 mV [open squares], from -30 mV equilibrium [closed squares] to -80 mV [half closed squares]). Dashed lines with arrows indicate the sequence of voltage- and time-dependent changes. (D) The time course for the reestablishment of equilibrium NP, values is shown after transitions from -30 to -80 mV (Dl) and from -80 to -30 mV (D2). The equilibrium NP, at -80 mV was 0.017; at -30 mV it was 0.035, determined from 3 min segments of recordings to minimize the variability introduced by the normal bursting behavior of the channels. Data points in the graphs were obtained by counting the number of events and measuring r0 for adjacent and/or overlapping 68 s intervals in each record. Symbols are placed 38 s into the interval. Normal variability of NP, at equilibrium can be seen in Dl between the 4th and 12th min.
Otherfactorsthat mayinfluenceoverall POwerealso examined briefly with respect to the voltage dependence, but the time course of the NP, change was not typically examined. Including ATP in the patch pipette may have decreased the rate of rundown of NMDA channel
activity
(MacDonald
et al., 1989),
but
its pres-
ence did not alter the voltage dependence of P, (n = 5). Extracellulardithiothreitol(1 mM) increased overall P, compared with untreated patches (n = 3), as expected from whole-cell responses (Aizenman and Lipton, 1989). Although both 70 and frequency of events increased in 1 mM dithiothreitol, the voltage dependence of NP, was similar in the dithiothreitol-treated patches and untreated patches. Again, an increase in
event frequency made the greater contribution to the increase in P, at positive potentials. Having observed the slow changes in P, in excised membrane patches exposed to simple saline solutions, with and without ATP present, it appears that the phenomenon is closely associated with NMDA receptor channel proteins. Discussion
While not nearly so dramatic as the voltage dependence seen in the presence of extracellular Mg2+, there is nonetheless an obvious sensitivity of NMDA responses to membrane voltage in the nominally Mg2+-
NWKWl 186
free solutions. The slowness of the voltage-dependent change in NMDA channel P, was exploited to demonstrate a hysteresis in whole-cell NMDA I-Vcurves (Figure IB). Thus, the observation that NMDA currents had not reached asteady-statevalueat most potentials within 2 min, coupled with the observation that voltage-dependent changes of currents in the control I-V curves had reached equilibrium within 20 s, led ustouse25-60sintervalsforvoltagechangesin Figure IB. Such slow changes in P, have important implications for interpretation of NMDA channel kinetics studies and for determination of the voltage sensitivityof association and dissociation rates of slow NMDA channel blockers like MK-801 (MacDonald and Nowak, 1990). The voltage dependence of P, is most remarkable because its slow time dependence gives the impression of a system with memory. Because much of the change in NP, occurs between -60 and f40 mV and its time course is slow, this phenomenon cannot be explained by a rapid block of channels by residual Mg 2+. In contrast, at -90 and -100 mV we did obtain evidence for the presence of residual Mg2+ in some recordings: openings were grouped in bursts containing brief closures (rc = 1.1 ms), and z,, was decreased. The apparent number of events also increased as predicted if the current through open channels was being interrupted by Mg2+. Together, these results suggest that a block by residual Mg2+ in the nominally Mg2+-free solution is a possible explanation for an outward rectification at large negative potentials. This result was not readily apparent in all patches. In our experiments the nominally”Mg*+-free” extracellular solutions typically contained I-2 PM Mg2+, as measured by atomic absorption spectroscopy (Wright et al., 1991). Further study is needed to elucidate the mechanism(s) underlying the voltage-dependent changes in NMDA channel P,. The increase in P, with depolarization occurs at considerably lower rates than would be anticipated for a rearrangement of dipoles in the protein(s), or the association/dissociation of ions, which might influencechannel gating, unless the process is linked to a particular state of the channel. For example, if the process underlyingchanging P,occurs in association with channel opening, it would be slow because NMDA channels open infrequently. In addition, if an NMDA channel must open in order to reset its activity level, it would take longer if the membrane is depolarized following a long period of hyperpolarization because the channel opening rates at negative potentials are lower. The process would be faster for hyperpolarization after a prolonged period at positive potentials because the channel activity would be equilibrated to a higher level. Alternatively, the slow increase in P, with depolarization may arise from a decrease in desensitization (Clark et al., 1990). Due to the relatively high permeability of NMDA channels to Ca2+(MacDermott et al., 1986; Mayer and Westbrook, 1987) and the link between Ca2+ entry and
excitotoxicity (Choi, 1987; Meldrum and Garthwaite, 1990), tight control of NMDA receptor channel activity appears vital for normal brain function. The slow increase in NP, was observed clearly at -30 mV, and the influence of holding patches at 0 mV was observed as an increase in NMDA channel activity that persisted for 4-5 min upon repolarization to -60 mV (n = 2 patches). Increasing NMDA channel activity in this potential range is important since glutamatergic synaptic activity tends to depolarize cells to near 0 mVand the increase in P, is likely to foster hyperexcitability in cells that are not voltage clamped. Thus, in contrast to voltage-gated Caz+channels, which exhibit voltagedependent and Ca2+-dependent inactivation with depolarization, NMDA channel activity is likely to increase following prolonged depolarization. The slow rise in NMDA channel open probability following sustained depolarization would result in a much higher degree of Ca*+ loading than would occur if NP, did not increase by between 2-and 3-fold near 0 mV, compared with NP, at resting membrane potential. Thus, persistent depolarization of cells in vivo by NMDA agonists leading to the increase in NMDA channel activity through the slow voltage-dependent increase in P, is likely to contribute to neurodegeneration. In several reports it appears that NMDA-induced excitotoxicity requires IO-15 min exposures to NMDA in the presence of extracellular Ca2+ to obtain a reliably high percentage of cell death in many preparations (Meldrum and Garthwaite, 1990); however, Hartley and Choi (1989) have observed that a majority of mouse cortical neurons in culture were killed by 3-5 min exposures to high concentrations of NMDA. It is difficult to compare the time scale of the changes in P, observed in our experiments with the time course of excitotoxic damage because low doses of NMDA (2-5 GM) were used for the P, studies. Thus, additional experiments directed at testing the effect of agonist concentration on the rate of change of P,, as well as other factors that influence overall P,, will be necessary in isolated patches and in intact cells in order to determine whether changes in P, play a role in regulating the physiological state of neurons in vivo.
General Methods Mouse forebrain neurons were prepared from 15 to I7-day old fetuses as described previously (Ascher et al., 1988). Recordings were performed at 20°C-25°C on neurons in culture for 4-43 days. Whole-cell and single-channel NMDA current responses were recorded in the presence of 1 u M glycine (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988) with a List EPC-7 patch-clamp amplifier using borosilicate glass patch pipettes (3-7MD; WPI TW150) containing the following: 140 m M CsCI, 10 m M K-EGTA/l m M CaCI,, and 10 m M K-HEPES (pH 7.2). NaNO, (75 mM) or 70 m M Cs-acetate was sometimes substituted for the equivalent amount of CsCl in intracellular solutions without any effect on NMDA channel 7, or conductance (Ascher et al., 1988; Wright et al., 1991). Mg-ATP (2-4 mM)was included in the pipette solutions for some experiments. Extracellular (bath) solutions contained the following: 150 m M NaCI, 2.8 m M KCI, 1.0 m M
NMDA 187
Channel
Open Probability
Varies with Voltage
CaCb, 10 m M Na-HEPES (pH 7.2) with 300 nM tetrodotoxin and 1 J&I glycine. NMDA (Cambridge Research Biochemicals), dithiothreitol (Sigma), and glycine (Sigma) were dissolved in the bath solution and applied by rapid local perfusion directed at the patch pipette through a large bore Pasteur pipette. Total bath solution was continually exchanged by slow perfusion of the 35 m m culturedish. Recordings were initially filtered (4 kHz; apole Bessel), digitized (44 kHz; Medical Systems, PCM-IB), and stored on cassettes by video (Sony Beta SL HF450) for later analysis. Single-Channel Methods Singlechannel data were reconverted to analog form, filtered at 2 kHz (Bessel), sampled at 10 kHz (Axolab, Axon Instruments), and analyzed on personal computers (AST Premium 286) using the CAP software (R. C. Electronics; Goleta, CA) and additional software developed in the laboratory (Wright et al., 1991). Channel ~~ was determined from histograms generated from events lists obtained from single channels recorded in patches where the channel openings to a second level represented less than 10% of openings in the list. In a few patches with many openings to a second level, open times were estimated from events lists obtained by the method of Jackson (1985). Open durations were fitted by the maximum likelihood method from unbinned data as described by Colquhoun and Sigworth (1983). Amplitude histograms were constructed as probability density functions of open NMDA channels using all of the points sampled in each segment of recording that was analyzed. Single-channel current amplitude was obtained by fitting a Gaussian function to the peak representing single-level openings. Additional Guassian functions were fitted to the amplitude peaks representing openings to second, third, and fourth levels as necessary to obtain the total charge per min (in pC.mirt-‘) by integrating the area under all of the Gaussian functions. The average current was determined by measuring total charge per minute from amplitude probability density functions standardized to represent 60 s of recordingand dividingthechargepermin by60sasdescribed previously(Wrightetal., 1991).This measureoftheaverage patch current determined at different potentials was used in constructing the I-V curves in Figure 2C and Figure 4C.
analysisof single-channel records. In Single-Channel E. Neher and B. Sakmann, eds. (New York: Plenum Corp.), pp. 191-263.
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We thank Philippe Ascher for his comments on an earlierversion of the manuscript, Paul A. Kline for writing the computer software, and Valerie S. Engle and Cornelia Poulopoulou for cell cultures. This project was supported by National Institutes of Health grant #NS24l67. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertiseme& in accordance with 18 USC Section 1734 solely to indicate this fact. Received
August
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30, 1991.
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