Screening for Activity Against AMPA Receptors Among Anticonvulsants-Focus on Phenytoin.

The interest in AMPA receptors as a target for epilepsy treatment increased substantially after the approval of perampanel, a negative AMPA receptor allosteric antagonist, for the treatment of partial-onset seizures and generalized tonic-clonic seizures. Here we performed a screening for activity against native calcium-permeable AMPA receptors (CP-AMPARs) and calcium-impermeable AMPA receptors (CI-AMPARs) among different anticonvulsants using the whole-cell patch-clamp method on isolated Wistar rat brain neurons. Lamotrigine, topiramate, levetiracetam, felbamate, carbamazepine, tiagabin, vigabatrin, zonisamide, and gabapentin in 100-µM concentration were practically inactive against both major subtypes of AMPARs, while phenytoin reversibly inhibited them with IC50 of 30 ± 4 µM and 250 ± 60 µM for CI-AMPARs and CP-AMPARs, respectively. The action of phenytoin on CI-AMPARs was attenuated in experiments with high agonist concentrations, in the presence of cyclothiazide and at pH 9.0. Features of phenytoin action matched those of the CI-AMPARs pore blocker pentobarbital, being different from classical competitive inhibitors, negative allosteric inhibitors, and CP-AMPARs selective channel blockers. Close 3D similarity between phenytoin and pentobarbital also suggests a common binding site in the pore and mechanism of inhibition. The main target for phenytoin in the brain, which is believed to underlie its anticonvulsant properties, are voltage-gated sodium channels. Here we have shown for the first time that phenytoin inhibits CI-AMPARs with similar potency. Thus, AMPAR inhibition by phenytoin may contribute to its anticonvulsant properties as well as its side effects.

Ligand Docking to the Acidic Pocket of the Proton-Gated Ion Channel Asic1A.

In this study, we performed the docking of ligands of the ASIC1a ion channel, which exert potentiating or inhibitory effects by stabilizing the open and closed states, respectively. It is shown for the first time that the direction of effect may depend on the three-dimensional structure of the ligand. Potentiators and inhibitors differently interact with the amino acid residues of the so-called "acidic pocket," where the binding of protons takes place. These results open up an opportunity for theoretical design of new pharmaceuticals.

Ligands of Acid-Sensing Ion Channel 1a: Mechanisms of Action and Binding Sites.

The proton-gated cationic channels belonging to the ASIC family are widely distributed in the central nervous system of vertebrates and play an important role in several physiological and pathological processes. ASIC1a are most sensitive to acidification of the external medium, which is the reason for the current interest in their function and pharmacology. Recently, the list of ASIC1a ligands has been rapidly expanding. It includes inorganic cations, a large number of synthetic and endogenous small molecules, and peptide toxins. The information on the mechanisms of action and the binding sites of the ligands comes from electrophysiological, mutational and structural studies. In the present review, we attempt to present a systematic view of the complex pattern of interactions between ligands and ASIC1a.

Ligands of histamine receptors modulate acid-sensing ion channels.

Recently we found that synthetic compounds containing amino group linked to hydrophobic or aromatic moiety are potent modulators of the proton-gated channels (ASICs). These structures have clear similarity with ligands of histamine receptors. We have also demonstrated that histamine potentiates homomeric ASIC1a by shifting its activation dependence to less acidic conditions. In the present work the action of a series of histamine receptors ligands on recombinant ASIC1a and ASIC2a was characterized. Two types of action were found for ASIC1a. 1-methylhistamine, N-alpha-methylhistamine, dimaprit and thioperamide caused significant potentiation, which was pH-dependent and voltage-independent. The H4R antagonist A943931 caused inhibition, which is likely due to voltage-dependent pore block. ASIC2a were virtually insensitive to the drugs tested. We conclude that ligands of histamine receptors should also be considered as ASIC modulators.

Computational Structural Pharmacology and Toxicology of Voltage-Gated Sodium Channels.

Voltage-gated sodium channels are targets for many toxins and medically important drugs. Despite decades of intensive studies in industry and academia, atomic mechanisms of action are still not completely understood. The major cause is a lack of high-resolution structures of eukaryotic channels and their complexes with ligands. In these circumstances a useful approach is homology modeling that employs as templates X-ray structures of potassium channels and prokaryotic sodium channels. On one hand, due to inherent limitations of this approach, results should be treated with caution. In particular, models should be tested against relevant experimental data. On the other hand, docking of drugs and toxins in homology models provides a unique possibility to integrate diverse experimental data provided by mutational analysis, electrophysiology, and studies of structure-activity relations. Here we describe how homology modeling advanced our understanding of mechanisms of several classes of ligands. These include tetrodotoxins and mu-conotoxins that block the outer pore, local anesthetics that block of the inner pore, batrachotoxin that binds in the inner pore but, paradoxically, activates the channel, pyrethroid insecticides that activate the channel by binding at lipid-exposed repeat interfaces, and scorpion alpha and beta-toxins, which bind between the pore and voltage-sensing domains and modify the channel gating. We emphasize importance of experimental data for elaborating the models.

[MOLECULAR EVOLUTION OF ION CHANNELS: AMINO ACID SEQUENCES AND 3D STRUCTURES].

An integral part of modern evolutionary biology is comparative analysis of structure and function of macromolecules such as proteins. The first and critical step to understand evolution of homologous proteins is their amino acid sequence alignment. However, standard algorithms fop not provide unambiguous sequence alignments for proteins of poor homology. More reliable results can be obtained by comparing experimental 3D structures obtained at atomic resolution, for instance, with the aid of X-ray structural analysis. If such structures are lacking, homology modeling is used, which may take into account indirect experimental data on functional roles of individual amino-acid residues. An important problem is that the sequence alignment, which reflects genetic modifications, does not necessarily correspond to the functional homology. The latter depends on three-dimensional structures which are critical for natural selection. Since alignment techniques relying only on the analysis of primary structures carry no information on the functional properties of proteins, including 3D structures into consideration is very important. Here we consider several examples involving ion channels and demonstrate that alignment of their three-dimensional structures can significantly improve sequence alignments obtained by traditional methods.

Analysis of inter-residue contacts reveals folding stabilizers in P-loops of potassium, sodium, and TRPV channels.

The family of P-loop channels includes potassium, sodium, calcium, cyclic nucleotide-gated and TRPV channels, as well as ionotropic glutamate receptors. Despite vastly different physiological and pharmacological properties, the channels have structurally conserved folding of the pore domain. Furthermore, crystallographic data demonstrate surprisingly similar mutual disposition of transmembrane and membrane-diving helices. To understand determinants of this conservation, here we have compared available high-resolution structures of sodium, potassium, and TRPV1 channels. We found that some residues, which are in matching positions of the sequence alignment, occur in different positions in the 3D alignment. Surprisingly, we found 3D mismatches in well-packed P-helices. Analysis of energetics of individual residues in Monte Carlo minimized structures revealed cyclic patterns of energetically favorable inter- and intra-subunit contacts of P-helices with S6 helices. The inter-subunit contacts are rather conserved in all the channels, whereas the intra-subunit contacts are specific for particular types of the channels. Our results suggest that these residue-residue contacts contribute to the folding stabilization. Analysis of such contacts is important for structural and phylogenetic studies of homologous proteins.

Statistical models suggest presence of two distinct subpopulations of miniature EPSCs in fast-spiking interneurons of rat prefrontal cortex.

Properties of excitatory synaptic responses in fast-spiking interneurons (FSIs) and pyramidal neurons (PNs) are different; however, the mechanisms and determinants of this diversity have not been fully investigated. In the present study, voltage-clamp recording of miniature excitatory post-synaptic currents (mEPSCs) was performed of layer 2-3 FSIs and PNs in the medial prefrontal cortex of rats aged 19-22days. The average mEPSCs in the FSIs exhibited amplitudes that were two times larger than those of the PNs and with much faster rise and decay. The mEPSC amplitude distributions in both cell types were asymmetric and in FSIs, the distributions were more skewed and had two-times larger coefficients of variation than in the PNs. In PNs but not in FSIs, the amplitude distributions were fitted well by different skewed unimodal functions that have been used previously for this purpose. In the FSIs, the distributions were well approximated only by a sum of two such functions, suggesting the presence of at least two subpopulations of events with different modal amplitudes. According to our estimates, two-thirds of the mEPSCs in FSIs belong to the high-amplitude subpopulation, and the modal amplitude in this subpopulation is approximately two times larger than that in the low-amplitude subpopulation. Using different statistical models, varying binning size, and data subsets, we confirmed the robustness and consistency of these findings.

The Effect of Hydrophobic Monoamines on Acid-Sensing Ion Channels ASIC1B.

Acid-sensing ion channels (ASICs) are widely distributed in both the central and peripheral nervous systems of vertebrates. The pharmacology of these receptors remains poorly investigated, while the search for new ASIC modulators is very important. Recently, we found that some monoamines, which are blockers of NMDA receptors, inhibit and/or potentiate acid-sensing ion channels, depending on the subunit composition of the channels. The effect of 9-aminoacridine, IEM-1921, IEM-2117, and memantine both on native receptors and on recombinant ASIC1a, ASIC2a, and ASIC3 homomers was studied. In the present study, we have investigated the effect of these four compounds on homomeric ASIC1b channels. Experiments were performed on recombinant receptors expressed in CHO cells using the whole-cell patch clamp technique. Only two compounds, 9-aminoacridine and memantine, inhibited ASIC1b channels. IEM-1921 and IEM-2117 were inactive even at a 1000 µM concentration. In most aspects, the effect of the compounds on ASIC1b was similar to their effect on ASIC1a. The distinguishing feature of homomeric ASIC1b channels is a steep activation-dependence, indicating cooperative activation by protons. In our experiments, the curve of the concentration dependence of ASIC1b inhibition by 9-aminoacridine also had a slope (Hill coefficient) of 3.8, unlike ASIC1a homomers, for which the Hill coefficient was close to 1. This finding indicates that the inhibitory effect of 9-aminoacridine is associated with changes in the activation properties of acid-sensing ion channels.

[Properties of spontaneous and miniature excitatory postsynaptic currents of rat prefrontal cortex neurons].

Quantum analysis of postsynaptic currents is important for fundamental and applied studies of synaptic transmission. In the present work, we investigated the possibility of using the characteristics of spontaneous excitatory postsynaptic currents (EPSCs) for estimation of quantum parameters of excitatory synaptic transmission in different types of neurons from rat prefrontal cortex slices. By blocking spontaneous spiking activity in slices by tetrodotoxin, we showed that spontaneous and miniature EPSCs in prefrontal cortex neurons did not differ by their properties. Thereby, both spontaneous and miniature responses can be used for estimation of quantum parameters of excitatory synaptic transmission in this preparation. We also revealed that excitatory spontaneous responses of pyramidal cells were 2 times lower by amplitude, had twice lower the coefficient of variation and exhibited much slower kinetics than responses of the fast-spiking and regular-spiking interneurons. Possible mechanisms of these differences are considered.

Specific mechanism of use-dependent channel block of calcium-permeable AMPA receptors provides activity-dependent inhibition of glutamatergic neurotransmission.

This study examined the blocking action of the selective channel blocker of calcium-permeable (CP) AMPA receptors, N1-(1-phenylcyclohexyl)pentane-1,5-diaminium bromide (IEM-1925), on excitatory postsynaptic currents in rat neostriatal and cortical neurons and in fly neuromuscular junctions. In both preparations, the blocking of CP-AMPA receptor currents increased along with the stimulation frequency. The continuous presence of kainate, which activates AMPA receptors, in the external solution also caused an enhanced blocking effect. Likewise, decrease of the synaptic release by lowering calcium concentration resulted in significant reduction of the blocking action. The activity dependence of the block is explained using the guarded receptor model. The drug molecule can only bind if the channel is open. After the channel has closed, the drug molecule remains trapped inside. However, the trapped molecule slowly egresses from closed channels to the cytoplasm. The total block effect is determined by the equilibrium between accumulation of the drug in the open channels and relief from the closed channels. Therefore, the conditions that favour the open state result in enhanced inhibition. This significant finding reveals a new way to modulate CP-AMPAR-mediated transmission using a physiologically relevant approach. Moreover, it allows the involvement of CP-AMPARs in the physiological and pathological processes ­ such as high-frequency synaptic activity or increase of the steady-state glutamate concentration ­ to be examined.

[The diversity of mechanisms of the ion channels blockade as a way towards designing new pharmacological agents].

Dysfunctions of glutamatergic synaptic neurotransmission often accompany various CNS disorders. Action of excessive glutamate, which causes excitotoxic effects by neuron depolarization and massive calcium influx can lead to cell death. Despite obvious importance of development of anti-glutamic neuroprotectors, among great number of known antagonists of ionotropic glutamate receptors only memantine is used in medicinal practice. One of the sources of numerous side effects caused by glutamate receptor antagonists is that the drugs usually inhibit receptors, which mediate both normal and pathological CNS processes. A possible approach to overcoming the problem is to develop the drugs whose action is enhanced in potentially pathological conditions such as high-frequency activation, high glutamate concentration, depolarized membrane, etc. Action of many classes of antagonists depends on pattern of receptor activation and on membrane voltage. In the present work, we discuss several peculiarities of channel blocking mechanisms from the viewpoint of neuroprotector development. In particular, we compare channel blockers which demonstrate different types of interaction with the channel gating machinery, we consider different types of voltage dependence and consider action of channel blockers, which can permeate through the channel. We conclude that meticulous analysis of the mechanism of action of the glutamate receptor channel antagonists could help to approach predicting of in vivo action using in vitro data.

[The effect of the environment ion composition upon the blockade of the AMPA receptor channels].

It is well known that ion composition significantly affects action of various pharmacological agents. In the present work we studied influence of external sodium on the block of Ca2+ -permeable AMPA receptor channels by dicationic derivative of phenylcyclohexyl IEM-1925. Experiments were performed on native receptors of giant striatal interneurones isolated from the rat brain slices. Registrations were done with the aid of whole-cell patch clamp technique. We found that partial substitution of external sodium by sucrose potentiated the blocking action of IEM-1925. The effect was voltage-dependent, being more pronounced at hyperpolarized voltages. The analysis of kinetics of the IEM-1925 action demonstrated that lowering of external sodium facilitated blocking action, whereas stability of the drug-channel complex remained unaffected. We conclude that the current carrying sodium ions compete with blocking cation IEM-1925 for an intrapore binding site.

Origin and molecular evolution of ionotropic glutamate receptors.

This article provides a review of approaches to identification of the pathways of the molecular evolution of glutamate receptors. Extensive evidence has now accumulated on the homology of glutamate-binding proteins with the ability to function as ligand-activated channels. However, knowledge of the amino acid sequences of the polypeptides forming these channels is a necessary but insufficient condition for identifying their origin and changes during evolution. Natural selection of protein molecules appears to have identified and fixed their functional nature. Molecular and functional approaches should therefore complement each other in studies of protein evolution. Studies of glutamate receptor channels in vertebrates and invertebrates provide an example showing how knowledge of the spatial organization and the details of the mechanisms of operation allows relationships to be identified and possible pathways of the molecular evolution of receptors to be established.

Characterization of ionotropic glutamate receptors in insect neuro-muscular junction.

Pharmacological properties of ionotropic glutamate receptors from Calliphora vicina larvae neuro-muscular junction (C. vicina iGlurs) were studied by two-electrode voltage-clamp technique. Characteristics of the ion channel pore were analyzed using a 26-member series of channel blockers, which includes mono- and dicationic derivatives of adamantane and phenylcyclohexyl. Structure-activity relationships were found to be markedly similar to the Ca2+-permeable AMPA receptors (AMPAR) but not NMDA receptors (NMDAR) channel subtype seen in vertebrates. Like AMPARs the channels of C. vicina iGlurs are sensitive mainly to dicationic compounds with 6-7 spacers between hydrophobic headgroup and terminal aminogroup. Study of the voltage dependence of block demonstrated that, like AMPARs, the C. vicina iGlur channels, are permeable to organic cations with dimensions exceeding 10 A. Concentration dependence of block suggests the presence of two distinct channel populations with approximately 20-fold different sensitivity to cationic blockers. The recognition domain properties are more complex. Besides glutamate, the channels can be activated by kainate, quisqualate and domoate. Competitive antagonists of AMPAR and NMDAR are virtually inactive against the C. vicina iGlurs as well as allosteric modulators GYKI 52466 and PEPA. Surprisingly, the responses were potentiated 3 times by 100 mkM of cyclothiazide. We conclude that the channel-forming domain of C. vicina iGlurs is AMPAR-like, whereas the recognition domain is specific.

[The origin and molecular evolution of ionotropic glutamate receptors].

A review of the main approaches to the revealing molecular evolution of glutamate receptors is presented. Large amount of evidences concerning the homology of glutamate-binding proteins forming the membrane channels has been accumulated. However, the knowledge of amino acid sequences of these proteins is the necessary but not sufficient condition for clarification of their origin and the changes in the course of molecular evolution. The natural selection estimated and secured the functional validity ofligand-gated channels. Therefore the functional and molecular approaches should supplement each other. It has been shown by and example of glutamate receptor channels of vertebrate and invertebrate animals that the combined analysis of the structure and function allows to reveal the main routes of molecular evolution of this kind of synaptic receptors.

Mechanisms of action of ligands of potential-dependent sodium channels.

Potential-dependent sodium channels play a leading role in generating action potentials in excitable cells. Sodium channels are the site of action of a variety of modulator ligands. Despite numerous studies, the mechanisms of action of many modulators remain incompletely understood. The main reason that many important questions cannot be resolved is that there is a lack of precise data on the structures of the channels themselves. Structurally, potential-dependent sodium channels are members of the P-loop channel superfamily, which also include potassium and calcium channels and glutamate receptor channels. Crystallization of a series of potassium channels showed that it was possible to analyze the structures of different members of the superfamily using the "homologous modeling" method. The present study addresses model investigations of the actions of ligands of sodium channels, including tetrodotoxin and batrachotoxin, as well as local anesthetics. Comparison of experimental data on sodium channel ligands with x-ray analysis data allowed us to reach a new level of understanding of the mechanisms of channel modulation and to propose a series of experimentally verifiable hypotheses.

Organic blockers escape from trapping in the AMPA receptor channels by leaking into the cytoplasm.

The voltage-dependent block of AMPA receptor (AMPAR) channels by a series of dicationic compounds was studied on native GluR2-lacking receptors of striatal giant interneurons isolated from rat brain slices. The dicationic derivatives of adamantane, dimethyladamantane, diphenyl, and phenylcyclohexyl were used. Voltage dependence of the blockade and of the unblocking rate suggests that the compounds permeate the open AMPAR channels. The permeation of adamantane derivatives was demonstrated previously. However, for derivatives of phenylcyclohexyl this finding is surprising because of the large dimensions of the phenylcyclohexyl moiety. All these compounds were found to get trapped in the closed state of the channel. However, time-dependent decrease of trapping was found. This effect is accelerated by hyperpolarization, suggesting that blockers can escape from trapping into the cytoplasm. Importantly, there is a correlation between permeation through the open channel and escape from trapping. Dicationic compounds were shown to block open and closed AMPAR channels from the inside of the cell. Thus, trapping of AMPAR channel blockers after agonist removal does not prevent escape of blockers into the cytoplasm. It is concluded that closure of the AMPAR channel gates at the extracellular vestibule is not coupled with plugging of the pathway between the selectivity filter and cytoplasm. Possible physiological importance of this blocking mechanism is discussed.

[Ion channels of glutamate receptors of nerve-muscle junction of the fly larva Calliphora vicina demonstrate a high structural homology with vertebrate AMPA-channels].

In experiments on the nerve-muscle junction of the fly larva Calliphora vicina, regularities of the blocking action of organic cations on ion channels of glutamate postsynaptic receptors have been studied. In total, 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 channel 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 characteristic of blockade of the AMPA channels, but not of the vertebrate type NMDA channels. Earlier it has been shown that differences in structural-functional relations during blockade of the AMPA and NMDA channels are 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 action of voltage-gated sodium channel ligands].

The voltage-gated sodium channels play a key role in the generation of action potential in excitable cells. Sodium channels are targeted by a number of modulating ligands. Despite numerous studies, the mechanisms of action of many ligands are still unknown. The main cause of the problem is the absence of the channel structure. Sodium channels belong to the superfamily of P-loop channels that also the data abowt includes potassium and calcium channels and the channels of ionotropic glutamate receptors. Crystallization of several potassium channels has opened a possibility to analyze the structure of other members of the superfamily using the homology modeling approach. The present study summarizes the results of several recent modelling studies of such sodium channel ligands as tetrodotoxin, batrachotoxin and local anesthetics. Comparison of available experimental data with X-ray structures of potassium channels has provided a new level of understanding of the mechanisms of action of sodium channel ligands and has allowed proposing several testable hypotheses.