Fast excitatory synaptic transmission in the mammalian central nervous system is mediated by glutamate-activated α-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA) receptors. In neurons, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs). Assembly with TARP γ8 alters the biophysical properties of the receptor, producing resensitization currents in the continued presence of glutamate. Using single-channel recordings, we show that under resensitizing conditions, GluA2 AMPA receptors primarily transition to higher conductance levels, similar to activation of the receptors in the presence of cyclothiazide, which stabilizes the open state. To study the conformation associated with these states, we have used single-molecule FRET and show that this high-conductance state exhibits tighter coupling between subunits in the extracellular parts of the receptor. Furthermore, the dwell times for the transition from the tightly coupled state to the decoupled states correlate to longer open durations of the channels, thus correlating conformation and function at the single-molecule level.

Glutamate is released from presynaptic nerve terminals in the central nervous system (CNS) and spreads excitation by binding to and activating postsynaptic iGluRs. Of the potential glutamate targets, tetrameric AMPA receptors mediate fast, transient CNS signaling. Each of the four AMPA subunits in the receptor channel complex is capable of binding glutamate at its ligand-binding domains and transmitting the energy of activation to the pore domain. Homotetrameric AMPA receptor channels open in a stepwise manner, consistent with independent activation of individual subunits, and they exhibit complex kinetic behavior that manifests as temporal shifts between four different conductance levels. Here, we investigate how two AMPA receptor-selective noncompetitive antagonists, GYKI-52466 and GYKI-53655, disrupt the intrinsic step-like gating patterns of maximally activated homotetrameric GluA3 receptors using single-channel recordings from cell-attached patches. Interactions of these 2,3-benzodiazepines with residues in the boundary between the extracellular linkers and transmembrane helical domains reorganize the gating behavior of channels. Low concentrations of modulators stabilize open and closed states to different degrees and coordinate the activation of subunits so that channels open directly from closed to higher conductance levels. Using kinetic and structural models, we provide insight into how the altered gating patterns might arise from molecular contacts within the extracellular linker-channel boundary. Our results suggest that this region may be a tunable locus for AMPA receptor channel gating.

Postsynaptic AMPA/glutamate receptors, essential for neuronal excitability, are important targets for anticonvulsant therapy. This single channel study of the selective noncompetitive AMPA receptor antagonist, perampanel, was performed on homotetrameric GluA3 receptor-channels that open in a stepwise manner to four distinct conductance levels through independent subunit activation. Previous structural studies show that perampanel binds to four sites located within the extracellular/transmembrane boundary of closed AMPA receptor-channel subunits. We found that channels exposed to 1 or 2 μM perampanel opened mainly to the two lower conductance levels in a dose-dependent manner. Comparison of the single channel results in the structures of the full length AMPA receptor in the closed state bound to perampanel, and the open state provide insights into the mechanism of allosteric reduction of AMPA-receptor-mediated excitation in epilepsy.

The kinetics of AMPA receptor activation, deactivation, and desensitization shape the synaptic current at a majority of synapses in the central nervous system; however, the sequence of molecular events that make up these receptors’ reaction mechanism is unknown. Information about reaction mechanism is best extracted from single-molecule observations, which for ion channels can be done in real time with the patch-clamp technique. AMPA receptors have complex single-channel behaviors, which have been difficult to organize into a comprehensive mechanism. Here we describe methodology to obtain lengthy recordings of currents from one AMPA receptor and show how kinetic analysis was applied to organize the signal into classes of activity or modes that follow similar kinetic patterns. This approach can provide valuable insights into the molecular transitions that produce the activation, deactivation, and desensitization of ionotropic glutamate receptors and of ligand-gated channels in general.

Ligand-gated ion channels undergo conformational changes that transfer the energy of agonist binding to channel opening. Within ionotropic glutamate receptor (iGluR) subunits, this process is initiated in their bilobate ligand binding domain (LBD) where agonist binding to lobe 1 favors closure of lobe 2 around the agonist and allows formation of interlobe hydrogen bonds. AMPA receptors (GluAs) differ from other iGluRs because glutamate binding causes an aspartate-serine peptide bond in a flexible part of lobe 2 to rotate 180° (flipped conformation), allowing these residues to form cross-cleft H-bonds with tyrosine and glycine in lobe 1. This aspartate also contacts the side chain of a lysine residue in the hydrophobic core of lobe 2 by a salt bridge. We investigated how the peptide flip and electrostatic contact (D655-K660) in GluA3 contribute to receptor function by examining pharmacological and structural properties with an antagonist (CNQX), a partial agonist (kainate), and two full agonists (glutamate and quisqualate) in the wildtype and two mutant receptors. Alanine substitution decreased the agonist potency of GluA3(i)-D655A and GluA3(i)-K660A receptor channels expressed in HEK293 cells and differentially affected agonist binding affinity for isolated LBDs without changing CNQX affinity. Correlations observed in the crystal structures of the mutant LBDs included the loss of the D655-K660 electrostatic contact, agonist-dependent differences in lobe 1 and lobe 2 closure, and unflipped D(A)655-S656 bonds. Glutamate-stimulated activation was slower for both mutants, suggesting that efficient energy transfer of agonist binding within the LBD of AMPA receptors requires an intact tether between the flexible peptide flip domain and the rigid hydrophobic core of lobe 2.

Several of the many steps in the mechanism of activation of tetrameric GluA receptor-channels have been explored in a variety of published structural and functional studies. The main findings from them are that 1) Agonist binding induces closure of the L1/L2 of the isolated ligand-binding domain (LBD) that is less for partial agonists than for glutamate. 2) Crystal structures of GluA LBDs bound to full agonists manifest a 180º rotation around an Asp-Ser transpeptide bond in a flexible part of L2 that leads to the formation of cross-interface H-bonds also hypothesized to play a role in receptor activation. 3) Channel conductance increases with increasing agonist concentration, consistent with multiple LBDs in the tetramer contributing to full channel activation. Here we show that the Asp of the Asp-Ser pair also makes an electrostatic contact with a Lys residue in helix-F, and that disrupting this salt-bridge in GluA3flip receptors with point mutations at D655 and K660 decreased agonist potency by 2-10 fold. In D655A where it was examined, this occurred without altering intrinsic receptor affinity for agonists in ligand binding assays or CNQX by Schild analysis. The D655A mutation also increased the efficacy of kainate relative to glutamate, and the isolated GluA-D655A LDB bound to kainate was 3° more closed than in wildtype. Pharmacological studies of the wildtype and mutant receptors expressed in human embryonic kidney cells employed whole cell patch recordings and structure data was from crystals of isolated wildtype and mutant LBDs bound to full agonists and kainate. Taken together, these studies showed that breaking the D655-K660 contact altered channel gating, indicating that tethering a flexible part of L2 to helix-F may contribute to the rapid activation of GluA receptors.

AMPA receptors are the major excitatory neurotransmitter receptors in the central nervous system and are involved in numerous neurological disorders. An agonist-binding site is present in each of four subunits that form a functional channel. Binding consists of three steps: docking of agonist to the bilobed ligand binding domain (LBD), closure of the LBD, and increased stability of the closed-lobe conformation through interlobe hydrogen bonding. We describe GluA3 single channel currents activated by nitrowillardiine (NO2W) and chlorowillardiine (ClW) in the presence of cyclothiazide, in conjunction with crystal structures of GluA2 and GluA3 LBDs bound to fluorowillardiine (FW), ClW, and NO2W. When bound to NO2W or ClW, the GluA3 channel opens to three conductance levels with comparable open probabilities and displays modal behavior similar to that obtained with glutamate and FW as agonists (Poon et al., 2010). At lower concentrations, ClW evoked an alternate kinetic behavior, consisting of high open probability in lower conductance states. The structure of ClW bound to GluA3 LBD exhibits a unique partially open hydrogen bonding structure that may be associated with these alternative kinetics. NO2W evoked longer open times than seen for other agonists in high and very high modes. The structure ofGluA2 LBD bound to NO2W exhibits fully closed lobes with additional interlobe interactions mediated by the nitro group. Beyond differences in efficacy between full and partial agonists, the complexities of the single channel behavior of AMPA receptors may also be associated with small interactions that modify the stability of various degrees of closure.

AMPA receptors play a major role in excitatory neurotransmission in the CNS and are involved in numerous neurological disorders. Agonists bind to each of four bilobed LBDs of this tetrameric receptor, and upon binding, the lobes close to envelope the agonist, leading to channel activation. However, AMPA receptors exhibit complex activation kinetics, the mechanism of which has not yet been determined. We report here single-channel studies of a homomeric AMPA receptor (GluA3) activated by the full agonist, glutamate, and a partial agonist, fluorowillardiine. Both agonists activate the channel to the same three open conductance levels but with different open probabilities in each level. The closed probability (Pc) varied within records, particularly at low agonist concentrations. By sorting discrete segments of the record according to Pc using the X-means algorithm, we defined five modes of activity. The kinetic behavior could then be analyzed for both agonists over a range of agonist concentrations with a relatively simple model (three closed states and two open states for each open conductance level). The structural mechanism underlying the modal behavior is not clear; however, it occurs on a timescale consistent with hydrogen bonding across the lobe interface in the LBD.

Ionotropic glutamate receptors (iGluR's) are ligand gated ion channels that mediate most of the fast excitatory neurotransmission in the CNS. Aberrant function of glutamate neurotransmission can lead to epilepsy and other neurodegenerative disorders. The extracellular ligand binding domain is a bilobal structure that binds an agonist and induces channel activation. Data from single channel recordings from homomeric AMPA receptor subtype (GluR3) in cell-attached patches were analyzed using QuB software to examine preliminary kinetic models of agonist dependent channel activity. Cell attached recordings were performed with both full and partial agonists on stably transfected HEK 293 cells. Amplitude analysis uncovered three conductance states, 15 pS, 27 pS, and 40 pS, in the presence of the full agonist, glutamate, as well as the partial agonists, fluorowillardiine, chlorowillardiine and nitrowillardiine. Different modes of activation ranging from low to high open probability exist for this channel. In the presence of the full agonist, glutamate, during a high mode of activation, the channel prefers to open to the intermediate and large conductance states. In the presence of the willardiine partial agonists, the channel opens more frequently to the smallest and intermediate conductance states. Kinetic modeling using maximum interval likelihood rate optimization revealed two time constants in each open state and at least three in the closed state for the partial and the full agonists. These data suggest the mode of channel activation is similar for both glutamate and willardiine compounds with varying rates of activation. Supported by NIH NS049223.

The voltage-dependent block of NMDA receptor channels by external Mg2+ (Mgo) was first reported in 1984 in two papers (Mayer et al. 1984; Nowak et al. 1984) that have had a great influence on our understanding of the functional role of NMDA receptors. However, the observations also offered a new way to explain the ‘gating’ of a voltage-dependent conductance, and our paper of 1988 published in The Journal of Physiology aimed primarily at a biophysical description of the Mgo block based on single channel records (Ascher & Nowak, 1988). To characterize the Mgo block we followed the approach used by Neher & Steinbach (1978) to study how QX222 transforms into ‘bursts’ the single openings of nicotinic ACh receptor channels. Assuming that Mgo enters the NMDA receptor channel, binds to a blocking site situated deep in the membrane and can only leave to the outside after unbinding, we evaluated the rates of Mg2+ binding and unbinding in various Mg2+ concentrations and at various potentials. We then deduced from the voltage dependence of these rates the depth of the blocking site in the membrane. This depth was evaluated by a coefficient ‘δ’ that could vary between 0 and 1. Our value of δ was close to 1, suggesting that the blocking site was actually very close to the inner limit of the membrane. This value was somewhat higher than the values obtained by analysis of the I–V relations of whole cell currents by Mayer & Westbrook (1985). We also characterized at the single channel level the Ca2+ permeability of the NMDA receptor channel. We measured the shifts of the reversal potential in different external Ca2+ concentrations and deduced the ratio of the permeabilities of Ca2+ and monovalent cations from the Goldman–Hodgkin–Katz voltage equation. Our results agreed with the values obtained by Mayer & Westbrook (1987) using I–V relations for whole cell current. We also observed that an increase in external Ca2+ reduced the single channel conductance, indicating that Ca2+ permeates the channel more slowly than monovalent cations. Our evaluation of the depth of the Mgo blocking site was soon put in doubt by the observation that the value of δ we deduced for Mgo block was not easily reconciled with the voltage dependence of the block by internal Mg2+ (Mgi) (Johnson & Ascher, 1990). The ‘crossing of the deltas’ paradox was solved by Jon Johnson and his collaborators, who showed that access of Mg2+ to the channel is prevented when permeant ions bind at the outer surface of the membrane. In the model of Antonov & Johnson (1999) the δ for Mgo is now equal to 0.5. We should also acknowledge that our single channel recordings made us miss the ‘slow Mgo unblock’ which was later described by Spruston et al. (1995) on whole cell current relaxations following voltage jumps, modelled by Vargas-Caballero & Robinson (2004) and by Kampa et al. (2004), and shown to be NR2 subunit dependent by Clarke & Johnson (2006). Recently the same authors (Clarke & Johnson, 2008) have shown that the slow block is the consequence of a voltage dependent gating which does not require Mgo. From 1991 onward, the cloning of the NMDA receptor subunits radically renewed the study of Mg2+ block and Ca2+ permeability. It rapidly led to the identification of the key amino acids involved in the binding of Mg2+ (reviewed by Dingledine et al. 1999), and it also revealed the heterogeneity of Mg block among NMDA receptors subtypes (not yet well understood). Concerning the Ca2+ permeability, the use of calcium indicators has allowed direct comparison of the Ca2+ influx and the total current and thus evaluation of the ‘fractional Ca2+ current’ (Pf). When appropriate corrections are made, the value of Pf agrees very well with the predictions of the GHK equation (Schneggenburger 1996). The molecular structures responsible for the Ca2+ permeability have been partially identified and comprise both a deep site, the N site of the NR1 subunit, and a superficial site at the entrance of the channel, the DRPEER motif, also specific to the NR1 subunit (Watanabe et al. 2002). Despite all these advances, one cannot yet say that either Mg2+ block or Ca2+ permeation are understood at the molecular level. We still lack a structural model of the NMDA receptor channel, but it may not be too far away.

Ionotropic glutamate receptors are members of a large family of plasma membrane proteins expressed by cells of the nervous system. Upon binding glutamate, the receptors transiently open transmembrane channels that allow the entry of sodium ions. The resulting changes in the transmembrane potential of the cell initiates a process that is involved in signal transmission to another cell. The binding of glutamic acid triggers the channel opening in the microsecond time domain and the reversible inactivation (desensitization) of the receptors in the millisecond time region. The channel-opening mechanism of glutamate receptors was investigated in rat hippocampal neurons voltage-clamped to -60 mV at room temperature and pH 7.4. Two rapid chemical reaction techniques were used: (1) a cell-flow method with a 4-10 ms time resolution to apply L-glutamate and (2) a laser-pulse photolysis technique to release glutamate from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (alphaCNB-caged L-glutamate) with a time constant of 30 micros. The rate and equilibrium constants for channel opening were determined. The results are consistent with the receptor binding two molecules of glutamic acid before the channel opens, with an apparent dissociation constant of 600 microM. Channel opening and closing rate constants, k(op) and k(cl), were determined to be (9.5 +/- 1) x 10(3) s(-1) and (1.1 +/- 0.1) x 10(3) s(-1), respectively. The value of the channel-opening equilibrium constant, Phi (=k(op)/k(cl)), was 8.6 when determined by laser-pulse photolysis and 6.6 in cell-flow experiments. The results suggest that there are at least two forms of glutamate receptors in rat hippocampal neurons that desensitize with different rates. At a concentration of 500 microM glutamate, 80% of the receptors desensitized with a rate of approximately 200 s(-1) and 20% with a rate of approximately 50 s(-1).

The effects of extracellularly applied 39-59 cyclic guanosine monophosphate (cGMP) on kainate responses from cultured cerebellar granule and Purkinje neurons were investigated using whole-cell and outside-out patch recording modes. Cerebellar granule cell responses to kainate were not homogeneous, nor were the effects of cGMP. Therefore, effects of cGMP are described for two groups of granule cells categorized on the basis of the underlying channel conductance estimated by variance analysis. Cells with high-noise kainate responses had average channel conductances of 5 to 7 picoseimens, whereas the average conductances of low-variance noise responses were 0.3 to 2.0 picoseimens. High-noise kainate responses were inhibited by externally applied cGMP (5–1000 mM) in a rapidly reversible and dose-dependent manner. IC50 values were estimated at ;150 mM cGMP for 25 mM kainate and ;500 mM cGMP for 100 mM kainate. Evidence that cGMP-mediated inhibition of high-noise kainate responses occurred by a competitive mechanism included the following: 1) cGMP-mediated inhibition was overcome by increasing agonist concentration. 2) The shape of kainate current-voltage (I-V) curves and their reversal potentials were unchanged in cGMP. 3) Neither the estimated conductance nor the kinetics of the kainate-activated channels was affected by cGMP. In contrast to the uniform effects of cGMP on the high-noise kainate responses, the effects on low-noise kainate responses were variable. Half of the low-noise kainate responses were inhibited by cGMP to a similar extent as the high-noise responses; however, the other 50% of cells exhibiting low-noise kainate responses appeared to be less sensitive to the cyclic nucleotide. Moreover, cGMP coapplication decreased the estimated conductances for some low-noise kainate responses and altered their noise kinetics, which suggests either that cGMP-sensitive and -insensitive kainate receptor channels are coexpressed in these cells or that cGMP-mediated inhibition is not competitive for this subgroup of glutamate receptor channels. Overall, these data indicate that there are direct inhibitory effects of extracellular cGMP on a large group of excitatory synapses in the CNS—effects that need to be taken into account when investigators utilize membrane-permeable cGMP analogs. Whether this cGMP-mediated inhibition has a functional role in brain is unknown. Studies of cGMP in mammalian brain begin with the early reports that activation of glutamate receptors in cerebellum causes a dramatic increase in the cGMP content of this tissue (Mao et al., 1974; Rubin and Ferrendelli, 1977; Garthwaite and Balázs, 1978). Subsequently, the signaling pathway mediating cGMP production in neurons was shown to involve an increase in intracellular Ca, leading to a Ca-calmodulin-dependent activation of NOS (Bredt and Snyder, 1989; Mayer et al., 1992) and NO stimulation of soluble guanylate cyclase typically located in cells in close proximity (Garthwaite, 1991). In the cerebellum, NOS appears to be localized to granule cells (Bredt et al., 1991) and the NO-sensitive soluble guanylate cyclase is localized to granule cells (Southam et al., 1992; Southam and Garthwaite, 1993) and to Purkinje neurons (Zwiller et al., 1981; Ariano et al., 1982; Matsuoka et al., 1992), which are thought to be largely responsible for the dramatic kainate-mediated increase in cerebellar cGMP (Rubin and Ferrendelli, 1977). The consequences of glutamate receptor-linked elevation of intracellular cGMP in Purkinje and granule neurons are poorly understood, but it is likely that cGMP activates GReceived for publication October 13, 1997. 1 This work was supported successively by NS 24467 and NS 33166 to L.M.N. 2 Present Address: Athens University Medical School, Department of Neurology, Egimition Hospital, Vas. Sofias 72, Athens, GREECE 11528. ABBREVIATIONS: AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; cGMP, 39,59 cyclic guanosine monophosphate; 8-Br-cGMP, 39,59 cyclic 8-bromo-guanosine monophosphate; FUDR, the mitotic inhibitors 5-fluoro-29deoxyuridine and uridine; LTD, long-term depression; MEM, minimal essential medium; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; g, estimated single-channel conductance; ie, elementary single-channel current amplitude; s , variance; pS, picosiemens; TTX, tetrodotoxin; VR, reversal potential of the agonist response; VH, membrane holding potential. 0022-3565/98/2861-0099$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 286, No. 1 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 286:99–109, 1998

The effects of extracellularly applied 3'-5' cyclic guanosine monophosphate (cGMP) on kainate responses from cultured cerebellar granule and Purkinje neurons were investigated using whole-cell and outside-out patch recording modes. Cerebellar granule cell responses to kainate were not homogeneous, nor were the effects of cGMP. Therefore, effects of cGMP are described for two groups of granule cells categorized on the basis of the underlying channel conductance estimated by variance analysis. Cells with high-noise kainate responses had average channel conductances of 5 to 7 picoseimens, whereas the average conductances of low-variance noise responses were 0.3 to 2.0 picoseimens. High-noise kainate responses were inhibited by externally applied cGMP (5-1000 microM) in a rapidly reversible and dose-dependent manner. IC50 values were estimated at approximately 150 microM cGMP for 25 microM kainate and approximately 500 microM cGMP for 100 microM kainate. Evidence that cGMP-mediated inhibition of high-noise kainate responses occurred by a competitive mechanism included the following: 1) cGMP-mediated inhibition was overcome by increasing agonist concentration. 2) The shape of kainate current-voltage (I-V) curves and their reversal potentials were unchanged in cGMP. 3) Neither the estimated conductance nor the kinetics of the kainate-activated channels was affected by cGMP. In contrast to the uniform effects of cGMP on the high-noise kainate responses, the effects on low-noise kainate responses were variable. Half of the low-noise kainate responses were inhibited by cGMP to a similar extent as the high-noise responses; however, the other 50% of cells exhibiting low-noise kainate responses appeared to be less sensitive to the cyclic nucleotide. Moreover, cGMP coapplication decreased the estimated conductances for some low-noise kainate responses and altered their noise kinetics, which suggests either that cGMP-sensitive and -insensitive kainate receptor channels are coexpressed in these cells or that cGMP-mediated inhibition is not competitive for this subgroup of glutamate receptor channels. Overall, these data indicate that there are direct inhibitory effects of extracellular cGMP on a large group of excitatory synapses in the CNS--effects that need to be taken into account when investigators utilize membrane-permeable cGMP analogs. Whether this cGMP-mediated inhibition has a functional role in brain is unknown.

Many single-channel studies rely on the assumption that the channels are functioning under steady-state conditions. In examining the basis for nonlinear whole-cell current-voltage curves in Mg2+-free solutions we discovered that N-methyl-D-aspartate (NMDA) channels in excised patches reversibly shifted their open-state probability (P(o)) in a voltage-dependent way, exhibiting approximately 3- to 4-fold greater P(o) at positive potentials than at rest. Changes in P(o) were mainly attributable to shifts in frequency of channel opening. P(o) changed remarkably slowly (2-15 min), explaining the hysteresis of whole-cell current-voltage curves obtained in nonequilibrium conditions. The slow increase in P(o) provides a mechanism by which NMDA channels can substantially increase Ca2+ influx in cells depolarized for prolonged periods of time and may play a role in excitotoxicity.

Bicuculline methiodide (BIC-Mel) (10-100 microM) altered the kinetics of N-methyl-D-aspartate (NMDA) responses in single-channel and whole-cell recordings. The principal effect of BIC-Mel (10-100 microM) on NMDA channels was a dose-dependent decrease in mean channel open time (tau o), accompanied by the introduction of a new closed time (tau B) of 14.0 +/- 3.5 msec (mean +/- standard deviation; n = 14) in closed time distributions, which was independent of BIC-Mel concentration. BIC-Mel (10-100 microM) increased the frequency of NMDA channel opening in a dose-dependent manner, offsetting the decrease in tau o, such that the total time spent in the open state per minute was unchanged, and thus the total charge/min through NMDA channels was unchanged. Similarly, the amplitudes of NMDA whole-cell current responses were not noticeably affected by 10-80 microM BIC-Mel, even though power spectra density analysis of the whole-cell NMDA-stimulated noise revealed changes in the underlying channel kinetics in the presence of BIC-Mel. Taken together, the effects of 10-80 microM BIC-Mel on NMDA responses were consistent with the predictions of the sequential block model; however, the effects of BIC-Mel exhibited no obvious voltage dependence. In addition to the low-dose effects of BIC-Mel, 100 and 200 microM BIC-Mel inhibited whole-cell NMDA responses. The inhibition by 100 microM BIC-Mel was not large, but it was augmented from 15% to 30% by increasing the NMDA concentration from 10 microM NMDA to 20 microM NMDA, indicating that channel activation was necessary for BIC-Mel-mediated inhibition. Preliminary single-channel experiments performed under conditions conducive to trapping of an open channel blocker at its binding site indicated that the effect of BIC-Mel on tau o persisted after the removal of the blocker, consistent with use dependence of the dissociation of BIC-Mel from the NMDA receptor-channel complex.

Annals of the New York Academy of SciencesVolume 648, Issue 1 p. 205-206 Comparative Pharmacology of Kainate-Stimulated Single-Channel Activity in Outside-Out Patches Is Consistent with a Multiplicity of Non-NMDA Receptors CORNELIA POULOPOULOU, CORNELIA POULOPOULOU Department of Pharmacology Cornell University Ithaca, New York 14853–6401Search for more papers by this authorJANET L. CHRISTIANSEN, JANET L. CHRISTIANSEN Department of Pharmacology Cornell University Ithaca, New York 14853–6401Search for more papers by this authorLINDA M. NOWAK, LINDA M. NOWAK Department of Pharmacology Cornell University Ithaca, New York 14853–6401Search for more papers by this author CORNELIA POULOPOULOU, CORNELIA POULOPOULOU Department of Pharmacology Cornell University Ithaca, New York 14853–6401Search for more papers by this authorJANET L. CHRISTIANSEN, JANET L. CHRISTIANSEN Department of Pharmacology Cornell University Ithaca, New York 14853–6401Search for more papers by this authorLINDA M. NOWAK, LINDA M. NOWAK Department of Pharmacology Cornell University Ithaca, New York 14853–6401Search for more papers by this author First published: May 1992 https://doi.org/10.1111/j.1749-6632.1992.tb24539.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume648, Issue1Neurotoxins and Neurodegenerative DiseaseMay 1992Pages 205-206 RelatedInformation

Annals of the New York Academy of SciencesVolume 648, Issue 1 p. 353-354 A Slow Voltage-Dependent Increase in N-Methyl-D-Aspartate Open-Channel Probabilitya J. M. WRIGHT, J. M. WRIGHT Department of Pharmacology Cornell University Ithaca, New York 14853Search for more papers by this authorL. M. NOWAK, L. M. NOWAK Department of Pharmacology Cornell University Ithaca, New York 14853Search for more papers by this author J. M. WRIGHT, J. M. WRIGHT Department of Pharmacology Cornell University Ithaca, New York 14853Search for more papers by this authorL. M. NOWAK, L. M. NOWAK Department of Pharmacology Cornell University Ithaca, New York 14853Search for more papers by this author First published: May 1992 https://doi.org/10.1111/j.1749-6632.1992.tb24580.x a This work was supported by NIH Grant NS24467. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume648, Issue1Neurotoxins and Neurodegenerative DiseaseMay 1992Pages 353-354 RelatedInformation

1. The mechanisms of tetraethylammonium (TEA) antagonism of N-methyl-D-aspartate (NMDA) responses were investigated in cultured mouse cortical neurons by analysing single-channel and whole-cell currents from patch clamp recordings. TEA (1-5 mM) decreased whole-cell NMDA responses. Kainate and quisqualate receptor-mediated responses were unaffected at these TEA concentrations. 2. In whole-cell recordings, increasing the NMDA concentration while keeping the TEA concentration constant resulted in greater inhibition by TEA. Thus, TEA-mediated inhibition of NMDA responses was not due to competitive antagonism, and the greater inhibition by a single dose of TEA as NMDA concentration was elevated indicated some form of non-competitive inhibition. In single-channel recordings, two inhibitory effects were seen in 1-5 mM-extracellular TEA: single-channel conductance (gamma) was decreased, and the frequency of channel events was decreased. These effects were not accompanied by any change in average channel open time. 3. Single-channel current-voltage (I-V) curves obtained in 2, 5, 10 and 30 mM-TEA indicated the decrease in NMDA channel conductance was voltage dependent with larger reduction occurring as patches were hyperpolarized. The data were well fitted by the Woodhull model with the dissociation constant (KD) showing an e-fold increase in inhibition for a 43-45 mV change in membrane potential. The 0 mV KD was 45 mM-TEA decreasing to about 11 mM at -60 mV. The TEA block site appeared to sense approximately 60% of the transmembrane potential field (delta = 0.6) for extracellular application of TEA. 4. The decrease in channel opening frequency seen in TEA was concentration dependent and generally more sensitive to extracellular TEA than the channel block effect. There was a 50% reduction in the number of NMDA channel openings observed in 5 mM-TEA. Increasing either NMDA or glycine concentrations in constant TEA concentration caused an additional decrease in the frequency of NMDA channel opening. In contrast to extracellular TEA, intracellular TEA had no noticeable effect on open-state probability. 5. NMDA single-channel currents were observed at positive potentials after completely replacing pipette Cs+ by 140 mM-TEA-Cl indicating TEA could serve as a current carrier through NMDA channels. Single channel I-V curves obtained with pipettes containing 70 or 140 nM-TEA in place of equivalent amounts of Cs+ were fitted by the Goldman-Hodgkin-Katz (GHK) equation over the range of -80 to +70 mV assuming a permeability of 0.45 compared with a Cs+ permeability of 1.0.(ABSTRACT TRUNCATED AT 400 WORDS)