Molecular size and hydrophobicity as factors which determine the efficacy of the blocking action of amino-adamantane derivatives on NMDA channels

Neurons isolated from the CA-1 region of rat hippocampal slices by the "vibrodissociation" method were voltage-clamped in the whole cell configuration. The currents through NMDA channels were recorded in response to rapid application (solution exchange time <30 ms) of 100 microM aspartate (ASP) in a Mg2+-free solution in the presence of 3 microM glycine. When added to the ASP solution, amantadine as well as other amino-adamantane derivatives (AAD) produced an open-channel blockade of NMDA channels. Membrane hyperpolarization enhanced the AAD block. The affinity between NMDA channels and AAD was different for various AAD. The analysis of the experimental data led us to conclude that this affinity depended both on the molecular size of the blocker (calculated using HyperChem molecular modeling program) and on the blocker's hydrophobicity (calculated according to Hansch and Leo, 1979). The affinity between NMDA channels and AAD diminished with an increase in molecular size and raised with an increase in blocker's hydrophobicity. We propose an empirical equation which describes the dependence of affinity on the size and hydrophobicity of the blocker. The estimated critical diameter of the NMDA channel pore where the AAD blocking site is located proved to be about 17 A.     

Structure of glutamate receptor ion channels and mechanisms of their blockade by organic cations

Structural determinants of blocking the glutamate receptors of AMPA and NMDA subtypes, were studied. Close location of hydrophobic and ammonium groups is necessary for affective blocking of the NMDA receptor channels, whereas blockers of the AMPA receptor channels have a distance of about 10 angstroms between these two groups. Models of the channels meeting these topographic data have been devised using a molecular mechanics approach. The accomplished studies revealed molecular basis of channel blockade of the NMDA and AMPA receptors. This may allow designing predictable new blocking compounds with a desired selectivity.     

Voltage-dependent block of fast chloride channels from rat cortical neurons by external tetraethylammonium ion

Tetraethylammonium ion (TEA) and its longer chain derivatives have been used extensively to block currents through K-selective ion channels. Substantial information has been gained about the structure and gating mechanisms of K and other cation channels from the analysis of the blocking interactions of TEA and other quaternary ammonium ions. We now present an analysis of blocking interactions between single Cl-selective ion channels from acutely dissociated rat cortical neurons and externally applied TEA. TEA applied to the extracellular membrane surface (TEAo) blocked Cl channels in a voltage-dependent manner, with hyperpolarizing potentials favoring block. The voltage dependence of block could be adequately fit assuming that TEA enters the channel pore and binds to a site located approximately 28% of the way through the membrane electrical field. The dose-response relationship between fractional current and [TEA]o at a fixed holding potential of -40 mV was well fit to a simple model with two blocking sites with dissociation constants (Kd) of approximately 2 and 70 mM. The dose-response relationship could also be fit by a mechanism where TEA only partially blocks the channels. At the bandwidth used in these experiments (1-2 kHz), both the mean open duration (composed of the open and blocked durations) and burst duration (composed of open, blocked, and short lifetime shut durations) increased with increased [TEA]o. This is expected if TEAo can bind and unbind only when the channel is in the open kinetic state. These results suggest that the structure of the permeability pathway of these anion-selective channels may be very similar to that of other channels that are blocked by TEA. Additionally, these results caution that a blocking effect by TEA cannot, by itself, be used as sufficient evidence for implicating the participation of K channels in a particular process.     

Quantitative analysis of tetrapentylammonium-induced blockade of open N-methyl-D-aspartate channels

The blockade of open N-methyl-d-aspartate (NMDA) channels by tetrapentylammonium (TPentA) in acutely isolated rat hippocampal neurons was studied using whole-cell patch-clamp techniques. TPentA prevented the closure of the NMDA channel following what is known as the foot-in-the-door mechanism. Hooked tail currents appearing after termination of the agonist (aspartate) and TPentA coapplication were analyzed quantitatively according to the corresponding sequential kinetic model. Studies of the hooked tail current amplitude and the degree of the stationary current inhibition dependence on the blocker concentration led to a new method for estimation of fast foot-in-the-door blocker binding/unbinding rate constants. The application of this method to the NMDA channel blockade by TPentA allowed finding the values of its binding (1.48 microM(-1)s(-1)) and unbinding (14 s(-1)) rate constants. An analysis of the dependence of the electric charge carried during the hooked tail current on the blocker concentration led to a new method for estimation of the maximum NMDA channel open probability, P(0). The value of P(0) found in experiments with TPentA was 0.04.     

Ion channels of glutamate receptors of nerve-muscle junction of larva of the fly <Emphasis Type="Italic">Calliphora vicina</Emphasis> demonstrate a high structural homology with vertebrate AMPA-channels

In experiments on the nerve-muscle junction of larvae of the fly Calliphora vicina, regularities of the blocking action of organic cations on ion channels of glutamate postsynaptic receptors have been studied. The measurements were performed by potential fixation on the muscle cell membrane. In total, effects of 26 compounds were studied. The following regularities of structural-functional relations have been revealed: (1) the channels are not blocked by monocation compounds; (2) bication derivatives block efficiently the channels with a certain distance between hydrophobic group and terminal amino group; (3) bication compounds with trimethylammonium terminal group are significantly more efficient than compounds with non-substituted amino group. All these regularities are characteristics of blockade of the AMPA channels, but not of the vertebrate-type NMDA channels. Earlier it was shown that differences in structural-functional relations during blockade of the AMPA and MNDA channels were determined by different location of the hydrophobic and hydrophilic components of the binding area as well as by different diameter of the channels. The fact that channels of the fly larva receptor demonstrate the same regularities of blockade as the vertebrate AMPA channels indicates their structural similarity that is a consequence of their high homology.     

Mechanisms of blockade of glutamate receptors channels: the significance for structural and physiological investigations

The mechanism of blocking effect of phenylcyclohexyl derivative, IEM-1925, on ionotropic glutamate receptors of the NMDA and AMPA types has been studied on the rat isolated brain neurons. The whole-cell configuration of patck clanp recording technique was used equilibrium conditions and -80 mV holding potential, the IEM-1925 manifests nonselective action on open channels of both receptors. However, the prominent differences in the mechanism of the blocking effect were revealed. Although IEM-1925 can not enter the closed channels of both types, its molecule are able to leave closed channels of the AMPA but not the NMDA receptors. Hyperpolarization reduces removal of blocker from the open channels of the NMDA receptors. Contrary to that, hyperpolarization facilitates going out of the IEM-1925 to cytozol from both open and closed channels. Evidently, the bloker can pass through the AMPA receptor channels into the cell, and the gating mechanism of these channels is located above the binding site for the blocker. The blocking action of the IEM-1925 on the NMDA and AMPA receptors was compared with its potency to weaken the tremor evoked by subcutaneous injection of arecoline to mice. The observed differences in the mechanisms of action help to explain the ambiguous effects of channel blocking drugs on experimental models of pathological processes.     

Two barium binding sites on a maxi K+ channel from human vas deferens epithelial cells.

Using the patch clamp technique, we have investigated the blockade of maxi-K+ channels present on vas deferens epithelial cells by extracellular Ba2+. With symmetrical 140 mM K+ solutions, Ba2+ produced discrete blocking events consisting of both long closings of seconds duration (slow block) and fast closings of milliseconds duration (flickering block). Kinetic analysis showed that flickering block occurred according to an "open channel blocking" scheme and was eliminated by reducing external K+ to 4.5 mM. Slow block showed a complex voltage-dependence. At potentials between -20 mV and 20 mV, blockade was voltage-dependent; at potentials greater than 20 mV, blockade was voltage-independent, but markedly sensitive to the extracellular K+ concentration. These data reveal that the vas deferens maxi-K+ channel has two Ba2+ binding sites accessible from the extracellular side. Site one is located at the cytoplasmic side of the gating region and binding to this site causes flickering block. Site two is located close to the extracellular mouth of the channel and binding to this site causes slow block.     

Glutamate receptor antagonists attenuate experimental catalepsy in rats

A long-term akinesia induced by haloperidol used as an experimental model of catalepsy helped to reveal that a dicationic derivatives adamantane (IEM-1754) and phenylcyclohexyl (IEM-1925) exerted different degrees of inhibition of the haloperidol effect: the IEM-1754 proved to be not inferior to the most effective NMDA antagonist MK-801. A relatively low potency of the IEM-1925 may be due to its obvious equal effects both on the NMDA and the AMPA receptor channels. A good correlation between the anticataleptic effects of the glutamate antagonists and the NMDA receptor blocking activity, were found. The AMPA receptor blockade might negatively affect the anticataleptic potency of the drugs under study.     

Activation kinetics reveal the number of glutamate and glycine binding sites on the N-methyl-D-aspartate receptor.

The activation kinetics of N-methyl-D-aspartate (NMDA) channels in outside-out patches from cultured hippocampal neurons were analyzed to determine the number of glutamate and glycine binding sites per channel. Following rapid steps into high concentrations of glutamate, the activation time course was concentration-independent and limited by transitions between the shut, but fully liganded state and the open state. At lower concentrations, ligand binding was rate-limiting. The resulting sigmoidal activation time course was best fitted by a kinetic model with two glutamate binding sites. Glycine concentration jumps in the continuous presence of glutamate were also best fitted with a two-site model. Agonist and co-agonist binding were better described by an independent, rather than a sequential model. We suggest that the NMDA receptor is at least a tetramer containing four ligand binding subunits, assuming a single binding site per subunit.     

Open channel block and alteration of N-methyl-D-aspartic acid receptor gating by an analog of phencyclidine.

We investigated inhibition of the N-methyl-D-aspartic acid (NMDA) receptor-channel complex by N-ethyl-1,4,9, 9alpha-tetrahydro-4alphaR-cis-4alphaH-fluoren-++ +4alpha-amine (NEFA), a structural analog of phencyclidine (PCP). Using the whole-cell recording technique, we demonstrated that NEFA inhibits NMDA responses with an IC50 of 0.51 microM at -66 mV. We determined that NEFA binds to the open channel, and subsequently the channel can close and trap the blocker. Once the channel has closed, NEFA is unable to dissociate until the channel reopens. Single-channel recordings revealed that NEFA reduces the mean open time of single NMDA-activated channels in a concentration-dependent manner with a forward blocking rate (k+) of 39.9 microM-1 s-1. A computational model of antagonism by NEFA was developed and constrained using kinetic measurements of single-channel data. By multiple criteria, only models in which blocker binding in the channel causes a change in receptor operation adequately fit or predicted whole-cell data. By comparing model predictions and experimental measurements of NEFA action at a high NMDA concentration, we determined that NEFA affects receptor operation through an influence on channel gating. We conclude that inhibition of NMDA receptors by PCP-like blockers involves a modification of channel gating as well as block of current flow through the open channel.     

Dependence of the concentration of the demi-maximal action of a channel blocker on the agonist concentration

Based on the analysis of kinetic scheme of blocking of open channels at any number of blocker binding sites, the dependence of current on blocker concentration was found. A variant of this dependence for a trapping blocker was also found. The restrictions of the applicability of the Hill equation and the necessity of taking into account the dependence of the concentration of demi-maximal blocker action (IC50) on the concentration of agonist were shown.     

Conserved structural and functional control of N-methyl-D-aspartate receptor gating by transmembrane domain M3

The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-d-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn(2+)). However, channels remain fully sensitive to Mg(2+) blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.     

Two-dimensional kinetic analysis suggests nonsequential gating of mechanosensitive channels in Xenopus oocytes

Xenopus oocytes express mechanosensitive (MS(XO)) channels that can be studied in excised patches of membrane with the patch-clamp technique. This study examines the steady-state kinetic gating properties of MS(XO) channels using detailed single-channel analysis. The open and closed one-dimensional dwell-time distributions were described by the sums of 2-3 open and 5-7 closed exponential components, respectively, indicating that the channels enter at least 2-3 open and 5-7 closed kinetic states during gating. Dependency plots revealed that the durations of adjacent open and closed intervals were correlated, indicating two or more gateway states in the gating mechanism for MS channels. Maximum likelihood fitting of two-dimensional dwell-time distributions to both generic and specific models was used to examine gating mechanism and rank models. A kinetic scheme with five closed and five open states, in which each closed state could make a direct transition to an open state (two-tiered model) could account for the major features of the single-channel data. Two-tiered models that allowed direct transitions to subconductance open states in addition to the fully open state were also consistent with multiple gateway states. Thus, the gating mechanism of MS(XO) channels differs from the sequential (linear) gating mechanisms considered for MS channels in bacteria, chick skeletal muscle, and Necturus proximal tubule.     

A peptide derived from the Shaker B K+ channel produces short and long blocks of reconstituted Ca(2+)-dependent K+ channels.

A 20 amino acid synthetic peptide, corresponding to the amino-terminal region of the Shaker B (ShB) K+ channel and responsible for its fast inactivation, can block large conductance Ca(2+)-dependent K+ channels from rat brain and muscle. The ShB inactivation peptide produces two kinetically distinct blocking events in these channels. At lower concentrations, it produces short blocks, and at higher concentrations long-lived blocks also appear. The L7E mutant peptide produces only infrequent short blocks (no long-lived blocks) at a much higher concentration. Internal tetraethylammonium competes with the peptide for the short block, which is also relieved by K+ influx. These results suggest that the peptide induces the short block by binding within the pore of Ca(2+)-dependent K+ channels. The long block is not affected by increased K+ influx, indicating that the binding site mediating this block may be different from that involved in the short block. The short block of Ca(2+)-dependent K+ channels and the inactivation of Shaker exhibit similar characteristics with respect to blocking affinity and open pore blockade. This suggests a conserved binding region for the peptide in the pore regions of these very different classes of K+ channel.     

Open channel block of NMDA receptor responses evoked by tricyclic antidepressants

The action of desipramine (DMI) and promazine on the response of mouse hippocampal neurons to the excitatory amino acid N-methyl-D-aspartic acid (NMDA) was investigated using whole-cell and single-channel recording. DMI at 20-50 microM was a potent, selective antagonist of responses to NMDA but not kainate or quisqualate. At -60 mV, the Kd for DMI block of responses to NMDA was 10 microM. The potency of DMI as an NMDA antagonist was highly voltage-dependent and behaved as though the Kd increased e-fold per 36 mV depolarization, reflecting an increase in the dissociation rate constant. Prior block of NMDA receptors with Mg2+ prevented binding of DMI, suggesting an action in the open channel. Single-channel analysis showed a decrease in the open time and burst length distributions, consistent with binding of DMI to open channels. We suggest that the action of DMI on NMDA receptor channels is similar to that of MK-801 and does not reflect binding to other domains, such as the regulatory sites for Zn2+ and glycine.     

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.     

Mechanism of blocking K+-channels with tetraethylammonium

Using the patch-voltage-clamp method action of tetraethylammonium on the fast (30 pS) and slow K+ channels was investigated. The slow K+ channels were presented by two types: with whole (30 pS) and decreased (20 pS) conductance. In all cases tetraethylammonium decreased the current magnitude and modified the channel kinetic parameters. Apparent blocking constants determined from the current decreasing are as 8-50 and 4-12 mM for the slow K+ channels with whole and decreased conductance, respectively, and 0.05-0.08 mM--for the fast K+ channel. The potential dependency of the blocking constants correlates with that of the channel conductance. Probability of the channel open state for the slow K+ channels decreases, and that for the fast K+ channel increases under application of tetraethylammonium. It is concluded that there are two sites of tetraethylammonium binding: the first site is into the channel pore, and the second one--into the regulatory centre responsible for the channel kinetic behaviour. Blocking of general conductance of the slow channels is accompanied by proportional decrease of the channel substate conductances without change of their number and cooperatively. Block of the fast K+ channel occurs without change of the channel elementary conductance but with decrease of the number of the channel substates and reversible violation of the channel transition cooperativity. The data are discussed from the point of the hypothesis on the channel clustery organization.     

State-independent block of BK channels by an intracellular quaternary ammonium

Intracellular blockade by quaternary ammonium (QA) molecules of many potassium channels is state dependent, where the requirement for channel opening is evidenced by a time-dependent component of block in the macroscopic record. Whether this is the case for Ca(2+)- and voltage-activated potassium (BK) channels, however, remains unclear. Previous work (Li, W., and R.W. Aldrich. 2004. J. Gen. Physiol. 124:43-57) tentatively proposed a state-dependent, trapping model, but left open the possibility of state-independent block. Here, we found BK channel blockade by a novel QA derivative, bbTBA, was time dependent, raising the possibility of state-dependent, open channel block. Alternatively, the observed voltage dependence of block could be sufficient to explain time-dependent block. We have used steady-state and kinetic measurements of bbTBA blockade in order to discriminate between these two possibilities. bbTBA did not significantly slow deactivation kinetics at potentials between -200 and -100 mV, suggesting that channels can close unhindered by bound bbTBA. We further find no evidence that bbTBA is trapped inside BK channels after closing. Measurements of steady state fractional block at +40 mV revealed a 1.3-fold change in apparent affinity for a 33-fold change in P(o), in striking contrast to the 31-fold change predicted by state-dependent block. Finally, the appearance of a third kinetic component of bbTBA blockade at high concentrations is incompatible with state-dependent block. Our results suggest that access of intracellular bbTBA to the BK channel cavity is not strictly gated by channel opening and closing, and imply that the permeation gate for BK channels may not be intracellular.     

Steady-State Interactions of Glibenclamide with CFTR: Evidence for Multiple Sites in the Pore

The objective of the present study was to clarify the mechanism by which the sulfonylurea drug, glibenclamide, inhibits single CFTR channels in excised patches from Xenopus oocytes. Glibenclamide blocks the open pore of the channel via binding at multiple sites with varying kinetics. In the absence of glibenclamide, open-channel bursts exhibited a flickery intraburst closed state (C1); this is due to block of the pore by the pH buffer, TES. Application of 25 microM glibenclamide to the cytoplasmic solution resulted in the appearance of two drug-induced intraburst closed states (C2, C3) of widely different duration, which differed in pH-dependence. The kinetics of interaction with the C3 state, but not the C2 state, were strongly voltage-dependent. The durations of both the C2 and C3 states were concentration-dependent, indicating a non-linear reaction scheme. Application of drug also increased the burst duration, which is consistent with an open-channel blocking mechanism. A kinetic model is proposed. These results indicate that glibenclamide interacts with open CFTR channels in a complex manner, involving interactions with multiple binding sites in the channel pore.