The diversity of AMPA receptor inhibition mechanisms among amidine-containing compounds.

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.

Probing the Proton-Gated ASIC Channels Using Tetraalkylammonium Ions.

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.

Mechanisms of dihydropyridine agonists and antagonists in view of cryo-EM structures of calcium and sodium channels.

Opposite effects of 1,4-dihydropyridine (DHP) agonists and antagonists on the L-type calcium channels are a challenging problem. Cryo-EM structures visualized DHPs between the pore-lining helices S6III and S6IV in agreement with published mutational data. However, the channel conformations in the presence of DHP agonists and antagonists are virtually the same, and the mechanisms of the ligands' action remain unclear. We docked the DHP agonist S-Bay k 8644 and antagonist R-Bay k 8644 in Cav1.1 channel models with or without π-bulges in helices S6III and S6IV. Cryo-EM structures of the DHP-bound Cav1.1 channel show a π-bulge in helix S6III but not in S6IV. The antagonist's hydrophobic group fits into the hydrophobic pocket formed by residues in S6IV. The agonists' polar NO2 group is too small to fill up the pocket. A water molecule could sterically fit into the void space, but its contacts with isoleucine in helix S6IV (motif INLF) would be unfavorable. In a model with π-bulged S6IV, this isoleucine turns away from the DHP molecule and its position is occupied by the asparagine from the same motif INLF. The asparagine provides favorable contacts for the water molecule at the agonist's NO2 group but unfavorable contacts for the antagonist's methoxy group. In our models, the DHP antagonist stabilizes entirely α-helical S6IV. In contrast, the DHP agonist stabilizes π-bulged helix S6IV whose C-terminal part turned and rearranged the activation-gate region. This would stabilize the open channel. Thus, agonists, but not antagonists, would promote channel opening by stabilizing π-bulged helix S6IV.

Possible Compensatory Role of ASICs in Glutamatergic Synapses.

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.

Analysis of residue-residue interactions in the structures of ASIC1a suggests possible gating mechanisms.

The gating mechanism of acid-sensitive ion channels (ASICs) remains unclear, despite the availability of atomic-scale structures in various functional states. The collapse of the acidic pocket and structural changes in the low-palm region are assumed to trigger activation. For the acidic pocket, protonation of some residues can minimize repulsion in the collapsed conformation. The relationship between low-palm rearrangements and gating is unknown. In this work, we performed a Monte Carlo energy optimization of known ASIC1a structures and determined the residue-residue interactions in different functional states. For rearrangements in the acidic pocket, our results are consistent with previously proposed mechanisms, although significant complexity was revealed for the residue-residue interactions. The data support the proposal of a gating mechanism in the low-palm region, in which residues E80 and E417 share a proton to activate the channel.

Derivatives of 2-aminobenzimidazole potentiate ASIC open state with slow kinetics of activation and desensitization.

The pharmacology of acid-sensitive ion channels (ASICs) is diverse, but potent and selective modulators, for instance for ASIC2a, are still lacking. In the present work we studied the effect of five 2-aminobenzimidazole derivatives on native ASICs in rat brain neurons and recombinant receptors expressed in CHO cells using the whole-cell patch clamp method. 2-aminobenzimidazole selectively potentiated ASIC3. Compound Ru-1355 strongly enhanced responses of ASIC2a and caused moderate potentiation of native ASICs and heteromeric ASIC1a/ASIC2a. The most active compound, Ru-1199, caused the strongest potentiation of ASIC2a, but also potentiated native ASICs, ASIC1a and ASIC3. The potentiating effects depended on the pH and was most pronounced with intermediate acidifications. In the presence of high concentrations of Ru-1355 and Ru-1199, the ASIC2a responses were biphasic, the initial transient currents were followed by slow component. These slow additional currents were weakly sensitive to the acid-sensitive ion channels pore blocker diminazene. We also found that sustained currents mediated by ASIC2a and ASIC3 are less sensitive to diminazene than the peak currents. Different sensitivities of peak and sustained components to the pore-blocking drug suggest that they are mediated by different open states. We propose that the main mechanism of action of 2-aminobenzimidazole derivatives is potentiation of the open state with slow kinetics of activation and desensitization.

Development of a quaternary ammonium photoswitchable antagonist of NMDA receptors.

NMDA receptors play critical roles in numerous physiological and pathological processes in CNS that requires development of modulating ligands. In particular, photoswitchable compounds that selectively target NMDA receptors would be particularly useful for analysis of receptor contributions to various processes. Recently, we identified a light-dependent anti-NMDA activity of the azobenzene-containing quaternary ammonium compounds DENAQ (diethylamine-azobenzene-quaternary ammonium) and DMNAQ (dimethylamine-azobenzene-quaternary ammonium). Here, we developed a series of light-sensitive compounds based on the DENAQ structure, and studied their action on glutamate receptors in rat brain neurons using patch-clamp method. We found that the activities of the compounds and the influence of illumination strongly depended on the structural details, as even minor structural modifications greatly altered the activity and sensitivity to illumination. The compound PyrAQ (pyrrolidine-azobenzene-quaternary ammonium) was the most active and produced fast and fully reversible inhibition of NMDA receptors. The IC50 values under ambient and monochromic light conditions were 2 and 14 µM, respectively. The anti-AMPA activity was much weaker. The action of PyrAQ did not depend on NMDA receptor activity, agonist concentration, or membrane voltage, making it a useful tool for photopharmacological studies.

Mechanisms of acid-sensing ion channels inhibition by nafamostat, sepimostat and diminazene.

Acid-sensing ion channels (ASICs) are blocked by many cationic compounds. Mechanisms of action, which may include pore block, modulation of activation and desensitization, need systematic analysis to allow predictable design of new potent and selective drugs. In this work, we studied the action of the serine protease inhibitors nafamostat, sepimostat, gabexate and camostat, on native ASICs in rat giant striatal interneurons and recombinant ASIC1a and ASIC2a channels, and compared it to that of well-known small molecule ASIC blocker diminazene. All these compounds have positively charged amidine and/or guanidine groups in their structure. Nafamostat, sepimostat and diminazene inhibited pH 6.5-induced currents in rat striatal interneurons at -80 mV holding voltage with IC50 values of 0.78 ± 0.12 µM, 2.4 ± 0.3 µM and 0.40 ± 0.09 µM, respectively, whereas camostat and gabexate were practically ineffective. The inhibition by nafamostat, sepimostat and diminazene was voltage-dependent evidencing binding in the channel pore. They were not trapped in the closed channels, suggesting "foot-in-the-door" mechanism of action. The inhibitory activity of nafamostat, sepimostat and diminazene was similar in experiments on native ASICs and recombinant ASIC1a channels, while all of them were drastically less active against ASIC2a channels. According to our molecular modeling, three active compounds bind in the channel pore between Glu 433 and Ala 444 in a similar way. In view of the relative safety of nafamostat for clinical use in humans, it can be considered as a potential candidate for the treatment of pathophysiological conditions linked to ASICs disfunction, including inflammatory pain and ischemic stroke.

Modulation of Slow Desensitization (Tachyphylaxis) of Acid-Sensing Ion Channel (ASIC)1a.

Among the proton-activated channels of the ASIC family, ASIC1a exhibits a specific tachyphylaxis phenomenon in the form of a progressive decrease in the response amplitude during a series of activations. This process is well known, but its mechanism is poorly understood. Here, we demonstrated a partial reversibility of this effect using long-term whole-cell recording of CHO cells transfected with rASIC1a cDNA. Thus, tachyphylaxis represents a slow desensitization of ASIC1a. Prolonged acidifications provided the same recovery from slow desensitization as short acidifications of the same frequency. Slow desensitization and steady-state desensitization are independent processes although the latter attenuates the development of the former. We found that drugs which facilitate ASIC1a activation (e.g., amitriptyline) cause an enhancement of slow desensitization, while inhibition of ASIC1a by 9-aminoacridine attenuates this process. Overall, for a broad variety of exposures, including increased calcium concentration, different pH conditions, and modulating drugs, we found a correlation between their effects on ASIC1a response amplitude and the development of slow desensitization. Thus, our results demonstrate that slow desensitization occurs only when ASIC1a is in the open state.

P-Loop Channels: Experimental Structures, and Physics-Based and Neural Networks-Based Models.

The superfamily of P-loop channels includes potassium, sodium, and calcium channels, as well as TRP channels and ionotropic glutamate receptors. A rapidly increasing number of crystal and cryo-EM structures have revealed conserved and variable elements of the channel structures. Intriguing differences are seen in transmembrane helices of channels, which may include π-helical bulges. The bulges reorient residues in the helices and thus strongly affect their intersegment contacts and patterns of ligand-sensing residues. Comparison of the experimental structures suggests that some π-bulges are dynamic: they may appear and disappear upon channel gating and ligand binding. The AlphaFold2 models represent a recent breakthrough in the computational prediction of protein structures. We compared some crystal and cryo-EM structures of P-loop channels with respective AlphaFold2 models. Folding of the regions, which are resolved experimentally, is generally similar to that predicted in the AlphaFold2 models. The models also reproduce some subtle but significant differences between various P-loop channels. However, patterns of π-bulges do not necessarily coincide in the experimental and AlphaFold2 structures. Given the importance of dynamic π-bulges, further studies involving experimental and theoretical approaches are necessary to understand the cause of the discrepancy.

Optical Control of N-Methyl-d-aspartate Receptors by Azobenzene Quaternary Ammonium Compounds.

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.

Computational Analysis of the Crystal and Cryo-EM Structures of P-Loop Channels with Drugs.

The superfamily of P-loop channels includes various potassium channels, voltage-gated sodium and calcium channels, transient receptor potential channels, and ionotropic glutamate receptors. Despite huge structural and functional diversity of the channels, their pore-forming domain has a conserved folding. In the past two decades, scores of atomic-scale structures of P-loop channels with medically important drugs in the inner pore have been published. High structural diversity of these complexes complicates the comparative analysis of these structures. Here we 3D-aligned structures of drug-bound P-loop channels, compared their geometric characteristics, and analyzed the energetics of ligand-channel interactions. In the superimposed structures drugs occupy most of the sterically available space in the inner pore and subunit/repeat interfaces. Cationic groups of some drugs occupy vacant binding sites of permeant ions in the inner pore and selectivity-filter region. Various electroneutral drugs, lipids, and detergent molecules are seen in the interfaces between subunits/repeats. In many structures the drugs strongly interact with lipid and detergent molecules, but physiological relevance of such interactions is unclear. Some eukaryotic sodium and calcium channels have state-dependent or drug-induced π-bulges in the inner helices, which would be difficult to predict. The drug-induced π-bulges may represent a novel mechanism of gating modulation.

Molecular mechanisms of action determine inhibition of paroxysmal depolarizing shifts by NMDA receptor antagonists in rat cortical neurons.

N-methyl-d-aspartate glutamate receptors (NMDARs) are involved in numerous central nervous system (CNS) processes, including epileptiform activity. We used a picrotoxin-induced epileptiform activity model to compare the action of different types of NMDAR antagonists in rat brain slices. Paroxysmal depolarizing shifts (PDS) were evoked by external stimulation in the medial prefrontal cortex (mPFC) slices and recorded in pyramidal cells (PC) and in fast-spiking interneurons (FSI). The NMDAR antagonists APV and memantine reduced the duration of PDS. However, the competitive antagonist APV caused similar effects on the PC and FSI, while the open-channel blocker memantine had a much stronger effect on the PDS in the FSI than in the PC. This difference cannot be explained by a corresponding difference in NMDAR sensitivity to memantine because the drug inhibited the excitatory postsynaptic current (EPSC) similarly in both cell types. Importantly, the PDS were significantly longer in the FSI than in the PC. The degree of PDS inhibition by memantine correlated with individual PDS durations in each cell type. Computer modeling of a synaptic network in the mPFC suggests that the different effects of memantine on the PDS in the PC and FSI can be explained by use dependence of its action. An open-channel blocking mechanism and competition with Mg2+ ions for the binding site result in pronounced inhibition of the long PDS, whereas the short PDS are weakly sensitive. Our results show that peculiarities of kinetics and the mechanism of action largely determine the effects of NMDAR antagonists on physiological and/or pathological processes.

Phenylalkylamines in calcium channels: computational analysis of experimental structures.

Experimental 3D structures of calcium channels with phenylalkylamines (PAAs) provide basis for further analysis of atomic mechanisms of these important cardiovascular drugs. In the crystal structure of the engineered calcium channel CavAb with Br-verapamil and in the cryo-EM structure of the Cav1.1 channel with verapamil, the ligands bind in the inner pore. However, there are significant differences between these structures. In the crystal structure the ligand ammonium group is much closer to the ion in the selectivity-filter region Site 3, which is most proximal to the inner pore, than in the cryo-EM structure. Here we used Monte Carlo energy minimizations to dock PAAs in calcium channels. Our computations suggest that in the crystal structure Site 3 is occupied by a water molecule rather than by a calcium ion. Analysis of the published electron density map does not rule out this possibility. In the cryo-EM structures the ammonium group of verapamil is shifted from the calcium ion in Site 3 either along the pore axis, towards the cytoplasm or away from the axis. Our unbiased docking reproduced these binding modes. However, in the cryo-EM structures detergent and lipid molecules interact with verapamil. When we removed these molecules, the nitrile group of verapamil bound to the calcium ion in Site 3. Models of Cav1.2 with different PAAs suggest similar binding modes and direct contacts of the ligands electronegative atoms with the calcium ion in Site 3. Such interactions explain paradoxes in structure-activity relationships of PAAs.

The pore domain in glutamate-gated ion channels: Structure, drug binding and similarity with potassium channels.

Ionotropic glutamate receptors in the CNS excitatory synapses of vertebrates are involved in numerous physiological and pathological processes. Decades of intensive studies greatly advanced our understanding of molecular organization of these transmembrane proteins. Here we focus on the channel pore domain, its selectivity filter and the activation gate, and the pore block by organic ligands. We compare findings from indirect experimental approaches, including site-directed mutagenesis, with recent crystal and cryo-EM structures of different channels in different functional states and complexed with different ligands. We summarize remaining uncertainties and unresolved problems related to the channel structure, function and pharmacology.

Intersegment contacts determine geometry of the open and closed states in P-loop channels.

Despite impressive progress in experimental studies of ion channels, determinants of their state-dependent geometry are not completely understood. Previous studies of P-loop channels suggested that the gating mechanism involves coupled movement of the S4-S5 cuff and S6 bundle and emphasized importance of specific intersegment contacts in stabilizing different states. However, it is unclear whether or not such contacts are sufficient to computationally reproduce gating rearrangements. Here we analyzed X-ray and cryo-EM structures of several channels in different functional states and selected structures with the wide cuff (open-state Kv1.2) and narrow cuff (closed-state MlotiK1) for detailed analysis. We revealed three categories of inter-residue contacts within the pore domain: (i) state-dependent and state-independent contacts between helices S4-S5 and S5, which provide integrity and state-dependent dimensions of the S4-S5 cuff; (ii) state-independent contacts between helices S4-S5 and S6 that enable their coupled movement during gating and (iii) state-dependent contacts between S6s that stabilize the open and/or closed activation gate. We imposed these contacts to transform the channels from the open to closed state and vice versa using Monte Carlo energy minimizations. In all cases, the target structures were reached with a good precision. Thus, a limited set of inter-residue contacts can be used to predict computationally state-dependent geometry of the pore domain in P-loop channels. Effects of various engineered and naturally occurring mutations (channelopathies) on the channel gating can be rationalized in view of the contacts.Communicated by Ramaswamy H. Sarma.

Extremely Potent Block of Bacterial Voltage-Gated Sodium Channels by µ-Conotoxin PIIIA.

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10-12 to 10-5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

Hydrophobic Amines and Their Guanidine Analogues Modulate Activation and Desensitization of ASIC3.

Acid-sensing ion channel 3 (ASIC3) is an important member of the acid-sensing ion channels family, which is widely expressed in the peripheral nervous system and contributes to pain sensation. ASICs are targeted by various drugs and toxins. However, mechanisms and structural determinants of ligands' action on ASIC3 are not completely understood. In the present work we studied ASIC3 modulation by a series of "hydrophobic monoamines" and their guanidine analogs, which were previously characterized to affect other ASIC channels via multiple mechanisms. Electrophysiological analysis of action via whole-cell patch clamp method was performed using rat ASIC3 expressed in Chinese hamster ovary (CHO) cells. We found that the compounds studied inhibited ASIC3 activation by inducing acidic shift of proton sensitivity and slowed channel desensitization, which was accompanied by a decrease of the equilibrium desensitization level. The total effect of the drugs on the sustained ASIC3-mediated currents was the sum of these opposite effects. It is demonstrated that drugs' action on activation and desensitization differed in their structural requirements, kinetics of action, and concentration and state dependencies. Taken together, these findings suggest that effects on activation and desensitization are independent and are likely mediated by drugs binding to distinct sites in ASIC3.

Lidocaine and carbamazepine inhibit while phenytoin and lamotrigine paradoxically enhance the insect neuromuscular transmission.

Primary mechanism of action of local anesthetics and various anticonvulsants is the voltage-gated sodium channel block. Many of these small molecules also have other targets in nervous system of vertebrates. However, little is known about their action on invertebrate nervous system. Nevertheless, insect-based models are suggested for high-throughput screening of antiepileptic drugs. In the present work, we characterized action of lidocaine, carbamazepine, lamotrigine, and phenytoin on the neuromuscular transition of Calliphora vicina fly larvae using conventional voltage-clamp approach. Carbamazepine and lidocaine caused inhibition of synaptic transmission, which has presynaptic origin. This action is in agreement with inhibition of voltage-gated sodium channels that reduces depolarization of nerve terminals and, thus, calcium entry. Surprisingly, phenytoin and lamotrigine produced a prominent increase in the evoked postsynaptic currents without any effect on frequency or amplitude of spontaneous miniature currents. Potassium channel blocker 4-aminopyridine affects synaptic transmission in similar way. Elevation of synaptic quantal content via increase in calcium concentration or via application of 1 mM 4-aminopyridine eliminates the enhancement effect or even turns it to modest inhibition. We propose that lamotrigine and phenytoin act as inhibitors of insect potassium channels that cause the membrane depolarization and thus facilitates calcium entry into the nerve terminal.