Maxim V. Nikolaev

Low concentrations of ouabain exert neuromodulatory effects beyond classical Na⁺/K⁺-ATPase inhibition, but the underlying mechanisms remain incompletely understood. Here, we investigated the involvement of Na⁺/Ca²⁺ exchangers (NCXs) in the modulation of spontaneous and evoked excitatory synaptic transmission by 1 nM ouabain. In cultured cortical neurons, ouabain reduced miniature excitatory postsynaptic current (mEPSC) frequency by approximately 4-fold without affecting amplitude. This effect was prevented by the broad-spectrum NCX inhibitor CB-DMB but persisted in the presence of the preferential NCX1 inhibitor SEA0400. In hippocampal CA1, ouabain increased the slope of evoked field excitatory postsynaptic potentials (fEPSPs) by approximately 34%. SEA0400 also potentiated fEPSPs, with additional potentiation observed upon sequential application of ouabain and SEA0400. In contrast, CB-DMB (NCX1–3 inhibitor) increased the fEPSP slope but prevented further potentiation by ouabain. These findings demonstrate differential modulation of spontaneous and evoked excitatory synaptic transmission by nanomolar ouabain and support the involvement of NCX-sensitive mechanisms, potentially including NCX2 and/or NCX3. Our results suggest that functional coupling between Na⁺/K⁺-ATPase and NCX-dependent Na⁺ and Ca²⁺ homeostasis contributes to the neuromodulatory effects of low ouabain concentrations.

The development of antiepileptic drugs remains a serious challenge for both academia and industry. Ionotropic glutamate receptors and voltage-gated sodium channels are among the primary targets of antiepileptic agents. Recent studies have revealed a unique property of phenytoin: unlike other sodium-channel blockers, it inhibits calcium-impermeable AMPA receptors at micromolar concentrations. In this study, we explored the structure–activity relationships of eight phenytoin derivatives. The effects of the compounds on neuronal voltage-gated Na⁺ channels and neuronal AMPA receptor channels were examined using the patch-clamp technique. For the Na⁺ channels, we analyzed tonic block, shifts of steady-state inactivation, and frequency-dependent block. For the AMPA receptors, we investigated kinetics, agonist dependence, and trapping effects. NH groups at positions 1 and 3, the carbonyl groups at positions 2 and 4, and the phenyl group at position 5 are important, since their replacement causes a decrease in activity. Replacement of oxygen with sulfur at position 2 results in a significant increase in activity on both types of channels. For the AMPA receptor, this increase is attributable to a more stable drug–channel complex. The enhanced action on sodium channels is due to an increase in tonic block, whereas the effects on inactivated and open channels remain unchanged. These results suggest a new possibility for tuning the activities of phenytoin derivatives against both primary targets to obtain anticonvulsants with novel properties.

Photopharmacology provides superior temporal and spatial resolution for studying the functions of ion channels within neuronal networks. Photomodulated channel blockers, such as azobenzene quaternary ammonium derivatives, QAQ (quaternary ammonium-azobenzene-quaternary ammonium), DENAQ (diethylamine-azobenzene-quaternary ammonium), and AAQ (acrylamide-azobenzene-quaternary ammonium), have been intensively studied in vertebrates, but their effects on insects remain unknown. In this paper we study their ability to modulate synaptic transmission at the neuromuscular junction of Calliphora vicina larvae. Despite having rather similar structures, these compounds cause dramatically different effects. QAQ in its trans-form effectively inhibits excitatory postsynaptic currents by blocking the ion pores of postsynaptic glutamate receptors at micromolar concentrations, but it becomes less effective when photoconverted to cis-form. In contrast, DENAQ behaves as a light-dependent potentiator of synaptic transmission that triggers the appearance of multispike responses, likely due to the blockage of presynaptic potassium channels, which enhances nerve excitability. The effect of AAQ demonstrates complex action due to a combination of these two mechanisms. The same targets for these compounds were previously identified in vertebrates, suggesting significant evolutionary conservativity of the targets. Our study highlights that even for structurally related compounds the targets, mode of action, and light-dependence can vary dramatically. Thus, the rational use of photochromic compounds requires careful analysis of their targets and molecular mechanism(s) of action.

A novel catalyst-free photochemical synthesis of benzoxazin-4-ones and benzoxazepin-5-ones is reported. The products were evaluated in an electrophysiological study on native AMPA receptors.

Background and Purpose: Photo‐switchable compounds acting on ion channels are valuable tools for neuroscience. Specific ligands have been developed for many channels, but selective compounds providing effective optical control for calcium‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors are still lacking. Because the difference between calcium‐permeable and impermeable receptors is localised in the channel selectivity filter, the only feasible approach to discriminate between them is to design photo‐switchable pore blockers. We have, here, synthesised azobenzene derivatives of spermine and investigated their actions.Experimental Approach: The biological activity of azobenzene–spermines was studied in electrophysiological experiments using isolated rat brain neurons, synaptic transmission in slices, fly larvae neuromuscular junction and recombinant AMPA receptors expressed in CHO cells. The experimental results were rationalised using computer modelling.Key Results: The most active compound, azobenzene–spermine‐6 (ABSP‐6), selectively blocked calcium‐permeable AMPA receptors in low nanomolar concentrations under ambient light, with an IC₅₀ under illumination, three orders of magnitude higher. Using rat brain slices, ABSP‐6 provided optical control of excitatory postsynaptic current (EPSC) mediated by AMPA receptors in giant interneurons of striatum, while EPSCs in cortical pyramidal neurons were unaffected. Computer modelling suggests that in the light‐induced cis configuration, formation of an intramolecular H bond reduces the effective length of polyamine chain, resulting in marked loss of activity.Conclusions and Implications: Our work provides the first effective optical control of calcium‐permeable AMPA receptors, suited to neurophysiological studies. It will be crucial in elucidating the localisation of glutamate receptor subtypes and clarifying their role in various processes.

The search for novel compounds with anticonvulsant properties remains a key focus in neuropharmacology. Recently, the diazepine-benzimidazole derivative, DAB-19, has emerged as a promising candidate due to its demonstrated anxiolytic and analgesic effects. In this study, we investigate the mechanisms underlying DAB-19’s activity, focusing on its impact on glutamatergic transmission, a key target in the pathophysiology of various central nervous system disorders. Intriguingly, while DAB-19 suppressed evoked glutamatergic transmission in rat brain slices, it simultaneously enhanced spontaneous neurotransmission. Further experiments on glutamatergic neuromuscular synapses in fly larvae revealed two distinct mechanisms: calcium-dependent potentiation of glutamate release and inhibition of spike propagation via blockade of voltage-gated sodium channels. The latter effect was directly confirmed in rat brain neurons. Given its action on sodium channels, we tested DAB-19 in the pentylenetetrazole model, where it delayed seizure onset but did not prevent seizures. These findings position DAB-19 as a multifaceted compound with significant therapeutic potential.

The action of tetraalkylammonium ions, from tetrametylammonium (TMA) to tetrapentylammonium (TPtA), on the recombinant and native acid-sensing ion channels (ASICs) was studied using the patch-clamp approach. The responses of ASIC1a, ASIC2a, and native heteromeric ASICs were inhibited by TPtA. The peak currents through ASIC3 were unaffected, whereas the steady-state currents were significantly potentiated. This effect was characterized by an EC₅₀ value of 1.22 ± 0.12 mM and a maximal effect of 3.2 ± 0.5. The effects of TPtA were voltage-independent but significantly decreased under conditions of strong acidification, which caused saturation of ASIC responses. Molecular modeling predicted TPtA binding in the acidic pocket of closed ASICs. Bound TPtA can prevent acidic pocket collapse through a process involving ASIC activation and desensitization. Tetraethylammonium (TEA) inhibited ASIC1a and native ASICs. The effect was independent of the activating pH but decreased with depolarization, suggesting a pore-blocking mechanism.

Glutamate ionotropic receptors mediate fast excitation processes in the central nervous system of vertebrates and play an important role in synaptic plasticity, learning, and memory. Here, we describe the action of two azobenene-containing compounds, AAQ (acrylamide–azobenzene–quaternary ammonium) and QAQ (quaternary ammonium–azobenzene–quaternary ammonium), which produced rapid and fully reversible light-dependent inhibition of glutamate ionotropic receptors. The compounds demonstrated voltage-dependent inhibition with only minor voltage-independent allosteric action. Calcium-impermeable AMPA receptors had weaker sensitivity compared to NMDA and calcium-permeable AMPA receptors. We further revealed that the compounds bound to NMDA and calcium-permeable AMPA receptors in different modes. They were able to enter the wide selectivity filter of AMPA receptors, and strong negative voltages caused permeation into the cytoplasm. The narrow selectivity filter of the NMDA receptors did not allow the molecules to bypass them; therefore, QAQ and AAQ bound to the shallow channel site and prevented channel closure by a foot-in-the-door mechanism. Computer simulations employing available AMPA and NMDA receptor structures readily reproduced the experimental findings, allowing for the structure-based design of more potent and selective drugs in the future. Thus, our work creates a framework for the development of light-sensitive blockers of calcium-permeable AMPA receptors, which are desirable tools for neuroscience.

Azobenzene-based quaternary ammonium compounds provide optical control of ion channels and are considered promising agents for regulation of neuronal excitability and for restoration of the photosensitivity of retinal cells. However, the selectivity of the action of these compounds remains insufficiently known. We studied the action of DENAQ (diethylamine-azobenzene-quaternary ammonium) and DMNAQ (dimethylamine-azobenzene-quaternary ammonium) on ionotropic glutamate receptors in rat brain neurons. In the dark, both compounds applied extracellularly caused fast and reversible inhibition of NMDA (N-methyl-d-aspartate) receptor-mediated currents with IC₅₀ values of 10 and 5 μM, respectively. Light-induced transformation of DENAQ and DMNAQ to their cis forms caused the IC₅₀ values to increase to 30 and 27 μM, respectively. Detailed analysis of this action revealed a complex nature consisting of fast inhibitory and slower potentiating effects. The AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors were only weakly affected independently on illumination. We conclude that, in addition to their long-lasting intracellular action, which persists after washout, azobenzene-based quaternary ammonium compounds should affect glutamatergic transmission and synaptic plasticity during treatment. Our findings also extend the list of soluble photoswitchable inhibitors of NMDA receptors. While the site(s) and mechanisms of action are unclear, the effect of DENAQ demonstrates strong pH dependence. At acidic pH values, DENAQ potentiates both NMDA and AMPA receptors.