N-methyl-D-aspartate (NMDA) receptors are inhibited by many amidine and guanidine compounds. In this work, we studied the mechanisms of their inhibition by sepimostat—an amidine-containing serine protease inhibitor with neuroprotective properties. Sepimostat inhibited native NMDA receptors in rat hippocampal CA1 pyramidal neurons with IC₅₀ of 3.5 ± 0.3 µM at −80 mV holding voltage. It demonstrated complex voltage dependence with voltage-independent and voltage-dependent components, suggesting the presence of shallow and deep binding sites. At −80 mV holding voltage, the voltage-dependent component dominates, and we observed pronounced tail currents and overshoots evidencing a “foot-in-the-door” open channel block. At depolarized voltages, the voltage-independent inhibition by sepimostat was significantly attenuated by the increase of agonist concentration. However, the voltage-independent inhibition was non-competitive. We further compared the mechanisms of the action of sepimostat with those of structurally-related amidine and guanidine compounds—nafamostat, gabexate, furamidine, pentamidine, diminazene, and DAPI—investigated previously. The action of all these compounds can be described by the two-component mechanism. All compounds demonstrated similar affinity to the shallow site, which is responsible for the voltage-independent inhibition, with binding constants in the range of 3–30 µM. In contrast, affinities to the deep site differed dramatically, with nafamostat, furamidine, and pentamidine being much more active.

Febrile seizures (FSs) are a relatively common early-life condition that can cause CNS developmental disorders, but the specific mechanisms of action of FS are poorly understood. In this work, we used hyperthermia-induced FS in 10-day-old rats. We demonstrated that the efficiency of glutamatergic synaptic transmission decreased rapidly after FS by recording local field potentials. This effect was transient, and after two days there were no differences between control and post-FS groups. During early ontogeny, the proportion of calcium-permeable (CP)-AMPA receptors in the synapses of the principal cortical and hippocampal neurons is high. Therefore, rapid internalization of CP-AMPA receptors may be one of the mechanisms underlying this phenomenon. Using the whole-cell patch-clamp method and the selective CP-AMPA receptor blocker IEM-1460, we tested whether the proportion of CP-AMPA receptors changed. We have demonstrated that FS rapidly reduces synaptic CP-AMPA receptors in both the hippocampus and the entorhinal cortex. This process was accompanied by a sharp decrease in the calcium permeability of the membrane of principal neurons, which we revealed in experiments with kainate-induced cobalt uptake. Our experiments show that FSs cause rapid changes in the function of the glutamatergic system, which may have compensatory effects that prevent excessive excitotoxicity and neuronal death.

Proton-gated channels of the ASIC family are widely distributed in central neurons, suggesting their role in common neurophysiological functions. They are involved in glutamatergic neurotransmission and synaptic plasticity; however, the exact function of these channels remains unclear. One problem is that acidification of the synaptic cleft due to the acidic content of synaptic vesicles has opposite effects on ionotropic glutamate receptors and ASICs. Thus, the pH values required to activate ASICs strongly inhibit AMPA receptors and almost completely inhibit NMDA receptors. This, in turn, suggests that ASICs can provide compensation for post-synaptic responses in the case of significant acidifications. We tested this hypothesis by patch-clamp recordings of rat brain neuron responses to acidifications and glutamate receptor agonists at different pH values. Hippocampal pyramidal neurons have much lower ASICs than glutamate receptor responses, whereas striatal interneurons show the opposite ratio. Cortical pyramidal neurons and hippocampal interneurons show similar amplitudes in their responses to acidification and glutamate. Consequently, the total response to glutamate agonists at different pH levels remains rather stable up to pH 6.2. Besides these pH effects, the relationship between the responses mediated by glutamate receptors and ASICs depends on the presence of Mg²⁺ and the membrane voltage. Together, these factors create a complex picture that provides a framework for understanding the role of ASICs in synaptic transmission and synaptic plasticity.

Febrile seizures (FS) are a relatively common early-life condition that can cause CNS developmental disorders, but the specific mechanisms of action of FS are poorly understood. In this work, we used hyperthermia-induced FS in 10-day-old rats. By recording local field potentials, we showed that the efficiency of glutamatergic synaptic transmission decreased 15 min after FS. This effect was transient, and after 2 days there were no differences between control and post-FS groups. During early ontogeny, the proportion of calcium-permeable (CP)-AMPA receptors in the synapses of principal cortical and hippocampal neurons is high. Therefore, rapid internalization of CP-AMPA receptors may be one of the mechanisms underlying this phenomenon. Using the whole-cell patch clamp method and the selective CP-AMPA receptor blocker IEM-1460, we tested whether the proportion of CP-AMPA receptors changes. We have demonstrated that FS rapidly reduces synaptic CP-AMPA receptors in both the hippocampus and the entorhinal cortex. This process was accompanied by a sharp decrease in calcium permeability of the membrane of principal neurons, which we revealed in experiments with kainate-induced cobalt uptake. Our experiments show that FSs cause rapid changes in the function of the glutamatergic system, which may have compensatory effects that prevent excessive excitotoxicity and neuronal death.

N-methyl-D-aspartate (NMDA) receptors are inhibited by many medicinal drugs. The recent successful repurposing of NMDA receptor antagonists ketamine and dextromethorphan for the treatment of major depressive disorder further enhanced the interest in this field. In this work, we performed a screening for the activity against native NMDA receptors of rat CA1 hippocampal pyramidal neurons among biguanide compounds using the whole-cell patch-clamp method. Antimalarial biguanides proguanil and cycloguanil, as well as hypoglycemic biguanide phenformin, inhibited them in micromolar concentrations, while another hypoglycemic biguanide metformin and antiviral biguanide moroxydine were practically ineffective. IC₅₀ values at −80 mV holding voltage were 3.4 ± 0.6 µM for cycloguanil, 9.0 ± 2.2 µM for proguanil and 13 ± 1 µM for phenformin. The inhibition by all three compounds was not competitive. Cycloguanil acted as an NMDA receptor voltage-dependent trapping channel blocker, while proguanil and phenformin acted as allosteric inhibitors. Our results support the potential clinical repurposing of biguanide compounds for the treatment of neurodegenerative disorders linked to glutamatergic excitotoxicity while also providing a better understanding of structural determinants of NMDA receptor antagonism by biguanides.

Amidine-containing compounds are primarily known as antiprotozoal agents (pentamidine, diminazene, furamidine) or as serine protease inhibitors (nafamostat, sepimostat, camostat, gabexate). DAPI is widely recognized as a fluorescent DNA stain. Recently, it has been shown that these compounds also act as NMDA receptor inhibitors. In this study, we examined the activity of these compounds and analyzed the mechanisms of action in relation to another important class of ionotropic glutamate receptors–calcium-permeable AMPA receptors (CP-AMPARs) and calcium-impermeable AMPA receptors (CI-AMPARs) – using the whole-cell patch-clamp method on isolated male Wistar rat brain neurons. Gabexate and camostat were found to be inactive. Other compounds preferentially inhibited calcium-permeable AMPA receptors with IC₅₀ values of 30–60 µM. DAPI and furamidine were also active against CI-AMPARs with IC₅₀s of 50–60 μM, while others showed poor activity. All active compounds acted as channel blockers, which are able for permeating into the cytoplasm on both CP- and CI-AMPARs. Specifically, sepimostat showed trapping in the closed CP-AMPAR channel. Furamidine and DAPI demonstrated a voltage-independent action on CI-AMPARs, indicating binding to an additional superficial site. While the majority of compounds inhibited glutamate-activated steady-state currents as well as kainate-activated currents on CI-AMPARs, pentamidine significantly potentiated glutamate-induced steady-state responses. The potentiating effect of pentamidine resembles the action of the positive allosteric modulator cyclothiazide although the exact binding site remains unclear. Thus, this study, together with our previous research on NMDA receptors, provides a comprehensive overview of this novel group of ionotropic glutamate receptors inhibitors with a complex pharmacological profile, remarkable diversity of effects and mechanisms of action.