Introduction

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Synaptic activity induces significant temporal and spatial fluctuations in interstitial pH of the brain (Chesler, 1990; Chesler and Kaila, 1992). Gross changes lasting milliseconds to minutes have been measured both in vivo and in vitro using pH-sensitive microelectrodes (Kraig et al., 1983; Chen and Chesler, 1992) and optical methods (Krishtal et al., 1987; Gottfried and Chesler, 1996). Local pH fluctuations in the synaptic cleft are believed to be both larger and briefer (Chesler and Kaila, 1992). Furthermore, pathophysiological insults such as spreading depression and seizures reduce brain pH by approximately 0.4 pH units (Kraig et al., 1983; Somjen, 1984; Siesjö et al., 1985), whereas ischemia or hypoxia can shift interstitial pH to 6.5 and below for prolonged periods (Siemkowicz and Hansen, 1981; Siesjö, 1988).

Many ligand-gated channels, including those gated by glutamate, are sensitive to extracellular pH. Previous studies examining modulation of mammalian glutamate receptors by extracellular pH have focused primarily on N-methyl-D-aspartate (NMDA) receptors, which are inhibited significantly even at pH 7.3 (Tang et al., 1990; Vyklicky et al., 1990; Traynelis and Cull-Candy, 1991). The inhibition of non-NMDA receptors by extracellular protons observed in these studies was modest in comparison, and was not considered physiologically relevant (Traynelis and Cull-Candy, 1991). However, because activation of a typical glutamatergic synapse produces < 200 µV depolarization (Andersen, 1990), a high degree of temporal and/or spatial summation is necessary to produce an action potential, and even small pH-mediated changes in AMPA receptor-mediated postsynaptic potentials could affect the efficacy of synaptic transmission in the central nervous system. Transient changes in pH in the synaptic cleft sufficient to significantly affect the function of postsynaptic AMPA receptors are likely to occur, particularly with high-frequency synaptic activity (Chesler, 1990).

Molecular receptor composition has previously been shown to affect proton modulation of NMDA and -aminobutyric acid (GABA) receptors (Traynelis and Cull-Candy, 1995; Krishek et al., 1996). Differences in the proton sensitivity of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors of varying composition has not been examined, but could be important for understanding the vulnerability of some neurons to excitotoxicity. Certain neuronal populations are selectively vulnerable to ischemia, including principal neurons in the hippocampus, striatum and cerebellum, although in these same regions interneurons may be spared (Cervos-Navarro and Diemer, 1991). This selective vulnerability may be explained by the particular subtypes of AMPA receptors that the different cells express. For example, the selective vulnerability of Purkinje cells may result from expression of AMPA receptors that exhibit less complete desensitization (Brorson et al., 1995) because they contain the flip isoforms of AMPA receptor subunits (Tomiyama et al., 1999).

AMPA receptor antagonists are neuroprotective in in vivo models of ischemia (Sheardown et al., 1990; Gill, 1994) and are also potent anticonvulsants (Chapman et al., 1991), suggesting that AMPA receptors play an important role in both ischemia and seizures, two pathophysiological conditions accompanied by significant acidification in brain. NMDA antagonists are less neuroprotective in models of ischemia (Buchan et al., 1991), presumably because NMDA receptors are already inhibited by acidic pH (Giffard et al., 1990; Tombaugh and Sapolsky, 1990). It was previously assumed that acidification would also protect against AMPA receptor-mediated neurotoxicity. In a recent article, however, McDonald et al. (1998) found that neurotoxicity mediated by AMPA receptors is enhanced rather than inhibited at acidic pH. Thus, modulation of AMPA receptors by protons seems to have significant consequences. A more detailed analysis is indicated, to identify the mechanisms responsible for the effects of protons on AMPA receptor function and to determine whether the molecular composition of AMPA receptors affects their sensitivity to proton modulation.

The experiments described below demonstrate significant modulation of AMPA receptors by pH changes typically associated with ischemia, and focus on identification of the mechanisms and molecular determinants of this modulation. The results identify several factors affecting the magnitude of proton-mediated modulation of AMPA receptors, including agonist properties and the molecular composition of receptors, and suggest that protons selectively interact with a desensitized state of the receptor.

	Experimental Procedures

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Cell Culture. Primary cultures of hippocampal neurons were prepared according to established methods (Patneau et al., 1993). Briefly, the hippocampi were dissected from the brains of newborn (P0-1) Sprague-Dawley rats, incubated in papain (Worthington Biochemical Corp., Lakewood, NJ) and dissociated by trituration with a glass pipette. The cells were suspended in 90% Dulbecco's modified Eagle's medium/10% FBS supplemented with 2 mM Glutamax (Life Technologies, Rockville, MD) and 1% penicillin/streptomycin, plated onto glass coverslips precoated with collagen and poly-L-lysine, and maintained at 37°C in a humidified 10% CO₂ incubator. Under these conditions glia proliferate and few neurons survive. After the glial monolayer became confluent, 33 µg/ml uridine and 13 µg/ml 5'-fluorodeoxyuridine was added to the culture medium to inhibit mitosis. Neurons from a second dissociation were plated onto the glial monolayer and maintained in 94% Dulbecco's modified Eagle's medium/5% horse serum/1% FBS, supplemented with 2 mM Glutamax, 5'-fluorodeoxyuridine, and a neuronal supplement. Experiments were performed at room temperature (23-25°C) using neurons after 2 to 15 days in culture.

Recording Solutions. Extracellular saline for neuronal experiments contained 166 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM BES (or HEPES), 10 mM glucose, and 0.01 mg/ml phenol red; the osmolarity was adjusted to 325 mOsM, and the pH titrated with NaOH to the desired value. When varying pH, the buffer species was selected to maintain optimal buffering capacity over the pH range examined and was held constant in each experiment because significant effects of varying buffer were observed in preliminary experiments (data not shown). Thus HEPES (pK_a = 7.48) buffered the solutions for experiments examining the effects of alkalinization, whereas BES (pK_a = 7.09) buffered solutions for all other experiments. Tetrodotoxin (400 nM; Calbiochem, La Jolla, CA), bicuculline (5 µM), and (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; 500 nM) were added to block voltage-gated sodium channels, GABA receptors, and NMDA receptors, respectively. MK-801 was also added to neuronal cultures in the incubator to allow spontaneous synaptic activity to effect the essentially irreversible activity-dependent block of NMDA receptors before recording. The standard intracellular solution for neurons contained 125 mM CsMeSO₃ (Aldrich Chemical Co., Milwaukee, WI), 15 mM CsCl, 10 mM HEPES, 5 mM Cs₄BAPTA (Molecular Probes, Eugene, OR), 0.5 mM CaCl₂, 3 mM MgCl₂ and 2 mM Na₂ATP; for recording from nucleated macropatches, 10 mM CsF replaced 10 mM CsMeSO₃. The osmolarity was adjusted to 305 to 310 mOsM, and the pH was titrated to 7.2 with CsOH. In some experiments, 35 mM CsMeSO₃ was replaced with 20 mM creatine phosphate (di-Tris salt) and an additional 2 mM Na₂ATP and 50 U/ml creatine phosphokinase were included (to regenerate ATP).

Electrophysiology. The recording chamber was continuously perfused with control extracellular saline at 0.2 to 0.5 ml/min. Recording electrodes were pulled from borosilicate glass, coated with Sylgard, and fire-polished; typical electrode resistance was 2 to 4 M when filled with intracellular solution. An isolated neuron or HEK 293 cell was voltage-clamped in the whole-cell configuration at 60 mV using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) and standard techniques. In experiments to examine the kinetics of desensitization, nucleated outside-out patches (macropatches) were used (Sather et al., 1992; Patneau et al., 1993). Resistance in series with the cell or macropatch was typically 4 to 8 M and was compensated by 60 to 90%. Data for on-line recording were filtered at 1 to 5 kHz and sampled at 2 to 20 kHz using pClamp (Axon Instruments).

Perfusion Techniques. For whole-cell recording, rapid agonist application was achieved using a glass flowpipe array (6-8 parallel barrels, each 400 µm in diameter) mounted on a `bimorph' ceramic wafer. The flowpipe array was placed near a voltage-clamped cell. Solutions were driven by a peristaltic pump (Minipuls3; Gilson, Middleton, WI) through three-way Isolatch valves (Parker Hannifin, Fairfield, NJ). Control solution (at the desired pH) from one barrel of the flowpipe continuously bathed the cell and its processes until agonist application was initiated; at this time the bimorph was charged, which rapidly moved an adjacent barrel containing agonist solution in front of the cell. Then, simultaneously, the valve regulating control solution flow was closed and the valve for agonist flow was opened. This system achieves solution exchange around a small neuron and its processes within approximately 10 ms. Other cells in the recording chamber were partially protected from pre-exposure to agonists by the inclusion of 0.5 mM kynurenate in the bath solution.

Data Analysis. Peak and steady-state amplitude and kinetic measurements were based on single or the average of a few (3-5) agonist-evoked responses. The majority of experiments examined the effects of protons on steady-state responses. Peak response amplitudes and kinetic measurements were only analyzed for macropatches, because the exchange time for whole-cell perfusion was too slow (see above) to make peak measurements reliable. Control responses preceded and followed an experimental manipulation. To correct for rundown, the experimental observation was normalized relative to the average of the two control responses. Numbers and statistical analyses in the text are based on these corrected responses. However, both control responses are illustrated in some bar graphs to demonstrate recovery (see Fig. 1). Because substantial rundown can indicate a compromised recording, data exhibiting more than a 15% (neurons) or 25% (macropatches and HEK 293 cells) decline in control responses were excluded from analysis.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Agonist-dependent modulation of AMPA receptors by protons. A, responses recorded from a hippocampal neuron to kainate (300 µM) and glutamate (1 mM) were inhibited by 19% and 44%, respectively, at pH 6.5 relative to pH 7.3. The solid bars above the traces indicate agonist application. The artifacts from the voltage-command switching the bimorph have been blanked (as indicated by gaps in the current traces) and the peak response to glutamate was truncated to better illustrate the change in the steady-state response. B, the bar graph compares the mean effect of acidic pH on responses to kainate and glutamate in the same neurons (n = 14). Responses at pH 6.5 were bracketed by control responses at pH 7.3 and data were normalized relative to the first response at pH 7.3. Responses to glutamate and kainate at pH 6.5 were significantly inhibited relative to both control responses (***P < .001), and responses to glutamate were inhibited significantly more than responses to kainate ( P < .001). The decline in kainate-evoked control responses, which reflects rundown, was significant (**P < .01).

Materials. Stock solutions of most drugs were prepared in the appropriate intracellular or extracellular solution, stored frozen, and added to experimental solutions immediately before use. When a very high final concentration ( 3 mM) of drug was required, the stock solution was made in NaCl-reduced saline to keep the final Na⁺ concentration constant across experimental solutions. Cyclothiazide was dissolved in dimethyl sulfoxide at 20 mM before dilution with extracellular solution, and an equivalent final concentration of dimethyl sulfoxide (0.5%) was added to all experimental and control solutions. The pK_a values of the carboxylic acid group of glutamate, quisqualate, homocysteate, kainate, and AMPA were determined by standard pH titration methods, with pK_a values of 4.41, 4.30, 2.79, 4.32, and 5.28, respectively. Thus, the concentrations of the unprotonated form of the first four of these agonists in solution at pH 6.5 and 7.3 were negligibly different and did not require adjustment; AMPA was not used because it would be partially protonated at pH 6.5. AMPA, bicuculline, kynurenate, MK-801, and quisqualate were purchased from Tocris Cookson (Ballwin, MO). Other reagents were obtained from Sigma Chemical (St. Louis, MO) unless noted otherwise.

	Results

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Modulation of AMPA Receptors by Protons Is Agonist-Dependent. Initial experiments focused on proton-mediated modulation of kainate- or glutamate-evoked responses in hippocampal neurons within a physiologically relevant pH range (pH 6.5-7.8). Alkaline pH (7.8) did not affect the amplitude of AMPA receptor-mediated responses to kainate or glutamate (data not shown). Because pathophysiological processes rarely alkalinize the interstitial milieu of the brain (Chesler, 1990) pH values greater that 7.8 were not tested. However, acidic pH (pH 6.8 or 6.5) did significantly affect responses to kainate and glutamate. This modulation was concentration-dependent, with larger effects observed at pH 6.5 than pH 6.8. The amplitudes of glutamate-evoked responses at pH 6.8 and pH 6.5 were 90 ± 3% and 72 ± 4% (P < .01, n = 6) of those at pH 7.3, respectively. Kainate-evoked responses at pH 6.8 and pH 6.5 were significantly reduced to 95 ± 2% and 86 ± 4% (P < .01, n = 10) of those at pH 7.3, respectively. To maximize the effect of protons while reproducing pathophysiologic conditions associated with ischemia, all further experiments compared agonist-evoked responses recorded at pH 7.3 (control) and pH 6.5.

Modulators of AMPA Receptor Desensitization Block Proton-Mediated Inhibition. Because kainate and glutamate differ in their desensitization properties (Patneau et al., 1993), the agonist-dependence of proton modulation suggested that protons may affect desensitization. One way in which protons could modulate desensitization is selective interaction with the desensitized state of the receptor. To determine whether modulation of AMPA receptors by protons was state-dependent, extracellular pH was varied as glutamate was applied to neurons in the presence of trichlormethiazide or cyclothiazide, benzothiadiazine modulators that block desensitization of AMPA receptors by glutamate (Patneau et al., 1993; Yamada and Tang, 1993).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Drugs that prevent AMPA receptor desensitization block proton-mediated inhibition. A, nondesensitizing responses to 1 mM glutamate (solid bar) recorded from a hippocampal neuron in the presence of 500 µM trichlormethiazide (open bar) are unaffected by pH. The traces recorded at pH 7.3 (thin trace) and pH 6.5 (thick trace) almost superimpose. B, the bar graph shows the mean effect of acidic pH on responses to glutamate recorded in the presence of 500 µM trichlormethiazide (n = 6) or 100 µM cyclothiazide (n = 8). Steady-state responses at pH 6.5 were bracketed by control responses and normalized relative to the first response at pH 7.3. There was no significant difference between responses at pH 6.5 and 7.3 in the presence of trichlormethiazide. The slight (3%) decline in responses at pH 6.5 in the presence of cyclothiazide was significant relative to the first control response (**P = 0.01), but not the second.

Protons Do Not Affect AMPA Receptor Desensitization or Deactivation Kinetics. The results of experiments described above suggested that protons modulate AMPA receptor desensitization. The effects of protons on the kinetics of desensitization were examined using macropatches pulled from hippocampal neurons and a piezo-based perfusion system to achieve submillisecond solution exchange. Responses to applications of three agonists, L-glutamate (3 mM), L-homocysteate (10 mM), and kainate (3 mM), were analyzed. Homocysteate is a strongly desensitizing full agonist with desensitization onset kinetics similar to those for glutamate, but with much lower affinity for the receptor (steady-state EC₅₀ value of 447 µM versus 19 µM for glutamate; Patneau and Mayer, 1990, 1991). Kainate is a weakly desensitizing partial agonist with desensitization kinetics that are significantly faster than glutamate and an intermediate affinity for the receptor (142 µM; Patneau et al., 1993).

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Modulation by protons does not affect the kinetics of AMPA receptor desensitization or deactivation. A, examples of desensitizing responses from hippocampal neuron macropatches evoked by 100-ms applications of glutamate (3 mM) and homocysteate (10 mM) or 40-ms application of kainate (3 mM) at pH 7.3 (thin traces) and 6.5 (thick traces). Agonist application in A and B is denoted by the junction potentials obtained after disrupting the macropatch. Amplitude scale bar denotes 300 pA (glutamate), 150 pA (kainate), or 400 pA (homocysteate). Steady-state responses to homocysteate at 10× amplification are also illustrated (indicated by the dashed traces). B, responses to 1-ms applications of glutamate and homocysteate were used to isolate deactivation from receptor desensitization. Note the very rapid deactivation of responses to the low affinity agonist homocysteate. C, desensitization onset was unaffected by acidic pH. The bar graph shows the mean time constants determined by fitting the decay of responses in the presence of agonist, as illustrated in A, with a single (kainate) or the sum of two exponentials (glutamate and homocysteate). D, deactivation kinetics were not affected by a change in extracellular pH for any of the three agonists. The bar graph shows mean time constants determined from single exponential fits to the decay of responses after removal of agonist, after a 1-ms application of glutamate or homocysteate (B), or after cessation of a 40-ms application of kainate (A). E, protons did not affect the kinetics of recovery from desensitization. The rate of recovery from desensitization after a 50-ms application of glutamate (open bar) was assessed with a paired-pulse protocol as illustrated in the inset. The arrows above the overlaid current traces for eight different intervals indicate 10-ms glutamate pulses. The graph is data from a single macropatch for paired-pulse intervals from 4 to 800 ms. The resulting recovery curves were fit with single exponentials, with time constants of 116 ms at pH 7.3 and 113 ms at pH 6.5. F, comparisons between inhibition of steady-state and peak amplitudes at pH 6.5 relative to pH 7.3 were made for agonist-evoked responses as illustrated in A. Only macropatches for which the steady-state amplitude of glutamate- and homocysteate-evoked responses at pH 7.3 was  10 pA were included in this analysis. Acidic pH significantly inhibited both the peak and steady-state amplitudes for all three agonists (**P < .01), but steady-state responses to glutamate and homocysteate were inhibited significantly more than peak responses (P < .01).

Protons Increase Steady-State Desensitization of AMPA Receptors. The rapid solution exchange possible on macropatches permitted comparison of the effects of protons on peak and steady-state responses. This analysis included all macropatches exposed to kainate. However, only macropatches with control steady-state responses to glutamate and homocysteate  10 pA were included because changes in the amplitude of smaller responses could not be distinguished from background noise. The peak amplitudes of responses to all three agonists were significantly inhibited at pH 6.5 relative to pH 7.3 (Fig. 3F). Inhibition of steady-state responses to glutamate (31 ± 13%) and kainate (22 ± 5%) in macropatches was comparable with that observed in whole-cell recording. Peak responses to glutamate and homocysteate were inhibited by approximately 10%, significantly less than steady-state responses (Fig. 3D); this indicates that the conformational states of the receptor occupied during instantaneous (peak) and equilibrium (steady-state) responses to glutamate are differentially sensitive to protons. The difference between inhibition of peak and steady-state responses to kainate at pH 6.5 was not significant.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Proton-mediated inhibition and desensitization of kainate-evoked responses covary. A, responses to 3 mM kainate recorded from two macropatches illustrate variability in the magnitude of desensitization between patches. Note that the responses on the left exhibit both more extensive kainate-evoked desensitization and greater proton-mediated inhibition at pH 6.5. B, percentage desensitization is plotted versus percentage inhibition of steady-state responses evoked by kainate for 11 macropatches. The solid line is a linear regression fit to the data (r = 0.89). C, leak-subtracted data for current-voltage determinations of kainate-evoked responses in a macropatch at pH 7.3 and 6.5 recorded at voltages from 80 to +80 mV. Note the apparent voltage-dependence of receptor desensitization but voltage-independence of proton-mediated inhibition. D, percentage desensitization of responses illustrated in C is plotted versus voltage. At all voltages kainate-evoked responses exhibited almost 10% more desensitization at pH 6.5 than at pH 7.3. Solid lines are linear regressions fit to the data with a correlation of r .97 for each fit.

Proton-Mediated Modulation Varies with AMPA Receptor Composition. Molecular composition has previously been shown to affect proton modulation of NMDA and GABA receptors (Traynelis et al., 1995; Krishek et al., 1996). The experiments described above suggest that AMPA receptor desensitization and proton modulation interact. Subunit (GluR-A to -D or 1-4; Hollmann and Heinemann, 1994) and isoform (flip or flop) composition of recombinant AMPA receptors determine desensitization time constants and the magnitude of steady-state desensitization of glutamate-evoked responses (Mosbacher et al., 1994; Partin et al., 1994). The flip (i) isoforms exhibit slower rates of desensitization and less extensive steady-state desensitization, whereas the flop (o) isoforms, and particularly Do, desensitize very rapidly (<1 ms) to a steady-state response that is 1% of the peak. We therefore tested the hypothesis that proton-mediated modulation would vary with the desensitization properties of recombinant AMPA receptors. HEK 293 cells were transiently transfected with either flip or flop isoforms of two subunits, and agonist-evoked responses were recorded at pH 7.3 and pH 6.5.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   The molecular composition of AMPA receptors determines their sensitivity to proton-mediated modulation. HEK 293 cells were transfected with cDNA for heterodimeric receptors varying in both subunit and isoform composition. A, kainate-evoked responses at pH 7.3 (thinner traces) and 6.5 (thicker traces) for the two compositions least (AiBi) and most (AoDo) sensitive to proton-mediated inhibition. Scale bars are 50 ms, 30 pA (AiBi); and 75 ms, 20 pA (AoDo). The bimorph switching artifacts have been blanked, as indicated by gaps in the current traces. B, mean ratios of responses evoked by kainate at pH 6.5 relative to pH 7.3 for HEK 293 cells transfected with the glutamate receptor subunits/isoforms GluR AiBi (n = 7), AoBo (n = 8), AiDi (n = 10), or AoDo (n = 7). Note the greater sensitivity to modulation by protons of flop isoforms (***P < .001), and that the receptor composition including the subunit exhibiting the fastest rate of onset of desensitization (Mosbacher et al., 1994), AoDo, was most strongly inhibited.

	Discussion

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These experiments have produced three novel and significant findings. First, proton-mediated inhibition of AMPA receptors is dependent on the type of agonist. Second, protons seem to specifically interact with receptor desensitization but do not affect the macroscopic kinetics of desensitization. Finally, molecular receptor composition determines the magnitude of proton-mediated inhibition.

Previous research on proton-mediated modulation of non-NMDA receptors concluded that protons increase the closed time of the channel (Christensen and Hida, 1990; Traynelis and Cull-Candy, 1991). However, these studies did not determine whether protons increased closed time by directly affecting the transitions between open and closed states or, more indirectly, by affecting receptor desensitization. The experiments described above indicate that protons affect receptor desensitization. However, because protons do not affect desensitization kinetics or the affinity of the desensitized state, our results are inconsistent with a simple model of AMPA receptor gating that includes a single open state accessible from a nondesensitized state (e.g., Patneau and Mayer, 1991). One way in which protons could affect steady-state receptor desensitization without affecting the macroscopic kinetics of desensitization is through modulation of a second open state accessible from a desensitized state. Inclusion of such an open state has been proposed previously by Raman and Trussell (1992) in their model for AMPA receptor gating in chick cochlear nucleus.

This cyclic model for AMPA receptor gating and desensitization is based on one originally proposed by Patneau and Mayer (1991), but includes a second open state that is accessible from the desensitized state. A represents the agonist; R and R_D represent the receptor in its unoccupied resting and desensitized states, respectively; A₂R is the agonist-bound, active (closed) state; A₂R_D is the agonist-bound, desensitized state; d₁ and d₁ represent the rate constants for entry into and recovery from the desensitized state, respectively;  and  designate rate constants of channel opening and closing for the first open state (Open), whereas ^' and ^' are the equivalent rate constants for the second open state (Open^').
Support for protons specifically modulating a "desensitized" open state comes from several findings. First, the smaller effect of protons on responses to the weakly desensitized agonist kainate, compared with the strongly desensitizing agonist glutamate, suggest that the receptor is more susceptible to proton-mediated modulation when it spends more time in the desensitized state. Second, benzothiadiazine drugs that prevent AMPA receptor desensitization also block proton-mediated modulation. Because protons do not affect the rate of entry into or recovery from desensitization, this suggests that protons affect another open state that is not available when desensitization is blocked. Third, inhibition of kainate-evoked responses in macropatches was correlated with the magnitude of desensitization, and protons increased equilibrium desensitization without affecting the macroscopic kinetics of desensitization. Fourth, in transfected cells, receptor compositions previously shown to exhibit the fastest and most extensive desensitization were most sensitive to modulation. Finally, the model is supported by the findings that protons did not affect the kinetics of receptor deactivation for any of the three agonists examined and that protons inhibited peak responses to glutamate and homocysteate significantly less than steady-state responses. If there were only a single open state, and protons increased its closed time, receptor deactivation should have been affected. Although the kinetics of deactivation after removal of glutamate may reflect multiple openings with agonist still bound (see Fig. 3B), the very rapid deactivation observed after removal of the low-affinity agonist homocysteate would seem to accurately reflect the true channel-closing rate, which was not altered by protons. This result therefore suggests that the conformational states of the receptor underlying the instantaneous (peak) and equilibrium (steady-state) responses to glutamate are differentially sensitive to protons.

Our experiments indicate that modulation of AMPA receptors by protons in vivo will depend on both receptor composition and the magnitude of the pH change, whether the acidification is global and persistent, as in ischemia, or local and transient, as may occur in the cleft during synaptic transmission. Although local fluctuations in pH at the synapse have not been directly quantified, the processes that may contribute are known. Glutamatergic vesicles of hippocampal neurons are more acidic than the interstitial milieu (pH 5.67; Miesenbock et al., 1998). Thus, a rapid acidic transient is predicted to occur simultaneously with the release of neurotransmitter (Krishtal et al., 1987) and could be further amplified by deprotonation of glutamate upon release into the less acidic cleft. In addition, proton ATPases from synaptic vesicles may be incorporated in the presynaptic membrane in vesicle fusion and could transport protons from the presynaptic terminal into the cleft (Krishtal et al., 1987). The receptors directly under the site of release should see the largest acidic transient.

The temporal and spatial dispersion of acidic transients will be determined by the kinetics and capacity of the endogenous CO₂/HCO₃ buffering system. Physiological levels of bicarbonate in the brain confer a high buffer capacity, but substantial experimental evidence suggests that synaptic activation produces transient changes in interstitial pH that are not rapidly buffered (Chesler, 1990). Thus, rapid acidic transients in the synaptic cleft sufficient to significantly affect the function of postsynaptic AMPA receptors are likely to occur, in particular with high frequency synaptic activity or at synapses with multivesicular release.

In animal models of ischemia, significant protection from excitotoxic cell death is conferred by AMPA receptor antagonists (Sheardown et al., 1990; Gill, 1994). The global acidification of the brain that occurs during ischemia is neuroprotective for NMDA receptor-mediated cell death, and it was assumed that it would also reduce AMPA receptor-mediated cell death (Christensen and Hida, 1990; Giffard et al., 1990). In contrast, McDonald et al. (1998) found that acidification similar to that occurring during ischemia (pH 6.6) significantly potentiated AMPA receptor-mediated neurotoxicity in neuronal cultures.

Although our findings do not provide an explanation for the results of McDonald et al. (1998), they suggest a mechanism for the selective vulnerability of principal neurons to ischemia (Cervos-Navarro and Diemer, 1991). Brorson et al. (1995) found that AMPA receptor-mediated responses of Purkinje cells, which are among those cell types most vulnerable to ischemia, exhibit less complete desensitization than those of other cerebellar neurons. This difference in desensitization properties has recently been related to higher levels of expression of flip isoforms of AMPA receptor subunits (Tomiyama et al., 1999). Similarly, cortical pyramidal cells exhibit slower and less extensive desensitization than interneurons (Hestrin, 1993). The desensitization properties of pyramidal neurons are also highly correlated with expression of AMPA receptor flip isoforms (Lambolez et al., 1996). Thus, AMPA receptors that exhibit less steady-state desensitization (e.g., flip-containing receptors) will disproportionately contribute to the neuronal depolarization resulting from tonic glutamate release and will also be less sensitive to the potentially inhibitory effects of interstitial protons. Neurons expressing these AMPA receptors are predicted to be more vulnerable to glutamate-mediated excitotoxicity in ischemia.