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Previous Article | Next Article
The Journal of Neuroscience, August 1, 2001, 21(15):5574-5586
Subunit Interactions and AMPA Receptor Desensitization
Antoine Robert1, Stacey N. Irizarry1, Thomas E. Hughes2, and James R. Howe1 Departments of 1 Pharmacology and 2 Ophthalmology, Yale University School of Medicine, New Haven, Connecticut 06520-8066
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Most AMPA-type glutamate receptors (GluRs) exhibit rapid and virtually complete desensitization when activated by glutamate, and at some central synapses it is largely desensitization that determines the decay of EPSCs. However, the mechanisms underlying the conformation change that results in desensitization are not fully understood. AMPA receptor subunits that contain a single amino acid substitution have been shown to form homomeric channels that show markedly reduced desensitization. We show here that the coexpression of wild-type GluR1 with one such mutant, GluR1(L497Y), results in heteromeric channels that show desensitization behavior that is intermediate between wild-type and mutant homomers. The relative amplitudes of the multiple exponential components present in the decay of glutamate-evoked currents depended on the relative abundance of wild-type and mutant subunits and were described by the combinatorial distribution of the two types of subunits into tetrameric, but not pentameric, assemblies. Our results are consistent with recent structural data suggesting that AMPA receptors are tetrameric assemblies composed of two dimers.
Key words: glutamate; AMPA receptor; desensitization; subunit interactions; allosteric; kinetics
INTRODUCTION
AMPA-type glutamate receptors (GluRs) rapidly desensitize on exposure to glutamate, and the kinetics of desensitization can determine the time course of synaptic events (Jonas and Spruston, 1994
; Jones and Westbrook, 1996
Although the above studies have identified domains within individual subunits that influence desensitization, the extent to which subunit-subunit interactions play a role is less clear. Allosteric models similar to those advanced to explain the behavior of tetrameric hemoglobin (Monod et al., 1965
We show here that the coexpression of wild-type GluR1flip with the non-desensitizing GluR1flip (L497Y) mutant results in what appears to be intermediate desensitization behavior. The relative amplitudes of the multiple components present in the decay of glutamate-evoked currents depended on the relative abundance of wild-type and mutant subunits. The results provide novel evidence that AMPA-type channels are tetrameric assemblies, and our findings are consistent with recent evidence suggesting that these assemblies are composed of two dimers.
MATERIALS AND METHODS
Cell culture. Human embryonic kidney (HEK) 293 cells were plated onto 12 mm glass coverslips that had been coated with poly-L-lysine (100 µg/ml) and were maintained in humidified 95% O2/5% CO2. The culture medium was modified Eagle's medium (MEM; Life Technologies) containing 10% fetal bovine serum. HEK 293 cells were transiently transfected using Lipofectamine 2000 (Life Technologies) with 0.2-0.6 µg of total cDNA per coverslip. The solution used for transfection consisted of 100 µl of Opti-MEM medium (Life Technologies), 3 µl of Lipofectamine 2000, 0.5 µg of a reporter cDNA encoding the green fluorescent protein (GFP) in pCMVsport, 0.1-1 µg of GluR1flip or GluR1flip (L497Y), or both, in a CMV-driven mammalian expression vector. The GluR1 plasmids were kindly provided by Derek Bowie (Emory University, Atlanta, GA), who introduced the L497Y mutation. The GluR1flip construct (original cDNA from Peter Seeburg, MPI Medical Research, Heidelberg, Germany) was modified previously by Mark Mayer and Kathy Partin (National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD) to enhance expression in mammalian cells. The transfection solution was first incubated at room temperature for 15 min, and 25 µl of this solution was added to each culture well. Electrophysiological recordings were performed 24 hr after transfection.
Cerebellar culture. The cerebella of Sprague Dawley rat pups (age 5-7 d) were removed and minced with a razor blade. Cells were dissociated into the culture medium with a fire-polished Pasteur pipette in the absence of enzymes. The resulting solution was then filtered through a nylon mesh, and the cells were plated onto acid-washed coverslips coated with poly-L-lysine (400 µg/ml for 1 hr). The culture medium was DMEM containing 25 mM KCl and 10% fetal bovine serum. The transfection technique was as described above. To minimize glutamate-mediated toxicity, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX; RBI/Sigma) was added at 1 µM to the transfected cultures.
Electrophysiology. Whole-cell patch-clamp recordings and recordings from excised outside-out patches were made with an EPC9 amplifier (HEKA). The cells were constantly superfused with normal external solution at a rate of 1 ml/min. Transfected cells were identified by epifluorescence imaging of GFP (Marshall et al., 1995
. The series resistance after going whole cell was 3-6 M
5 mV at the holding potential of
90 mV used for all recordings. We found that series resistance compensation (60-80%) had no effect on the amplitude or kinetics of the glutamate-evoked currents, but did often introduce high-frequency noise; therefore it was not used in most recordings. Most currents were analog low-pass filtered at 2.9 kHz (four-pole Bessel-type,
The external medium was (in mM): 150 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES (pH adjusted to 7.4 with NaOH). Pipettes were filled with a solution containing (in mM): 120 CsF, 33 KOH, 2 MgCl2, 1 CaCl2, 0.1 spermine, 10 HEPES, and 11 EGTA (pH adjusted to 7.4 with CsOH). Glutamate was added to the external solution. Cyclothiazide was prepared in DMSO and diluted in external solution to a final concentration of 100 µM (final DMSO concentration 0.5%). All chemicals were purchased from Sigma (St. Louis, MO).
Fast perfusion. In most experiments, glutamate was applied with a rapid perfusion system made from a pulled theta capillary. The tip of the theta glass was cleanly broken with a diamond cutter to a tip diameter of ~300 µm, and its septum was snapped back at the tip with a sharp needle so that the flow of solution leaving the tip from either side of the theta capillary overlapped. Four capillaries (outer diameter 450 µm; Polymicro Technologies) were introduced into each barrel at the back of the theta glass and secured with Sylgard (Dow Corning). Each capillary was connected to plastic tubing that branched to a solenoid valve (The Lee Company). The valves were opened and closed by a analog-to-digital (A/D) interface (TIB 14, HEKA) that was controlled by the acquisition software of the EPC9. The speed of the solution exchanges obtained with this system was estimated with an open patch pipette by measuring the current deflections induced by a change in the NaCl concentration. The mean 10-90% rise times of the open-tip responses were 275 ± 25 µsec (n = 5).
The valve-controlled system described above produced rapid solution exchanges, but it did not allow us to make applications briefer than ~50 msec. For measurements of deactivation time constants, and for experiments to determine the effect of cyclothiazide on peak currents, glutamate was applied to excised patches using a theta glass pipette mounted on a piezoelectric bimorph. The theta glass pipettes were pulled and broken to a tip diameter of ~300 µm. The width of the septum separating the two barrels was reduced by etching with hydrofluoric acid to increase the sharpness of the interface created between solutions flowing out of each barrel. Patches were positioned near the solution interface, and the interface was moved by applying voltage across the bimorph with a constant voltage source (Winston Electronics Co.) that was triggered with one of the A/D outputs on the EPC9. The voltage pulses were resistance-capacitive filtered to minimize mechanical oscillations of the piezoelectric device. The 10-90% rise times of the open-tip responses obtained with this system were 100-200 µsec, and glutamate applications as brief as 1 msec could be delivered reproducibly.
Data analysis. The digitized records were transferred to IGOR software (Wavemetrics). The decays of glutamate-evoked currents were fitted with functions consisting of the mixture of multiple exponential components and a steady-state plateau current. Usually 5-10 responses were averaged, and the fits were performed on the averaged currents. The segment of the decay to be fitted was defined by placing cursors on the data trace, where the first cursor was placed near the peak of the current and the other cursor ~700 msec after the peak. Fits were performed by sequentially including from one to four exponential components. At each stage, the quality of the fit was evaluated by visual inspection of the residual current (obtained by subtracting the fit from the data). If the residual current was not flat, an additional exponential component was added. Two exponential components and a plateau current gave adequate fits in a few cases, and functions containing three exponentials were sufficient to obtain a flat residual current in all cases. Not only did the inclusion of a fourth component not improve the fits as assessed by visual inspection of the residual currents, but repeated four-component fits of the same data gave parameter values that were not reproducible. In most cases, the amplitude of one component converged to a value near zero, two of the time constants converged to very similar values, or one of the time constants converged to a value that was very brief or very long. Inclusion of a fourth component typically reduced the sum of squared deviations by <1% and rarely improved the goodness of fit significantly as assessed by a standard test for comparison of nested models (Horn, 1987
The recovery from desensitization was estimated with two-pulse protocols in which a constant 100 msec application of 500 µM glutamate was followed by test pulses applied at variable intervals. Each two-pulse protocol was separated by a 5 sec interval to allow complete recovery from desensitization. The decays of the currents evoked by the test pulses were fitted as described above, and the amplitudes of the fast and intermediate exponential components were plotted as a function of recovery time. Each set of data was fitted with a single exponential function: It/I
= 1
rec), where It is the amplitude of the current for a given test pulse, I
Fluctuation analysis of steady-state currents evoked by a range of glutamate concentrations was used to estimate the unitary conductance and open probability of GluR1flip (L497Y) homomeric channels. These results were compared with the corresponding results obtained for wild-type GluR1flip channels in the presence of 100 µM cyclothiazide (to slow desensitization). Values for the current variance were obtained from spectral density analysis of steady-state currents evoked at different glutamate concentrations, and these values were plotted against the corresponding mean currents to give estimates of Popen and the apparent unitary conductance (
noise). For these experiments, the data were sampled at 9.4 kHz and low-pass filtered at 2 kHz. Power spectra were obtained and fitted with bi-Lorentzian functions as described previously (Howe, 1996
It was assumed that the assembly of wild-type and mutant subunits was random and combinatorial. The probabilities for each possible stoichiometric combination of wild-type and mutant subunits were calculated from the binomial relationship: Pj = (N!/(j!(N
j, where Pj is the probability that a channel composed of N subunits will contain j mutant subunits, and pmut is the amount of GluR1flip (L497Y) cDNA as a proportion of the total GluR-encoding cDNA (wild type plus mutant) used for the transfection. For each wild-type/mutant cDNA ratio studied, the results were compared with the stoichiometric predictions for channels containing N = 4 and N = 5 subunits (tetramers and pentamers, respectively). Similar calculations were done, assuming that the channels assembled first as dimers, to obtain the probabilities that a channel contained at least one dimer in which both subunits were the L497Y mutant.
Simulated glutamate-evoked currents were generated using QUB software (Qin et al., 1996
RESULTS
Desensitization of GluR1flip and GluR1flip (L497Y) homomeric channels
The transient transfection of HEK 293 cells with the cDNAs encoding the GluR1flip and the GluR1flip (L497Y) subunits resulted in high-level expression of each type of homomeric channel. This facilitated our studies of desensitization kinetics because we were able to measure large ensemble currents in outside-out patches from transfected cells. Although the superfusion systems used produced solution exchanges of <300 µsec, the rise times of whole-cell currents were much slower (2-5 msec), presumably because of diffusion barriers. Therefore, although qualitatively similar data were obtained from whole-cell recordings, all quantitative analysis reported here was limited to results obtained in outside-out patches in which the rise times of the currents were <500 µsec.
Typical currents evoked by 5 mM glutamate in outside-out patches are shown in Figure 1. Currents through wild-type GluR1flip homomers showed rapid and virtually complete desensitization. Although the decay of the currents proceeded largely monophasically, we found that these decays were consistently better fitted by two, rather than one, exponential component. Figure 1Aa shows the current evoked by the sustained application of 5 mM glutamate in a patch from a GluR1flip-transfected cell. The two-exponential fit is superposed on the data, and the corresponding residual current is also shown. An expanded view of the same current with the best single exponential fit superposed is shown in Figure 1Ab. As was the case in all patches studied, single exponential fits failed to describe well the tail of the glutamate-evoked currents and also gave significant residual current near the peak response. The source of the small slower component is uncertain, but we believe it arose because a few of the receptors in the patches had restricted access to the agonist because of physical barriers to diffusion. To limit the effect of this small tail on the time constants measured for the major component of decay, the slow component was routinely included in the fitting. On average, the currents through GluR1flip homomers decayed with a large fast component (
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Figure 1. Desensitization kinetics of wild-type GluR1flip and GluR1flip (L497Y). Aa, Current elicited by the rapid and sustained application of 5 mM glutamate (bar) in an outside-out patch from a HEK 293 cell transfected with wild-type GluR1flip. The bi-exponential fit (smooth solid line) to the decay of the current, as well as the residual trace (res; obtained by subtracting the recording from the fit), are shown. The fit gave time constants of 2.25 and 14 msec for the two components. The amplitude of the fast component was 99% of the total peak current. Ab, Enlargement of the same current fitted with a single exponential. Note that the fit is poor in the tail of the current, as well as the presence of substantial residual current near the peak. Ba, Response to 5 mM glutamate in a patch from a cell expressing GluR1flip (L497Y). The single-exponential fit to the decay and the corresponding residual current are shown. Bb, The rises of the currents in A (GluR1flip) and B [GluR1flip (L497Y)] are superposed after the peak amplitudes are scaled. Note that the rises are virtually identical.
Typical currents evoked by glutamate pulses in patches from cells expressing GluR1flip (L497Y) channels are illustrated in Figure 1B. As shown by Stern-Bach et al. (1998)
The L497Y mutation did not appear to affect the speed of activation. The currents in Figure 1, A and Ba, have been normalized and superposed in Figure 1Bb to illustrate the similar rise of the currents through wild-type and mutant channels.
Heteromeric channels display intermediate desensitization kinetics
To determine the relative dominance of the wild-type and mutant phenotypes, we recorded currents activated by 5 mM glutamate in outside-out patches from HEK 293 cells that were cotransfected with equal amounts of the plasmids encoding GluR1flip and GluR1flip (L497Y). A typical example of the recordings obtained in these experiments is presented in Figure 2. In each of six patches, three exponential components were required to obtain adequate fits to the decays of the glutamate-activated currents. The three individual components are shown in Figure 2B as dotted lines. The mean values obtained for the time constant of the fast (
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Figure 2. Coexpression of GluR1flip and GluR1flip (L497Y) gives an intermediate component of decay. Aa, Current elicited by 5 mM glutamate (bar) in a patch from a cell transiently cotransfected with equal amounts of wild-type GluR1flip and the GluR1flip (L497Y) mutant. The decay of the current contains three resolvable exponential components. The triple exponential fit (smooth solid line) and the corresponding residual current (res) are shown. Ab, The same current and the fit are shown on an expanded time scale, together with each of the individual exponential components (dotted lines). The three components decay at fast (f;
The results of these coexpression experiments indicated that the channel population generated by mixing wild-type and non-desensitizing subunits displays three distinct phenotypes that are characterized by different rates and extents of desensitization. Under conditions in which the expression of the two types of subunits is approximately equal (1:1 cDNA ratio), the three components that are distinguishable in the decay of large ensemble currents have similar amplitudes. Approximately one-third of the channels desensitize with a rapid time course that is slightly slower than that of homomeric wild-type channels, approximately one-third desensitize at a rate (
20 msec) that is intermediate between wild-type and mutant homomers, and the remaining one-third desensitize incompletely and at a rate that is similar to that of homomeric mutant channels.
The amplitude of each phenotype depends on the ratio of wild-type and mutant cDNAs
One interpretation of the results obtained in the coexpression experiments is that the rate and extent of desensitization depends on the stoichiometry of wild-type and mutant subunits in individual channel assemblies. To investigate this possibility further, we tested different mutant/wild-type cDNA ratios to vary the relative abundance of mutant and wild-type subunits. With the exception of the L497Y mutation, the expression plasmids used in our experiments were identical. Figure 3 shows typical results obtained in outside-out patches from cells transfected with cDNA mixtures at mutant/wild-type ratios of 1:6 (A), 1:3 (B), and 3:1 (C). Each panel of Figure 3 shows the current evoked by a 700 msec application of 5 mM glutamate, the fit to its decay, and the corresponding residual current. The insets show the same recordings on an expanded time scale with the individual components (fast, intermediate, and slow) that were detected in the decays shown as dotted lines.
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Figure 3. The relative amplitudes of the fast, intermediate, and slow components of decay vary systematically with the ratio of wild-type and mutant cDNAs. Aa, Glutamate-evoked current in a patch from a cell cotransfected with plasmids encoding GluR1flip (L497Y) and GluR1flip at a cDNA ratio of 1:6. The decay of the current was fitted with two exponential components and a small sustained current. The fit (smooth solid line) and the residual current (res) are shown. Ab, Same trace as in Aa on an enlarged time scale together with the three individual components (dotted lines). The values for
Three main findings were evident from the coexpression experiments. First, as the mutant/wild-type cDNA ratio was systematically increased, the relative amplitude of the fast component of decay decreased and the relative amplitude of the slow component increased. At a mutant/wild-type ratio of 3:1, the fast component was very small or absent. Second, concomitant with these changes, the time constant of the fast component became gradually slower. Third, at each cDNA ratio tested, there was a component of decay that proceeded with a time constant of ~20 msec. The fits to the currents evoked in patches from cells transfected with mutant and wild-type cDNAs at a ratio of 1:6 gave mean
Comparison of the results with combinatorial predictions
The results above show that the relative amplitudes of the three components distinguished in the decay of currents evoked by sustained applications of glutamate changed in a graded fashion as the mutant/wild-type cDNA ratio was altered. To test further the hypothesis that the three components arise from channels with different stoichiometries of mutant and wild-type subunits, we calculated the proportions of each possible subunit combination, assuming that channel assembly is combinatorial.
The combinatorial analysis that we performed rests on several simplifying assumptions. First, we assumed that the relative abundance of wild-type and mutant subunits accurately reflects the ratio of the two cDNAs used for the transfection. Because the expression levels were high, and the two cDNA constructs were identical except for the point mutation, we believe that this assumption is reasonable. Second, our analysis assumed that the point mutation does not affect subunit assembly. Although this assumption is untested, the levels of expression seen when each cDNA was expressed alone were similar, and some channel properties, e.g., the apparent rate of activation, were unaltered by the mutation.
The combinatorial analysis also assumes that the unitary conductance and probability of channel opening were similar for wild-type and mutant channels. It has been shown that the corresponding leucine-to-tyrosine mutation in GluR3 (L507Y) does not alter either the main conductance level or the open probability (Popen) of homomeric channels formed from this subunit (Rosenmund et al., 1998
Finally, our combinatorial analysis implicitly assumed that the channels in the excised patches are representative of the population expressed in a given cell. The patches used for the analysis gave peak inward currents that were always >300 pA and were typically ~1 nA (at the routine holding potential of
With the above assumptions, the probability of each possible wild-type/mutant stoichiometry can be predicted from the binomial equation (see Materials and Methods). The calculated probabilities for the possible wild-type and mutant subunit combinations are given in Figure 4 for each of the four mutant/wild-type cDNA ratios investigated. Figure 4A gives the combinatorial predictions assuming that the receptor is a tetramer, whereas the probabilities in Figure 4B assume the channel is a pentamer. These probabilities can then be compared with the relative amplitudes of the three exponential components distinguished in the current decays. If AMPA receptors are tetramers, the results obtained with a 1:1 cDNA ratio (approximately equal amounts of fast, intermediate, and slow current) can be accounted for if the rapidly decaying component corresponds to channels containing three or four wild-type subunits (0.25 + 0.06 = 0.31), the intermediate component corresponds to channels with two wild-type and two mutant subunits [0.37 (Fig. 4A, shaded row)], and the slow component arises from channels with three or four mutant subunits (0.06 + 0.25 = 0.31). This partitioning of the probabilities also matches well our observation that the relative amplitude of the intermediate component is similar for mutant/wild-type ratios of 1:3 and 3:1 (0.17 and 0.19 vs 0.21 predicted). The relative amplitude of the intermediate component at a mutant/wild-type ratio of 1:6 also agrees with the predictions (0.08 observed vs 0.09 predicted).
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Figure 4. The results support the idea that AMPA receptors are tetramers. A, Table showing the probability of occurrence of each possible subunit combination if the channels are tetramers for the different cDNA ratios examined. Each row corresponds to channels with a different number of mutant subunits (0, 1, 2, 3, 4) as indicated. The shaded row corresponds to channels containing two mutant subunits and predicts well the measured relative amplitude of the intermediate component. The relative proportions of the fast component of desensitization match well the predicted proportion of channels containing no or one mutant subunit. The relative proportions of the slow component match well with the sum of homomeric mutants and channels containing one wild-type subunit. B, Probability of occurrence of all subunit combinations if the channels are pentamers. No partitioning of the subunit combinations matches the proportion of fast, intermediate, and slow phenotypes over the range of cDNA ratios tested.
In contrast to the results obtained assuming the channel is a tetramer, there is no partitioning of the probabilities in Figure 4B (channels assumed to be pentamers) that matches well the relative amplitudes of the three components over the range of cDNA ratios tested. This is so despite the fact that there are more ways to partition the results, which might be expected to increase the chance of a spurious match. In particular, there is no partitioning that can account for the roughly equal amplitudes of each component at a cDNA ratio of 1:1, nor our observation that the amplitude of the intermediate component was similar at mutant/wild-type ratios of 1:3 and 3:1. For example, if the intermediate component is identified with channel assemblies containing three mutant subunits [(Fig. 4B, shaded row) 0.31 probability at a 1:1 ratio], then it is predicted that the fast component should be more than twice the amplitude of the slow component (0.5 vs 0.19). This way of partitioning the pentameric probabilities also predicts that the intermediate component should have been very small (0.02) at a mutant/wild-type ratio of 1:6 and that the relative amplitudes of the intermediate component at cDNA ratios of 1:3 and 3:1 should have differed substantially (0.26 vs 0.09). None of these predictions is borne out by the results.
The above comparisons indicate that the results can be accounted for if the channels are tetramers and the three distinguishable components correspond to channel assemblies with different stoichiometries of mutant and wild-type subunits. As noted, this analysis rests on the assumption that the relative amplitude of each component accurately reflects the relative abundance of the corresponding channel population. The rise of the currents was substantially faster than the decays, even for the fast component, making it unlikely that we were significantly underestimating the peak amplitudes because the solution exchanges were insufficiently fast. However, because desensitization appears to proceed via closed states (Vyklicky et al., 1991
Responses to brief applications of 5 mM glutamate in the absence and presence of 100 µM cyclothiazide are shown in Figure 5A. The currents were measured in the same patch from a cell transfected with wild-type GluR1flip. In each of four patches studied, cyclothiazide caused an increase in the peak current through homomeric wild-type channels (mean percentage increase 34 ± 2%; n = 4). This result is predicted for models of the type shown in Figure 6D, where activation and desensitization occur independently from the same closed state. Although the available results are insufficient to define some of the rate constants for the model in Figure 6D, at high agonist concentrations the channels spend most of their time in fully liganded states, and this model predicts behavior similar to simpler models proposed previously. Simulations of channel activity evoked by 5 mM glutamate (using the model in Fig. 6D) showed that the decay of wild-type currents during sustained applications of glutamate could be reproduced with values for channel opening (
4) and channel closing (
4) of 7000 s
4) and 1 s
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Figure 5. The effect of cyclothiazide and comparison of the results with combinatorial predictions. A, Currents evoked by 5 mM glutamate in a patch from a cell transected with wild-type GluR1flip. Preexposure of the patch to cyclothiazide (100 µM) greatly slows desensitization and potentiates the peak current amplitude. B, Simulated currents generated with the model shown in Figure 6D in response to a sustained application of 5 mM glutamate. The number of channels was set to 1000 for each simulation. The analysis was performed to estimate the extent to which the peak current amplitude underestimated the number of channels as the result of some channels desensitizing without ever opening. The trace labeled 1 mimics the kinetics of wild-type GluR1flip homomers. The following values were used for the rate constants: k1 = 1 × 107 M ), intermediate (
), and slow (
), at the four cDNA ratios tested. Each point is the mean value from measurements made in five to seven patches. The bars indicate the SD, some of which were less than half the symbol height. The black curves show the summed probability of homomeric GluR1flip channels and channels with one GluR1flip (L497Y) subunit (solid line), the probability that channels will contain two GluR1flip (L497Y) subunits (dotted line), and the summed probability of having homomeric GluR1flip (L497Y) and channels with one wild-type GluR1flip subunit (dashed line). The probabilities are plotted as a function of the proportion of wild-type subunits present, assuming that the subunit assemblies are tetramers. The gray curves show the corresponding probabilities after adjusting the results for the fast and intermediate components (for missing channels that never open) using the relative amplitudes of the peak currents predicted from the simulated results in B (fast and intermediate components underestimated by 35 and 12%, respectively, relative to the slow component).
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Figure 6. The results are consistent with the conclusion that AMPA receptors are dimers of dimers. A, The relative amplitude of the slow component is plotted as a function of the proportion of wild-type cDNA included in the transfection mixture. Each point is the mean value from measurements made in five to seven patches. The bars indicate the SD. The black curve shows the expected proportion of tetramers that contain at least one dimer with two mutant subunits. The gray curve shows the corresponding prediction after correcting for missing channels that never open using the simulated data in Figure 5B. B, Diagram illustrating the partitioning of tetrameric assemblies assumed to display wild-type and mutant desensitization for the combinatorial analysis shown in A. C, Deactivation kinetics of wild-type GluR1flip (left) and homomeric GluR1flip (L497Y) channels (right). The currents were evoked by 1 msec applications (bars) of 5 mM glutamate. The decays of the currents (dotted traces) were fitted with single exponential functions (solid curves) with the indicated time constants. Da, Possible kinetic scheme illustrating some general features of AMPA receptor activation and desensitization suggested by previous work. It is assumed that the channel is a tetramer where each subunit can bind a single agonist molecule (A) and can exist in three distinct conformations: closed (C), open (O), and desensitized (D). The states in the top row are open, those in the middle row are closed, and those in the bottom row are desensitized. For closed and desensitized states, each subunit is constrained to adopt the same conformation. The affinity of agonist binding is higher affinity to desensitized states than to closed states (k
Figure 5C is a plot of the mean relative amplitudes of the fast, intermediate, and slow components of desensitization as a function of the proportion of wild-type cDNA included in the transfection mixture. The smooth curves show the respective probabilities that channel assemblies will contain 0 or 1, 2, and 3 or 4 mutant subunits, assuming that the channels are tetramers. The black curves show the combinatorial predictions. The gray curves show the result of correcting the combinatorial predictions for the likely underestimation of the fast and intermediate components that would occur if some channels desensitize without ever opening. There is good agreement between the combinatorial predictions and the results at each cDNA ratio tested, supporting the notion that the kinetically distinct components of desensitization arise from channels with different numbers of mutant subunits. Although the cyclothiazide results suggest that the peak fast and intermediate currents underestimate the number of channels by ~35 and 12%, respectively, this is predicted to have only modest effects on the results. The potential influence of such errors is greatest when all three components are of similar amplitude.
Although we believe that the combinatorial analysis supports the view that AMPA receptors are tetramers, two further observations are worthy of emphasis. First, the time constant of the fast component detected in the coexpression experiments was slower than the corresponding value measured for homomeric wild-type channels (
The results are consistent with dimeric subunit assembly
Armstrong and Gouaux (2000)
Although Armstrong and Gouaux (2000)
Slow desensitization versus slow deactivation
Although the "dominant dimer" interpretation accounts for the dependence of the size of the slow component on the relative abundance of wild-type and mutant subunits, it does not explain the intermediate component of decay. However, as emphasized by Partin et al. (1996)
Two commonly agreed upon features in previously published models of AMPA receptor kinetics are that desensitization occurs exclusively from closed states and that the rate constant for channel opening is greater than the rate constant for entry into desensitization (Vyklicky et al., 1991
To gain some insight into the effect of the L497Y on the various microscopic rate constants that could influence operational measurements of the time constants of deactivation and desensitization, we performed channel simulations and systematically altered the rate constants for transitions into and out of fully liganded channel states. The analysis indicated that the slowing of deactivation could be largely accounted for if it was assumed that it arose from alterations in
Intermediate channels recover faster from desensitization
It has been proposed that the entry into, and recovery from, desensitization may proceed via different paths (Patneau and Mayer, 1991
A typical example of the results obtained in these experiments is shown in Figure 7A. The decays of the currents evoked by the conditioning and test pulses were each fitted with three exponential components. The amplitudes of the fast and intermediate components in the decay of the current evoked by the test pulse were then expressed as a proportion of the amplitude of the corresponding component in the decay of the current evoked by the conditioning pulse. Figure 7B shows a plot of the relative amplitudes of the fast and intermediate components as a function of recovery time, where each point is the mean value obtained from five patches. Single exponential fits to each set of results gave recovery time constants (
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Figure 7. The intermediate component recovers faster from desensitization and shows reduced apparent affinity for glutamate. A, Six consecutive sweeps obtained in a patch from a cell that was cotransfected with GluR1flip (L497Y) and GluR1flip cDNAs at a ratio of 1:3. Glutamate (0.5 mM) was applied for 100 msec, and then a second application was made at a varying interval (40, 80, 120, 150, 200, 300 msec). The decays of the currents evoked by the second application of each pair were fitted with three exponential components, and their amplitudes were expressed as a proportion of the corresponding component in the decay of the current evoked by the preceding conditioning pulse. B, Graph showing the recovery from desensitization of the fast (
At high agonist concentrations, recovery from desensitization could occur directly to non-desensitized states or it might proceed initially via unbinding transitions, with exit from desensitization only occurring after agonist has dissociated from one or more subunits. Thus the somewhat faster recovery of the intermediate component could either reflect modest destabilization of desensitized channel states (increase in
The expression of GluR1flip (L497Y) in neurons also gives an intermediate phenotype
From studies in oocytes, Thalhammer et al. (1999)
Typical currents evoked by the application of 2 mM glutamate in patches from control and transfected cells are shown in Figure 8. As illustrated in Figure 8A, currents in patches pulled from control cells showed rapid and virtually complete desensitization. The decays of these currents contained two exponential components. The fits to results obtained in eight control patches gave mean time constants for these components of 1.05 ± 0.09 and 3.82 ± 0.15 msec. On average, the amplitude of the fast and slower components represented 66 ± 1.4 and 34 ± 1.8% of the total peak current. Granule cells express predominantly the GluR2 and GluR4 subunits (Monyer et al., 1991
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Figure 8. The assembly of GluR1flip (L497Y) with native subunits also generates intermediate desensitization. Aa, Current activated by glutamate (2 mM) in an outside-out patch from a control granule cell maintained in culture for 7 d. The decay of the current was fitted with a function consisting of two exponentials (smooth solid line). Ab, Same current on an expanded time scale with the individual components superposed (dotted lines). The time constants of the two components are given on the figure. Ba, Current activated by 2 mM glutamate in a patch from a granule cell that was maintained in vitro for 7 d and transiently transfected 24 hr before with GluR1flip (L497Y). The decay of the current was fitted with a function consisting of three exponentials and a sustained current (smooth solid line). Bb, The current, the fit, and the three individual exponential components are shown on an enlarged time scale. The time constants for the fast and intermediate components are given. Note that the fast component is slower than the fast component in the control neuron.
As shown in Figure 8B, the currents evoked by glutamate in patches pulled from neurons expressing the GluR1flip (L497Y) subunit displayed a large sustained component that was clearly not present in the patches pulled from control granule cells. The decays of these currents contained three exponential components with time constants of 3.61 ± 0.24, 26.5 ± 2.4, and 180 ± 20 msec (n = 5 patches). In all the patches, both the slow component (including the plateau current) and the intermediate component represented >20% of the total peak current. The similarity of the intermediate component of decay in transfected neurons to the intermediate component seen in the cotransfection experiments strongly supports the conclusion that it arises from heteromeric assembly of GluR1flip (L497Y) with native AMPA receptor subunits. Indeed, the decays of the responses in transfected neurons also showed a slowing of the fast component of decay, and they were strikingly similar to those observed in HEK 293 cells (compare Figs. 3 and 8). This similarity suggests that our observations with recombinant channels are not restricted to GluR1 homomeric channels, nor are they likely to be unique to channels composed solely of flip splice variants.
DISCUSSION
The coexpression of GluR1flip with the non-desensitizing mutant subunit GluR1flip (L497Y) produced channels that appear to desensitize at three kinetically distinct rates, one of which is intermediate between that of wild-type and mutant homomeric channels. The relative amplitudes of the three components were described well by simple combinatorial predictions if the channels were assumed to be tetramers. In contrast, there was no partitioning of the combinatorial probabilities that accounted well for the results if the channels were assumed to be pentamers.
Allosteric models of desensitization and subunit dominance
Allosteric models of the Monod-Wyman-Changeaux (MWC) type (Monod et al., 1965
It has been noted that the desensitization of AMPA receptors shows features reminiscent of such allosteric models. First, concentrations of glutamate insufficient to activate AMPA receptors can produce nearly complete AMPA receptor desensitization (Trussell and Fischbach, 1989
One interpretation of our results is that the rate and extent of desensitization titrates with the number of non-desensitizing subunits. Although this interpretation conflicts with the notion of subunit dominance, graded stoichiometric changes do not necessarily argue against allosteric mechanisms. If the conformational change corresponding to desensitization is concerted, then it might become less likely as the number of "reluctant" subunits in the oligomer is increased and thereby result in a progressive slowing of the rate at which the population of channels desensitizes.
The results are described just as well, however, by the "dominant dimer" analysis presented in Figure 6. This interpretation is consistent with recent structural data (Armstrong and Gouaux, 2000
The crystallographic data on the ligand binding domain of GluR2 demonstrate that the dimers show twofold symmetry (Armstrong and Gouaux, 2000
The L497Y mutation appears to destabilize desensitized states
Although the slowing of deactivation accounts substantially for the intermediate component of decay, it cannot account for the substantial plateau current generated by channels containing three or four mutant subunits. This greatly reduced steady-state desensitization must reflect alterations in the rate constants for entry into, or recovery from, desensitized channel states. The L497Y mutation in GluR1 does not remove desensitization completely. Currents through homomeric mutant channels decayed with a time constant of ~100 msec to 85-90% of their peak amplitude (Fig. 1Ba). We found that reproducing this phenotype in channel simulations required substantial changes in the values of both
Armstrong and Gouaux (2000)
Effects on apparent affinity
For cyclic models of the type shown in Figure 6D, it is expected that altering desensitization will alter binding affinity (and vice versa). This is so because to maintain microscopic reversibility the
The EC50 value that we determined for glutamate activation of homomeric GluR1flip (L497Y) channels (28 µM; data not shown) is ~30-fold smaller than the corresponding value for wild-type GluR1 channels when desensitization is intact (Partin et al., 1996
The intermediate component of decay showed hastened recovery from desensitization, and the apparent affinity of glutamate for desensitized channel states was reduced to a similar extent as recovery from these states was speeded. These results suggest that unbinding steps precede exit from desensitization for the channels underlying the fast and intermediate components. Studies on native channels support a similar conclusion (Patneau and Mayer, 1991
Allosteric versus subunit independent gating
Our results support the idea that AMPA receptor desensitization proceeds via an allosteric mechanism, and they provide functional evidence consistent with dimeric receptor assembly. AMPA receptors also show concentration-dependent substate gating (Rosenmund et al., 1998
Individual AMPA receptors show as many as four conductance levels, the frequency of occurrence of which depends on the number of subunits occupied by agonist (Smith and Howe, 2000
FOOTNOTES Received Feb. 14, 2001; revised May 9, 2001; accepted May 21, 2001.
This work was supported by National Institutes of Health Grant NS 37904. We thank Derek Bowie for providing the GluR1 constructs.
Correspondence should be addressed to James R. Howe, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066. E-mail: james.howe{at}yale.edu.
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