Different arrangement of hydrophobic and nucleophilic components of channel binding sites in N-methyl-d-aspartate and AMPA receptors of rat brain is revealed by channel blockade (original) (raw)

Two Blocking Sites of Amino-Adamantane Derivatives in Open N-Methyl- d-Aspartate Channels

Biophysical Journal, 1998

Using whole-cell patch-clamp techniques, we studied the blockade of open N-methyl-D-aspartate (NMDA) channels by amino-adamantane derivatives (AADs) in rat hippocampal neurons acutely isolated by the vibrodissociation method. The rapid concentration-jump technique was used to replace superfusion solutions. A kinetic analysis of the interaction of AAD with open NMDA channels revealed fast and slow components of their blockade and recovery. Mathematical modeling showed that these kinetic components are evidence for two distinct blocking sites of AADs in open NMDA channels. A comparative analysis of different simplest models led us to conclude that these AAD blocking sites can be simultaneously occupied by two blocker molecules. The voltage dependence of the AAD block suggested that both sites were located deep in the channel pore.

Block of open channels of recombinant AMPA receptors and native AMPA/kainate receptors by Adamantane derivatives

The Journal of Physiology, 1997

University of Nottingham, Nottingham NG 7 2RD, UK 1. The effects of two adamantane derivatives, 1-trimethylammonio-5-(1-adamantane-methylammoniopentane dibromide) (IEM-1460) and 1-ammonio-5-(1-adamantane-methylammoniopentane dibromide) (IEM-1754) on kainate-induced currents were studied in Xenopus oocytes expressing recombinant ionotropic glutamate receptors and in freshly isolated neurones from rat hippocampal slices. 2. The adamantane derivatives caused use-and voltage-dependent block of open channels of recombinant AMPA receptors. This antagonism was dependent on receptor subunit composition; channels gated by recombinant, homomeric GluR1 and GluR3 receptors exhibited a higher sensitivity to block than those gated by receptors containing edited GluR2 subunits. In the former cases, IEM-1460 had an IC50 of 1P6 uM at a holding potential (Vh) of -80 mV and IEM-1754 was 3 8 times less potent than IEM-1460. In contrast, 100 /M IEM-1460 inhibited responses to 100 JM kainate of receptors containing edited GluR2 subunits by only 7-8 + 2 4% (n = 5 oocytes) at a Vh of -80 mV. 3. Native AMPA/kainate receptors in isolated hippocampal cells were inhibited by adamantane derivatives in a use-and voltage-dependent manner. This antagonism was dependent on cell type: pyramidal neurones were less sensitive to IEM-1460 (IC50 = 1617 FM at Vh = -80 mV) than interneurones (IC50 = 1-6 /M at Vh = -80 mV). IEM-1460 and IEM-1754 were equipotent when applied to pyramidal neurones, but IEM-1754 was less potent (-3 times) than IEM-1460 when applied to interneurones. 4. It is concluded that the presence of the edited GluR2 subunit in recombinant AMPA receptors and native AMPA/kainate receptors inhibits channel block by organic cations and that adamantane derivatives are potentially valuable tools for identifying classes of AMPA/kainate receptors and their roles in synaptic transmission.

Structural determinants of the blocker binding site in glutamate and NMDA receptor channels

Neuropharmacology, 1998

Glutamate receptor channels of the NMDA-type (N-methyl-D-aspartate) and non-NMDA-type (GluR) differ in their pore properties. The N-site in the M2 transmembrane segment of NMDA receptors (NMDAR), or the corresponding Q/R-site in GluRs, is a pivotal structural determinant of their permeation and blockade characteristics. Substitutions at a second site in M2, the L-site (L577) in GluR1, drastically alter the receptor selectivity to divalent cations. Here we report that M2 mutants carrying an asparagine or a threonine residue at the Q-site of GluR1, along with a tryptophan residue at the L-site, form homomeric GluR1 channels that are highly sensitive to structurally diverse, uncompetitive NMDA antagonists such as arylcyclohexylamines, dibenzocycloheptenimines, and to morphinian and adamantane derivatives. Analysis of the voltage dependence of channel blockade locates the blocker binding site 0.65 partway into the transmembrane electric field in both GluR1 mutants and NMDAR channels. Our results suggest that the homomeric GluR1 double mutants, L577W/Q582N and L577W/Q582T, fairly approximate the pore properties of the heteromeric NMDA receptor and support the structural kinship of their permeation pathways.

Voltage‐dependent block of native AMPA receptor channels by dicationic compounds

British Journal of Pharmacology, 2000

The kinetics of open channel block of GluR2‐containing and GluR2‐lacking AMPA receptors (AMPAR) by dicationic compounds (IEM‐1460, IEM‐1754, and IEM‐1925) have been studied in rat hippocampal neurones using whole‐cell patch clamp recording and concentration‐jump techniques. Neurones were isolated from hippocampal slices by vibrodissociation. The dicationic compounds were approximately 100–200 times more potent as blockers of GluR2‐lacking AMPAR than as blockers of GluR2‐containing AMPAR. The subunit specificity of channel block is determined by the blocking rate constant of a dicationic compound, whereas differences in unblocking rate constants account for differences in potency. Hyperpolarization may decrease the block produced by IEM‐1460 and IEM‐1754 block due to the voltage‐dependence of the unblocking rate constants for these compounds. This suggests that dicationic compounds permeate the AMPAR channel at negative membrane potentials. The effect was particularly apparent for Gl...

Determinants of trapping block of N-methyl-d-aspartate receptor channels

Journal of Neurochemistry, 2003

Open channel blockers of NMDA receptors interact with the channel gate in different ways. Compounds like MK-801 and phencyclidine exhibit pronounced trapping block, whereas 9-aminoacridine and tetrapentylammonium cannot be trapped. Some blockers such as memantine and amantadine exhibit intermediate properties, so called 'partial trapping'. To analyze the determinants of trapping we have synthesized a series of mono-and dicationic derivatives of phenylcyclohexyl. The blocking action of these compounds as well as that of amantadine has been studied on native NMDA receptors of hippocampal pyramidal neurons. Use-dependence and kinetics of the blockade have been analyzed to estimate the degree of trapping. Dimensions of the blocking molecules apparently do not correlate with their trapping. However, the degree of trapping is voltage-dependent and correlates with the kinetics of unblock. For instance, amantadine behaved as non-trapping blocker at positive voltages, but demonstrated significant trapping at negative voltages. The data may be explained by the model in which the NMDA receptor channel has two binding sites: the shallow and deep ones. Binding to the deep but not to the shallow site allows trapping of the blockers.

[Structural characteristics of ionotropic glutamate receptors revealed by channel blockade]

Rossiĭskii fiziologicheskiĭ zhurnal imeni I.M. Sechenova / Rossiĭskaia akademiia nauk

Glutamate is far from the only (in terms of the number of synapses at which it acts) neurotransmitter in the nervous systems of vertebrates and invertebrates. Transmission of excitatory stimuli in the brains of vertebrates and in the neuromuscular system of arthropods is mediated by activation of postsynaptic glutamate receptors of the ionotropic type, i.e., receptors which are ligand-controlled ion channels. The interaction of glutamate with the recognition site on the extracellular side of the receptor converts the channel, for a short period of time, into the open state, this being the basis of the generation of the excitatory postsynaptic potential.

Structural characteristics of ionotropic glutamate receptors as identified by channel blockade

2002

Blockade of glutamate- and cholinergic ion channels by amantadane derivatives

Neuroscience and Behavioral Physiology, 1996

It has been demonstrated that members of a homologous series of adamantane derivatives of the general formula Ad-Chz-N § where Ad is adamantane, while R varies from H (hydrogen) to t-Bu (tertiary butyl), possess the capacity to block the open state of postsynaptic ion channels. The decay of cholinergic postsynaptic currents could be fitted by two exponentials in experiments on nerve-muscle preparations of the frog during the action of the agents investigated; however, the rate constants of the interaction of the blocker and channel essentially did not depend on the structure of the cationic head or the membrane potential. The rate of blockade of the glutamatergic postsynaptic currents in the nerve-muscle junction of the insect increased as heavier radicals appeared at the nitrogen atom, but also did not depend on the membrane potential. The substances investigated proved to be powerful and "rapid" blockers of glutamate channels of the NMDA type, recorded by the method of local clamping on the membrane of cultivated rat cortical neurons. The afffmity of the substances for the open channel increased e-fold with hyperpolarization to 25 mV. All of the substances prevented convulsions induced by the introduction of NMDA into the lateral ventricle of the mouse. At the same time, substance II~M-1754, which had proven to be the most powerful in experiments in isolated ion channels, exhibited a six-fold greater anticonvulsant activity than dizocilpine (MK-801). The molecular mechanism of the interaction of the blockers with different types of postsynaptic ion channels is discussed. One of the most important components of the mechanism of signal transmission through the chemical synapse is the alteration of the state of ion channels in the plasma membrane of the effector cell. A macromolecule (an integral oligomeric protein), which performs both the function of recognition of a specific mediator and the function of regulation of the ion channel, may act as the postsynaptic receptor. Accordingly, the pharmacological modulation of the functioning of these systems which are key in the activity of the nervous system, whether pursuing investigatory or therapeutic aims, may be accomplished along at least two pathways: 1) by competitive effect on the "recognition particle" of the postsynaptic receptor in order to imitate or prevent the mediator effect; 2) by noncompetitive effect on the channel function of the postsynaptic receptor. Among the diverse noncompetitive modulation pathways, the pharmacological blockade of the open channel state is most frequently used. At the same time, the mediator, by activating the receptor, freely switches the channel into the open state, while a blocker, by interacting with structures of the open channel which have become accessible, disrupts the transport of the penetrating ions, and thereby blocks the function of the activated receptor. The sequence of the kinetic stages of this process is usually illustrated by the following schema [16] kt Aj~ .-, Aj~* + B-, A,R*B, where A.R is the complex of the receptor with agonist molecules that corresponds to the closed channel state; A.R* is the activated receptor complex which provides for the open channel state; B, blocker molecule; A,R*B, the complex of the activated receptor and blocker molecule; k~ and ~, the rate constants of association and dissociation of the blocker with an open channel, respectively.

Different arrangement of hydrophobic and nucleophilic components of channel binding sites in N-methyl-d-aspartate and AMPA receptors of rat brain is revealed by channel blockade (original) (raw)

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