Role of membrane GM1 on early neuronal membrane actions of Aß during onset of Alzheimer's disease.

The ability of beta-amyloid peptide (Aß) to disrupt the plasma membrane through formation of pores and membrane breakage has been previously described. However, the molecular determinants for these effects are largely unknown. In this study, we examined if the association and subsequent membrane perforation induced by Aß was dependent on GM1 levels. Pretreatment of hippocampal neurons with D-PDMP decreased GM1 and Aß clustering at the membrane (Aß fluorescent-punctas/20µm, control=16.2±1.1 vs. D-PDMP=6.4±0.4, p<0.001). Interestingly, membrane perforation with Aß occurred with a slower time course when the GM1 content was diminished (time to establish perforated configuration (TEPC) (min): control=7.8±2 vs. low GM1=12.1±0.5, p<0.01), suggesting that the presence of GM1 in the membrane can modulate the distribution and the membrane perforation by Aß. On the other hand, increasing GM1 facilitated the membrane perforation (TEPC: control=7.8±2 vs. GM1=6.2±1min, p<0.05). Additionally, using Cholera Toxin Subunit-B (CTB) to block the interaction of Aß with GM1 attenuated membrane perforation significantly. Furthermore, pretreatment with CTB decreased the membrane association of Aß (fluorescent-punctas/20µm, Aß: control=14.8±2.5 vs. CTB=8±1.4, p<0.05), suggesting that GM1 also plays a role in both association of Aß with the membrane and in perforation. In addition, blockade of the Aß association with CTB inhibited synaptotoxicity. Taken together, our results strongly suggest that membrane lipid composition can affect the ability of Aß to associate and subsequently perforate the plasma membrane thereby modulating its neurotoxicity in hippocampal neurons.

Mutations at F637 in the NMDA receptor NR2A subunit M3 domain influence agonist potency, ion channel gating and alcohol action.

BACKGROUND AND PURPOSE: NMDA receptors are important molecular targets of ethanol action in the CNS. Previous studies have identified a site in membrane-associated domain 3 (M3) of the NR1 subunit and two sites in M4 of the NR2A subunit that influence alcohol action; the sites in NR2A M4 also regulate ion channel gating. The purpose of this study was to determine whether mutations at the site in the NR2A subunit corresponding to the NR1 M3 site influence alcohol action and ion channel gating. EXPERIMENTAL APPROACH: We investigated the effects of mutations at phenylalanine (F) 637 of the NR2A subunit using whole-cell and single-channel patch-clamp electrophysiological recording in transiently-transfected HEK 293 cells. KEY RESULTS: Mutations at F637 in the NR2A subunit altered peak and steady-state glutamate EC(50) values, maximal steady-state to peak current ratios (I(ss):I(p)), mean open time, and ethanol IC(50) values. Differences in glutamate potency among the mutants were not due to changes in desensitization. Ethanol IC(50) values were significantly correlated with glutamate EC(50) values, but not with maximal I(ss):I(p) or mean open time. Ethanol IC(50) values were linearly and inversely related to molecular volume of the substituent. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that NR2A(F637) influences NMDA receptor affinity, ion channel gating, and ethanol sensitivity. The changes in NMDA receptor affinity are likely to be the result of altered ion channel gating. In contrast to the cognate site in the NR1 subunit, the action of ethanol does not appear to involve occupation of a critical volume at NR2A(F637).

Intersubunit interactions at putative sites of ethanol action in the M3 and M4 domains of the NMDA receptor GluN1 and GluN2B subunits.

BACKGROUND AND PURPOSE: The NMDA receptor is an important target of alcohol action in the brain. Recent studies in this laboratory have demonstrated that alcohol-sensitive positions in the intersubunit interfaces of the M3 and M4 domains of GluN1 and GluN2A subunits interact with respect to ethanol sensitivity and receptor kinetics and that alcohol-sensitive positions in the M domains of GluN2A and GluN2B subunits differ. In this study, we tested for interactions among alcohol-sensitive positions at the M domain intersubunit interfaces in GluN1/GluN2B NMDA receptors. EXPERIMENTAL APPROACH: We used whole-cell patch-clamp recording in tsA201 cells expressing tryptophan substitution mutants at ethanol-sensitive positions in the GluN1 and GluN2B NMDA receptor subunits to test for interactions among positions. KEY RESULTS: Six pairs of positions in GluN1/GluN2B significantly interacted to regulate ethanol inhibition: Gly(638) /Met(824) , Gly(638) /Leu(825) , Phe(639) /Leu(825) , Phe(639) /Gly(826) , Met(818) /Phe(637) and Val(820) /Phe(637) . Tryptophan substitution at Met(824) or Leu(825) in GluN2B did not alter ethanol sensitivity but interacted with positions in the GluN1 M3 domain to regulate ethanol action, whereas tryptophan substitution at Gly(638) , which is the cognate of an ethanol-sensitive position in GluN2A, did not alter ethanol sensitivity or interact with positions in GluN1. Two and three pairs of positions interacted to regulate glutamate steady-state and peak current EC50 , respectively, and one pair interacted with respect to macroscopic desensitization. CONCLUSIONS: Despite highly-conserved M domain sequences and similar ethanol sensitivity in the GluN2A and GluN2B subunits, the manner in which these subunits interact with the GluN1 subunit to regulate ethanol sensitivity and receptor kinetics differs.

Alcohols inhibit N-methyl-D-aspartate receptors via a site exposed to the extracellular environment.

N-Methyl-D-aspartate (NMDA) receptors are important CNS target sites of alcohols, but the site and mechanism of action of alcohols on NMDA receptors remains unclear. In CHO-K1 cells transfected with NR1/NR2B NMDA receptor subunits, ethanol inhibited NMDA-activated current with an IC(50) of 138 mM. Truncation of the intracellular C-terminal domain of the NR1 subunit (NR1T) did not alter ethanol sensitivity when combined with the NR2B subunit, but a similar truncation of the NR2B subunit (NR2BT) slightly enhanced ethanol sensitivity of receptors formed from coexpression with either NR1 or NR1T subunits. 1-Pentanol applied externally inhibited NMDA receptors with an IC(50) of 9.9 mM, but intracellular application of 1-pentanol (25 mM) did not alter NMDA receptor inhibition by externally applied ethanol or 1-pentanol. In addition, the amplitude of NMDA-activated current did not decrease during the time required for 1-pentanol (25 mM) to diffuse throughout the cytoplasm. Ethanol did not inhibit NMDA receptors when bath-applied in cell-attached patches or when applied to the cytoplasmic face of inside-out membrane patches. These results appear to be best explained by an action of alcohols on the NMDA receptor-channel protein, at a site located in a domain exposed to, or only accessible from, the extracellular environment.

Inhibition of NMDA-gated ion channels by the P2 purinoceptor antagonists suramin and reactive blue 2 in mouse hippocampal neurones.

1. The action of suramin and reactive blue 2 on N-methyl-D-aspartate (NMDA)-activated ion current was studied in mouse hippocampal neurones in culture by use of whole-cell patch-clamp recording. 2. Suramin and reactive blue 2 inhibited steady-state current activated by 25 microM NMDA with IC50 values of 68 and 11 microM, respectively. 3. Reactive blue 2 produced a gradual decline of NMDA-activated current to a steady-state, but this slow onset was not an indication of use-dependence, as it could be eliminated by exposure to reactive blue 2 before NMDA application. In addition, NMDA-activated current recovered completely from inhibition by reactive blue 2 in the absence of agonist. 4. The slow onset of inhibition by reactive blue 2 was not apparently due to an action at an intracellular site, as inclusion of 250 microM reactive blue 2 in the recording pipette did not alter inhibition by 25 microM reactive blue 2 applied externally. 5. Reactive blue 2 and suramin inhibited NMDA-gated channels in a voltage-independent manner. 6. Reactive blue 2, 25 microM, decreased the maximal response to NMDA from 1441 to 598 pA without changing its EC50. In contrast, 75 microM suramin increased the EC50 for NMDA from 13 to 35 microM, and decreased the maximal response to NMDA from 1822 to 1498 pA. Schild analysis of suramin inhibition of NMDA-activated current yielded a nonlinear plot. 7. Both agents decreased the maximal response to glycine without altering its EC50. 8. Suramin and reactive blue 2 appear to inhibit NMDA receptor-channels in a manner that is noncompetitive with respect to both NMDA and glycine. However, inhibition by suramin differed from that by reactive blue 2, in that suramin significantly increased the EC50 of NMDA.

Inhibition of excitatory amino acid-activated currents by trichloroethanol and trifluoroethanol in mouse hippocampal neurones.

1. The effects of the active metabolite of chloral derivative sedative-hypnotic agents, 2,2,2-trichloroethanol (trichloroethanol), and its analog 2,2,2-trifluoroethanol (trifluoroethanol), were studied on ion current activated by the excitatory amino acids N-methyl-D-aspartate (NMDA) and kainate in mouse hippocampal neurones in culture using whole-cell patch-clamp recording. 2. Both trichloroethanol and trifluoroethanol inhibited excitatory amino acid-activated currents in a concentration-dependent manner. Trichloroethanol inhibited NMDA- and kainate-activated currents with IC50 values of 6.4 and 12 mM, respectively, while trifluoroethanol inhibited NMDA- and kainate-activated currents with IC50 values of 28 and 35 mM, respectively. 3. Both trichloroethanol and trifluoroethanol appeared to be able to inhibit excitatory amino acid-activated currents by 100 per cent. 4.Concentration-response analysis of NMDA- and kainate-activated current revealed that trichloroethanol decreased the maximal response to both agonists without significantly affecting their EC50 values. 5. Both trichloroethanol and trifluoroethanol inhibited excitatory amino acid-activated currents more potently than did ethanol. The inhibitory potency of trichloroethanol and trifluoroethanol appears to be associated with their increased hydrophobicity. 6. The observation that trichloroethanol inhibits excitatory amino acid-activated currents at anaesthetic concentrations suggests that inhibition of excitatory amino acid receptors may contribute to the CNS depressant effects of chloral derivative sedative-hypnotic agents.

Trichloroethanol potentiation of gamma-aminobutyric acid-activated chloride current in mouse hippocampal neurones.

1. The action of 2,2,2-trichloroethanol on gamma-aminobutyric acid (GABA)-activated Cl- current was studied in mouse hippocampal neurones in tissue culture by use of whole-cell patch-clamp recording. 2. Trichloroethanol increased the amplitude of currents activated by 1 microM GABA or 0.1 microM muscimol. Trichloroethanol, 1-25 mM, potentiated current activated by 1 microM GABA in a concentration-dependent manner with an EC50 of 3.0 +/- 1.4 mM and a maximal response (Emax) of 576 +/- 72% of control. 3. Trichloroethanol potentiated currents activated by GABA concentrations < 10 microM, but did not increase the amplitude of currents activated by concentrations of GABA > or = 10 microM. Despite marked potentiation of currents activated by low concentrations of GABA, trichloroethanol did not significantly alter the EC50, slope, or Emax of the GABA concentration-response curve. 4. Trichloroethanol, 5 mM, potentiated GABA-activated current in neurones in which ethanol, 10-500 mM, did not. The effect of trichloroethanol was not altered by the putative ethanol antagonist, Ro 15-4513. Trichloroethanol did not potentiate currents activated by pentobarbitone. 5. In the absence of exogenous GABA, trichloroethanol at concentrations > or = 2.5 mM activated a current that appeared to be carried by Cl- as its reversal potential changed with changes in the Cl- gradient and as it was inhibited by the GABAA antagonists, bicuculline methiodide and picrotoxin. 6. Since trichloroethanol is thought to be the active metabolite of chloral hydrate and other chloral derivative anaesthetics, potentiation of the GABA-activated current in central nervous system neurones by trichloroethanol may contribute to the sedative/hypnotic effects of these agents.

Ethanol-induced inhibition of a neuronal P2X purinoceptor by an allosteric mechanism.

Ethanol inhibits a neuronal P2X purinoceptor by shifting the ATP concentration-response curve to the right in an apparently competitive manner. However, the underlying mechanism has not been determined. We investigated the effects of ethanol on the activation and deactivation time constants for ATP-activated current in bullfrog dorsal root ganglion neurones. Ethanol decreased the time constant of deactivation of ATP-gated ion channels without affecting the time constant of activation. The observations are not consistent with a competitive mechanism of inhibition by ethanol, but may be explained by an allosteric action of ethanol to decrease apparent agonist affinity. This represents a novel mechanism of action of ethanol on a neurotransmitter-gated ion channel.

Ethanol inhibition of N-methyl-D-aspartate-activated current in mouse hippocampal neurones: whole-cell patch-clamp analysis.

1. The action of ethanol on N-methyl-D-aspartate (NMDA)-activated ion current was studied in mouse hippocampal neurones in culture using whole-cell patch-clamp recording. 2. Ethanol inhibited NMDA-activated current in a voltage-independent manner, and did not alter the reversal potential of NMDA-activated current. 3. Concentration-response analysis of NMDA- and glycine-activated current revealed that ethanol decreased the maximal response to both agonists without affecting their EC50 values. 4. The polyamine spermine (1 microM) increased amplitude of NMDA-activated current but did not alter the percentage inhibition of ethanol. 5. Compared to an extracellular pH of 7.0, pH 6.0 decreased and pH 8.0 increased the amplitude of NMDA-activated current, but these changes in pH did not significantly alter the percentage inhibition by ethanol. 6. The sulphydryl reducing agent dithiothreitol (2 mM) increased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 7. Mg2+ (10, 100, 500 microM), (5, 20 microM) or ketamine (2, 10 microM) decreased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 8. The observations are consistent with ethanol inhibiting the function of NMDA receptors by a non-competitive mechanism that does not involve several modulatory sites on the NMDA receptor-ionophore complex.

Volatile general anaesthetic actions on recombinant nACh alpha 7, 5-HT3 and chimeric nACh alpha 7-5-HT3 receptors expressed in Xenopus oocytes.

The effect of halothane and isoflurane was studied on the function of recombinant neurotransmitter receptors expressed in Xenopus oocytes. Both anaesthetics inhibited nicotinic acetylcholine type alpha 7 (nACh alpha 7) receptor-mediated responses, potentiated 5-hydroxytryptamine type 3 (5-HT3) receptor-mediated responses at low agonist concentrations, and inhibited the function of a chimeric receptor (with the N-terminal domain from the nACh alpha 7 receptor and the transmembrane and C-terminal domains from the 5-HT3 receptor) in a manner similar to that of the nACh alpha 7 receptor. Since the N-terminal domain of the chimeric receptor was from the nACh alpha 7 receptor, the observations suggest that the inhibition involves the N-terminal domain of the receptor.

Gamma-aminobutyric acidA (GABAA) receptor modulation of morphine inhibition of norepinephrine release.

Agents that enhance gamma-aminobutyric acid (GABA) neurotransmission can modulate certain effects of opioids, such as analgesia. In this study, the interaction between morphine and GABAergic agents on the release of [3H]norepinephrine ([3H]NE) from rat frontal cortical slices was examined. GABA (10(-4) M), enhanced potassium-stimulated [3H]NE release and reversed the inhibitory effect of 10(-6) M morphine. GABA and muscimol modulated the inhibitory effect of morphine in a noncompetitive manner. Bicuculline methiodide (10(-4) M) reduced the effect of GABA in the absence of morphine, and appeared to reduce the effect of GABA in the presence of morphine, although the latter effect was not statistically significant from the controls. While the GABAA agonist muscimol mimicked the effect of GABA, the GABAB agonist baclofen did not affect the release of [3H]NE in the absence or the presence of 10(-6) M morphine. These results support the involvement of GABAA receptors in modulating the action of opioids on the noradrenergic system in the cerebral cortex of the rat.

Gamma-aminobutyric acid enhancement of potassium-stimulated release of [3H]norepinephrine by multiple mechanisms in rat cortical slices.

Gamma-Aminobutyric acid (GABA) and GABAA agonists enhance stimulated release of [3H]norepinephrine [( 3H]NA) in several regions of the rat brain. In this study, the mechanisms by which GABA and GABAergic agonists augment potassium-stimulated release of [3H]NA from rat frontal cortical slices were examined. GABA enhanced potassium-stimulated [3H]NA release, but did not alter release of [3H]NA evoked by the calcium ionophore A23187, 10(-5) M, either in the presence or the absence of extracellular calcium. The effect of GABA on potassium-stimulated [3H]NA release was apparently reduced by the GABAA antagonist bicuculline methiodide, 10(-4) M, and by the selective inhibitor of GABA uptake SKF 89976A, 10(-5) M, but was abolished only when bicuculline methiodide and SKF 89976A were present in combination. The GABAA agonist muscimol enhanced potassium-stimulated release of [3H]NA in a manner similar to GABA. In addition, nipecotic acid, a substrate for GABA uptake, enhanced potassium-stimulated [3H]NA release. Thus, GABA appears to enhance potassium-stimulated [3H]NA release by acting upon both GABA uptake and GABAA receptors. The GABAA receptors involved in this effect may be a subtype of GABAA receptors since they are not modulated by benzodiazepines. These results support the involvement of the GABA uptake carrier and the GABAA receptor in mediating the enhancement by GABA of potassium-stimulated [3H]NA release in the cortex of the rat.

Identification of functional beta-adrenergic receptors on AC glioma cells.

AC glioma cells, a clonal cell line derived from a rat glioma, responded to 1 mM dibutyryl-cyclic AMP and isobutylmethylxanthine with a change to stellate morphology. A concentration-related morphological change was induced by beta 1- and beta 2-adrenergic agonists with the order of potency being isoproterenol greater than soterenol greater than norepinephrine. Propranolol (nonselective, beta-antagonist), butoxamine (beta 2-antagonist) and metoprolol (beta 1-antagonist) significantly decreased the cell response to isoproternol. Schild analysis of the response, using the competitive antagonist metoprolol, gave pA2 values of 7.5 and 8.5 for the agonists norepinephrine and soterenol, respectively, with slopes of the curves being less than unity. These observations indicate that both beta 1- and beta 2-adrenergic receptors mediate the change in cellular morphology.

Alcohol action on membrane ion channels gated by extracellular ATP (P2X receptors).

Extracellular adenosine 5'-triphosphate (ATP) has been reported to produce excitatory actions in the nervous system, such as excitatory postsynaptic potentials or currents in both central and peripheral neurons, via activation of a class of ATP-gated membrane ion channels designated P2X receptors. This article reviews studies of alcohol effects on these receptor-channels. Ethanol has been found to inhibit ATP-gated ion channel function by shifting the agonist concentration-response curve to the right in a parallel manner, increasing the EC50 without affecting Emax of this curve. To distinguish whether this inhibition involves competitive antagonism of agonist action or a decrease in the affinity of the agonist binding site, the kinetics of activation and deactivation of agonist-activated current were studied. Ethanol was found to decrease the time-constant of deactivation of ATP-gated ion channels without affecting the time-constant of activation, indicating that ethanol inhibits the function of these receptors by an allosteric decrease in the affinity of the agonist binding site. The inhibition of ATP-gated ion channel function by a number of alcohols was found to exhibit a distinct cutoff effect that appeared to be related to the molecular volume of the alcohols. For alcohols with a molecular volume of < or = 42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. This cutoff effect has been interpreted as evidence that alcohols inhibit the function of ATP-gated ion channels by interacting with a hydrophobic pocket of circumscribed dimensions on the receptor protein. To evaluate the localization of this presumed alcohol binding site, the effect of the intracellular application of ethanol was studied on the inhibition of ATP-activated current by extracellularly applied ethanol. The intracellular application of 100 mM ethanol did not affect the inhibition of current by 100 mM extracellular ethanol, suggesting that the alcohol inhibition of ATP-gated ion channel function involves the extracellular domain of the receptor. Finally, recent studies suggest that the alcohol sensitivity of ATP-gated channels may be regulated by physiological mechanisms.

Alcohol and anesthetic actions on excitatory amino acid-activated ion channels.

The actions of alcohol and anesthetics have been studied on excitatory amino acid activated ion channels in mammalian neurons. Ethanol inhibits NMDA-activated current over a concentration range that produces intoxication, and the potency of several alcohols for inhibiting the NMDA-activated current is correlated with their intoxicating potency, suggesting that alcohol-induced inhibition of responses to NMDA receptor activation may contribute to the neural and cognitive impairments associated with intoxication. Studies on the mechanism of ethanol inhibition of NMDA-activated current indicate that ethanol does not appear to block the ion channel, alter the ion selectivity of the channel, or interact with previously described binding sites on the NMDA receptor/ionophore complex. The linear relation between the potency of several alcohols for inhibiting the NMDA-activated current and the hydrophobicity of the alcohols suggests that ethanol may inhibit the NMDA-activated ion current by a novel type of interaction with a hydrophobic site associated with the NMDA channel. In addition, different types of general anesthetic agents exhibit different inhibitory actions on NMDA-, kainate-, and quisqualate-activated currents, suggesting that differences in the profile of inhibition of excitatory amino acid neurotransmission in the CNS among different classes of general anesthetics may contribute to the differences in their behavioral and physiological effects.

Differential alcohol modulation of GABA(A) and NMDA receptors.

NMDA and GABA(A) receptors are believed to be important CNS targets of alcohol action. In mouse hippocampal neurons, n-alcohols from ethanol to dodecanol enhanced GABA-activated ion current, whereas higher alcohols had no effect. Alcohols below pentanol affected NMDA receptors more potently than GABA(A) receptors. Increasing alcohol carbon chain length produced a greater average change in apparent binding energy and potency for modulation of GABA(A) than of NMDA receptor-channels, with the result that alcohols above pentanol affected GABA(A) receptors more potently than NMDA receptors. The anesthetic potency of n-alcohols in rats more closely reflected NMDA receptor modulatory potency for lower alcohols and GABA(A) receptor modulatory potency for higher alcohols. The results suggest that there may be fundamental differences in the sites through which alcohols affect NMDA and GABA(A) receptor function.

Acid pH augments excitatory action of ATP on a dissociated mammalian sensory neuron.

ATP stimulates nociceptive neurons via an action on ligand-gated ion channels. Since tissue injury and inflammation result in both localized acidosis and release of ATP, we studied the effect of acid pH on ATP-gated ion channels in rat nodose ganglion neurons. Lowering pH dramatically increased membrane depolarization and action potential firing elicited by ATP. ATP-activated current was enhanced by acid pH and suppressed by alkaline pH. A pH of 7.2 produced the half-maximal effect. Acidification increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. The observations suggest that the localized acidosis associated with tissue injury may enhance pain perception via an action on ATP-gated ion channels on mammalian sensory neurons.

Ethanol inhibition of N-methyl-D-aspartate-activated ion current in rat hippocampal neurons is not competitive with glycine.

The interaction of ethanol with glycine at the N-methyl-D-aspartate (NMDA)-activated ion channel was investigated in voltage-clamped rat cultured hippocampal neurons. As shown previously, glycine increased, and ethanol inhibited, the NMDA-activated current in these cells. Concentration-response data for glycine (0.1-100 microM) indicate that the inhibition of NMDA-activated current by ethanol does not involve a competitive interaction with glycine. Thus, ethanol appears to inhibit NMDA-activated current at a locus different from the glycine modulatory site.

Single-channel and whole-cell analysis of ethanol inhibition of NMDA-activated currents in cultured mouse cortical and hippocampal neurons.

The effects of 0.1 to 500 mM ethanol on NMDA-activated currents were studied in primary cultures of mouse cortical and hippocampal neurons. In whole-cell recordings the IC50S for inhibition of NMDA-activated currents by ethanol were 129 mM +/- 20 mM in hippocampal neurons and 126 +/- 18 mM in cortical neurons. In single-channel recordings from excised outside-out patches of cortical neurons, ethanol inhibited total charge per minute with an IC50 of 174 +/- 23 mM, which was not significantly different from the IC50S for inhibition of whole-cell current. The reduction in mean open channel lifetime by ethanol was fit by the logistic equation with an apparent IC50 of 340 +/- 28 mM. Analysis of single-channel data indicated that ethanol inhibition of NMDA currents did not involve substantial changes in fast closed state kinetics, changes in open channel conductance, or block of the open channel. At the whole-cell IC50 of ethanol, mean open channel lifetime would decrease by 28% and frequency of opening would decline by 31% to account for the reduction in current. Single-channel data were consistent with ethanol being an allosteric modulator of gating which reduces agonist efficacy.

Inhibition of N-methyl-D-aspartate activated ion current by desmethylimipramine.

The tricyclic antidepressant desmethylimipramine (DMI) interacts with the NMDA receptor/ionophore complex; however, the site of the interaction has not been clearly established. Although evidence from receptor binding assays suggests that DMI interacts with the Zn2+ binding site, other binding studies and electrophysiological studies suggest otherwise. Using the whole-cell patch clamp technique to record from cultured hippocampal neurons, we report that recovery of NMDA-activated current from block by DMI is time-dependent and this time-dependent component was not observed following preexposure of neurons to Zn2+. These observations favor the hypothesis that DMI interacts at a binding site within the NMDA receptor/complex channel pore and not at the Zn2+ binding site.