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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 CPAMPARs 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. [ABSTRACT FROM AUTHOR]

Structure-activity relationships of 56 pentamethylenbis-ammonium compounds, the blockers of the neuronal nicotinic acetylcholine receptor (nAChR) ion channel, have been studied to estimate the cross-sectional dimensions of the channel pore. The cat superior cervical sympathetic ganglion in situ and isolated guinea pig ileum were used to evaluate the potency of the compounds to block ganglionic transmission. Minimum-energy conformations of each compound were calculated by the molecular mechanics method. A topographic model of the binding site of the blockers was proposed. It incorporates two narrowings, a large and a small one. The small narrowing is located between the large one and the cytoplasmic end of the pore. The cross-sectional dimensions of the large and small narrowings estimated from the dimensions of the blockers are 6.1 x 8.3 A and 5.5 x 6.4 A, respectively, the distance between the narrowings along the pore being approximately 7 A. Most potent blockers would occlude the pore via binding to the channel at the levels of both narrowings. Less potent blockers are either too large or too small to bind to both narrowings simultaneously: large blockers would occlude the pore at the level of large narrowing, while small blockers would pass the large narrowing and occlude the pore at the level of small narrowing only. A comparison of the topographic model with a molecular five-helix bundle model of nAChR pore predicts Serine and Threonine rings to be the most probable candidates for the large and small narrowings, respectively. [ABSTRACT FROM AUTHOR]

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.

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., (Copyright © 2017. Published by Elsevier Inc.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

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.

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.

Using whole cell patch-clamp recording from pyramidal cells and interneurons in the CA1 area of hippocampal slices, the effect of IEM-1460, a selective channel blocker of Ca2+ permeable AMPA receptors (AMPARs), on postsynaptic currents (PSCs) was studied. Excitatory postsynaptic currents (EPSCs) were evoked by stimulation of Schaffer collaterals (SCs) in the presence of APV and bicuculline to pharmacologically isolate the EPSCs mediated by AMPAR activation. IEM-1460 (50 microM) did not affect the amplitude of EPSCs in CA1 pyramidal cells but reversibly decreased their amplitude in interneurons of pyramidal layer (15 cells), radiatum (37 cells) and border radiatum-lacunosum-moleculare (R-LM) (55 cells) layers. The ability of IEM-1460 to decrease EPSC amplitude correlated with EPSC rectification properties in CA1 interneurons, providing evidence for synaptic localization of Ca2+ permeable AMPARs at the SC synaptic input. Independent of their localization, the majority of interneurons studied exhibited only modest sensitivity to IEM-1460 (EPSC amplitude decreased by less than 30%), while in 15% of interneurons IEM-1460 induced more than 50% reduction in EPSC amplitude. To reveal possible afferent-specific localization of Ca2+ permeable AMPARs on R-LM interneurons, the effect of IEM-1460 on EPSCs evoked by stimulation of SC was compared with that of perforant path (PP). Although average sensitivities did not differ significantly, in 61% of R-LM layer interneurons, the SC-evoked EPSCs exhibited higher sensitivity to IEM-1460 than the PP-evoked EPSCs. Moreover, in 54% of R-LM layer interneurons the EPSCs evoked by SC stimulation were complex, having an initial peak followed by one or several late components. Kinetics, latency distribution and reversal potential of late components suggest di- and polysynaptic origin of the late components. Late EPSCs were strongly and reversibly inhibited by IEM-1460 indicating that Ca2+ permeable AMPARs are involved in the indirect excitation of R-LM layer interneurons. Despite the ability to decrease the excitatory synaptic input to interneurons, IEM-1460 did not affect interneuron-mediated inhibitory postsynaptic currents (IPSCs) evoked in pyramidal neurons by SC stimulation. These data suggest that interneurons with a synaptic input highly sensitive to IEM-1460 do not contribute specifically to the feed-forward inhibition of hippocampal pyramidal neurons.

Transient proton-activated currents induced by rapid shifts of the extracellular pH from 7.4 to < or =6.8 were recorded in different neurons freshly isolated from rat brain (hypoglossal motoneurons, cerebellar Purkinje cells, striatal giant cholinergic interneurons, hippocampal interneurons, CA1 pyramidal neurons and cortical pyramidal neurons) using whole-cell patch clamp technique. Responses of hippocampal CA1 pyramidal neurons were weak (100-300 pA) in contrast to other types of neurons (1-3 nA). Sensitivity of neurons to rapid acidification varied from pH(50) 6.4 in hypoglossal motoneurons to 4.9 in hippocampal interneurons. Proton-activated currents were blocked by amiloride (IC(50) varied from 3.6 to 9.5 microM). Reversal potential of the currents was close to E(Na), indicating that the currents are carried by sodium ions. The data obtained suggest that the proton-activated currents in the neurons studied are mediated by acid-sensitive ion channels. Strong acidification (pH<4) induced biphasic responses in all neuron types: the transient current was followed by a pronounced sustained one. Sustained current was not blocked by amiloride and exhibited low selectivity for sodium and cesium ions. Slow acidification from pH 7.4 to 6.5 did not induce detectable whole-cell currents. At pH 6.5, most of the channels are desensitized and responses to fast pH shifts from this initial level are decreased at least 10 times. This suggests that slow acidification which is well known to accompany some pathological states should rather desensitize than activate acid-sensitive ion channels and depress their function. Our results provide evidence for a widespread and neuron-specific distribution of acid-sensitive ion channels in the brain. The large amplitudes and transient character of currents mediated by these channels suggest that they could contribute to fast neuronal signaling processes.

In order to investigate the topography of the channel binding site in (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) types of glutamate receptors, we have compared the blocking actions of mono- and dicationic derivatives of adamantane and phenylcyclohexyl. The potencies of dicationic derivatives to block AMPA receptor channels are about 1000 times higher than those of monocationic ones, whereas NMDA receptors are equally sensitive to both mono- and dicationic derivatives. The dependence of the activity of dicationic compounds on the length of the polymethylene chain between ammonium groups has a pronounced maximum for AMPA receptor channel block. For NMDA receptor channel dicationic compounds with various internitrogen distances produce similar blocking effects. The results show that hydrophobic and nucleophilic components of the binding site are located close to each other in the NMDA receptor channel but are separated by approximately 10 A in the AMPA receptor channel.