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.

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.

Apamin structure and pharmacology revisited.

Apamin is often cited as one of the few substances selectively acting on small-conductance Ca2+-activated potassium channels (KCa2). However, published pharmacological and structural data remain controversial. Here, we investigated the molecular pharmacology of apamin by two-electrode voltage-clamp in Xenopus laevis oocytes and patch-clamp in HEK293, COS7, and CHO cells expressing the studied ion channels, as well as in isolated rat brain neurons. The microtitre broth dilution method was used for antimicrobial activity screening. The spatial structure of apamin in aqueous solution was determined by NMR spectroscopy. We tested apamin against 42 ion channels (KCa, KV, NaV, nAChR, ASIC, and others) and confirmed its unique selectivity to KCa2 channels. No antimicrobial activity was detected for apamin against Gram-positive or Gram-negative bacteria. The NMR solution structure of apamin was deposited in the Protein Data Bank. The results presented here demonstrate that apamin is a selective nanomolar or even subnanomolar-affinity KCa2 inhibitor with no significant effects on other molecular targets. The spatial structure as well as ample functional data provided here support the use of apamin as a KCa2-selective pharmacological tool and as a template for drug design.

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.

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.

Discovery of a Recombinant Human Monoclonal Immunoglobulin G Antibody Against α-Latrotoxin From the Mediterranean Black Widow Spider (Latrodectus tredecimguttatus).

Widow spiders are among the few spider species worldwide that can cause serious envenoming in humans. The clinical syndrome resulting from Latrodectus spp. envenoming is called latrodectism and characterized by pain (local or regional) associated with diaphoresis and nonspecific systemic effects. The syndrome is caused by α-latrotoxin, a ~130 kDa neurotoxin that induces massive neurotransmitter release. Due to this function, α-latrotoxin has played a fundamental role as a tool in the study of neuroexocytosis. Nevertheless, some questions concerning its mode of action remain unresolved today. The diagnosis of latrodectism is purely clinical, combined with the patient's history of spider bite, as no analytical assays exist to detect widow spider venom. By utilizing antibody phage display technology, we here report the discovery of the first recombinant human monoclonal immunoglobulin G antibody (TPL0020_02_G9) that binds α-latrotoxin from the Mediterranean black widow spider (Latrodectus tredecimguttatus) and show neutralization efficacy ex vivo. Such antibody can be used as an affinity reagent for research and diagnostic purposes, providing researchers with a novel tool for more sophisticated experimentation and analysis. Moreover, it may also find therapeutic application in future.

Modulation of Proton-Gated Channels by Antidepressants.

The chemical structures of some antidepressants are similar to those of recently described amine-containing ligands of acid-sensing ion channels (ASICs). ASICs are expressed in brain neurons and participate in numerous CNS functions. As such, they can be related to antidepressant action or side effects. We therefore studied the actions of a series of antidepressants on recombinant ASIC1a and ASIC2a and on native ASICs in rat brain neurons. Most of the tested compounds prevented steady-state ASIC1a desensitization evoked by conditioning acidification to pH 7.1. Amitriptyline also potentiated ASIC1a responses evoked by pH drops from 7.4 to 6.5. We conclude that amitriptyline has a twofold effect: it shifts activation to less acidic values while also shifting steady-state desensitization to more acidic values. Chlorpromazine, desipramine, amitriptyline, fluoxetine, and atomoxetine potentiated ASIC2a response. Tianeptine caused strong inhibition of ASIC2a. Both potentiation and inhibition of ASIC2a were accompanied by the slowdown of desensitization, suggesting distinct mechanisms of action on activation and desensitization. In experiments on native heteromeric ASICs, tianeptine and amitriptyline demonstrated the same modes of action as on ASIC2a although with reduced potency.

Complex action of tyramine, tryptamine and histamine on native and recombinant ASICs.

Proton-gated channels of the ASIC family are widely distributed in the mammalian brain, and, according to the recent data, participate in synaptic transmission. However, ASIC-mediated currents are small, and special efforts are required to detect them. This prompts the search for endogenous ASIC ligands, which can activate or potentiate these channels. A recent finding of the potentiating action of histamine on recombinant homomeric ASIC1a has directed attention to amine-containing compounds. In the present study, we have analyzed the action of histamine, tyramine, and tryptamine on native and recombinant ASICs. None of the compounds caused potentiation of native ASICs in hippocampal interneurons. Furthermore, when applied simultaneously with channel activation, they produced voltage-dependent inhibition. Experiments on recombinant ASIC1a and ASIC2a allowed for an interpretation of these findings. Histamine and tyramine were found to be inactive on the ASIC2a, while tryptamine demonstrated weak inhibition. However, they induce both voltage-dependent inhibition of open channels and voltage-independent potentiation of closed/desensitized channels on the ASIC1a. We suggest that the presence of an ASIC2a subunit in heteromeric native ASICs prevents potentiation but not inhibition. As a result, the inhibitory action of histamine, which is masked by a strong potentiating effect on the ASIC1a homomers, becomes pronounced in experiments with native ASICs.

TRPV1 activation power can switch an action mode for its polypeptide ligands.

TRPV1 (vanilloid) receptors are activated by different types of stimuli including capsaicin, acidification and heat. Various ligands demonstrate stimulus-dependent action on TRPV1. In the present work we studied the action of polypeptides isolated from sea anemone Heteractis crispa (APHC1, APHC2 and APHC3) on rat TRPV1 receptors stably expressed in CHO cells using electrophysiological recordings, fluorescent Ca2+ measurements and molecular modeling. The APHCs potentiated TRPV1 responses to low (3-300 nM) concentrations of capsaicin but inhibited responses to high (>3.0 µM) concentrations. The activity-dependent action was also found for TRPV1 responses to 2APB and acidification. Thus the action mode of APHCs is bimodal and depended on the activation stimuli strength-potentiation of low-amplitude responses and no effect/inhibition of high-amplitude responses. The double-gate model of TRPV1 activation suggests that APHC-polypeptides may stabilize an intermediate state during the receptor activation. Molecular modeling revealed putative binding site at the outer loops of TRPV1. Binding to this site can directly affect activation by protons and can be allosterically coupled with capsaicin site. The results are important for further investigations of both TRPV1 and its ligands for potential therapeutic use.

Determinants of action of hydrophobic amines on ASIC1a and ASIC2a.

Acid-sensing ion channels (ASICs) are involved in numerous physiological and pathological processes in the central nervous system. Development of pharmacological tools capable to inhibit or potentiate these channels is important for our knowledge about roles of ASICs in the neuronal network and can be promising for treatment of some disorders. Recently we described four hydrophobic monoamines that potentiate and inhibit ASICs depending on subunit composition of the channel and peculiarities of the drug structure. In the present work we performed structure-activity relationship analysis using derivatives of adamantane, phenylcyclohexyl and 9-aminoacridine to reveal the main determinants of action of amine-containing compounds on recombinant ASIC1a and ASIC2a homomers expressed in CHO cells. We found that the most active compounds are monocations with protonatable aminogroup. In general, potentiators and inhibitors of ASIC1a we found, but only potentiators for ASIC2a. Flat aromatic structure of the headgroup determines inhibition of ASIC1a while "V-shape" structure of the hydrophobic moiety favors potentiation of ASIC2a. Moreover, for some series of monoamines there was a correlation between action on ASIC1a and ASIC2a, the weaker ASIC1a inhibition, the stronger ASIC2a potentiation. Decay of response was accelerated by ASIC1a inhibitors as well as by potentiators. All compounds potentiating ASIC2a slowed down desensitization. Our results suggest that hydrophobic amines cause complex action on ASICs.

Influence of external magnesium ions on the NMDA receptor channel block by different types of organic cations.

The NMDA type of ionotropic glutamate receptors plays a unique role in synaptic functions because of high permeability for calcium and because of a voltage-dependent block by endogenous Mg(2+). Activity and voltage dependence of the NMDA receptor channel block by organic cations are strongly affected by competition with magnesium ions for the binding site in the channel pore. It complicates prediction of action of NMDA receptor channel blockers in vivo. In the present work we studied the NMDA receptor channel block in the presence of Mg(2+) by several organic blockers with different characteristics of voltage dependence and mechanism of action. The action of NMDA receptor channel antagonists was studied in native NMDA receptors of hippocampus CA1 pyramidal neurons isolated from rat brain slices. It was demonstrated that the IC(50) values of NMDA receptor channel blockers at -30 mV are increased 1.5-5 times compared with magnesium-free conditions. The voltage dependence of the channel block is decreased, abolished or even inversed in the presence of magnesium. Although simple competition between magnesium ion and organic channel blockers provides a general explanation of the observed effects, certain disagreements were revealed. Diversity in Mg(2+) effects on the NMDAR channel block by different organic cations reported herein likely reflects interaction of NMDAR channel blockers with additional binding site(s) and suggests that individual analysis in the presence of Mg(2+) is required for newly developed NMDAR channel blocking drugs.

Muscarinic M1 modulation of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) are ligand-gated cation channels that are highly expressed in nervous system. Little is known about the regulation of these channels. Therefore, we tested whether muscarinic M1 receptors can modulate ASICs. The muscarinic agonist oxotremorine methiodide applied to the bath solution strongly inhibited the whole-cell current in Chinese hamster ovary cells heterologously expressing ASIC1a and M1 receptors. Maximal current was inhibited 30% during muscarinic receptor stimulation. These effects were fast, fully reversible and subunit specific. The acid-sensing current in population of isolated rat hippocampus CA1 and striatum interneurons, thought to be carried primarily by ASIC1a, was similarly inhibited by oxotremorine methiodide. Thus, the current study identifies ASIC1a as a novel target for muscarinic signaling.