Konstantin V. Bolshakov

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.

The interaction of TRPV1-receptors agonists (capsaicin and protons) has been studied on cultured CHO cells transfected by TRPV1-receptors. Using the whole-cell patch-clamp approach, it was shown that summation of the currents induced by agonist application was dependent on the membrane potential. The TRPV1-mediated currents induced by the pH and Capsaicin demonstrated arithmetical summation at potentials between 40--40 mV, while they were potentiated at potentials below -40 mV. Currents induced by the pH and Capsaicin combined were higher in comparison with the arithmetic sum of the currents induced by the pH and Capsaicin applied separately at such potentials. Such a potential dependence seems to be a base of the sensitization that is induced by inflammation or pain, when concentrations of proinflammatory mediators acting as TRPV1 agonists are increasing. Further depolarization induced by TRPV1 activation doesnt generate potentiation, which might serve as a protective mechanism to restrict their activity.

Nitric oxide donors were studied on acid‐sensing channels (ASICs) of rat striatal neurons and CHO cells expressing recombinant receptors. S‐nitroso‐acetyl‐penicillamine (SNAP) potentiate or inhibit ASICs depending on their functional state. It potentiated ASICs after pretreatment with extracellular pH 7.4. When ASICs were partially desensitized by pretreatment with pH 7.0, application of SNAP led to increase of their desensitisization. The potentiating effect of NO on ASICs is explained by an increase of ASIC proton sensitivity, whereas inhibition of ASICs by NO is due to an increase of their steady‐state desensitization. Potentiation and inhibition produced by SNAP had different subunit dependence. The most sensitive to potentiation were ASIC1a/2a and ASIC2a channels while ASIC2a and ASIC3 were the most sensitive to inhibition. Potentiation, produced by SNAP, was prevented by sulphhydryl reagent mercaptoethanol, but it did not affect inhibition produced by SNAP. Thus potentiation and inhibition seems to be mediated by different binding sites. We suppose both effects of SNAP are most probably due to nitrosylation, but not to soluble guanylate cyclase activation, because they were unaffected by ODQ, inhibitor of NO‐sensitive guanylate cyclase, and by 8‐Br‐cGMP, an activator of cGMP‐dependent PKG cascades. Work was supported by grant RFBR/STGI 09‐04‐92662‐Ind‐a.

It is well known that not all effects of such classical NMDA receptor channel blockers as spermine and phylantotoxins are madiated via channel blockade. To determine if these side effects have any functional role we studied inhibition of NMDA receptors by derivatives of phenylcyclohexyl and clinically approved memantine. We showed that these drugs not only blocked the channel pore but also potentiated desensitization of NMDA receptors. Apparently, these effects are not interrelated because there was no correlation between a degree of “trapping” and a level of channel desensitization induced by these blockers. Yet, in contrast to channel blockade, which was strongly voltage-dependent, these compounds affected NMDA receptor desensitization in a voltage-independent manner. Based on these facts, we suppose the existence of two different binding sites for memantine and functionally related compounds, one being responsible for channel blockade and another being coupled to channel desensitization. This possibility should be taken into account when interpreting experimental and clinical effects of these channel blockers.

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.

The inhibitory action of non‐steroid anti‐inflammatory drugs was investigated on acid‐sensing ionic channels (ASIC) in isolated hippocampal interneurons and on recombinant ASICs expressed in Chinese hamster ovary (CHO) cells. Diclofenac and ibuprofen inhibited proton‐induced currents in hippocampal interneurons (IC₅₀ were 622 ± 34 μM and 3.42 ± 0.50 mM, respectively). This non‐competitive effect was fast and fully reversible for both drugs. Aspirin and salicylic acid at 500 μM were ineffective. Diclofenac and ibuprofen decreased the amplitude of proton‐evoked currents and slowed the rates of current decay with a good correlation between these effects. Simultaneous application of acid solution and diclofenac was required for its inhibitory effect. Unlike amiloride, the action of diclofenac was voltage‐independent and no competition between two drugs was found. Analysis of the action of diclofenac and ibuprofen on activation and desensitization of ASICs showed that diclofenac but not ibuprofen shifted the steady‐state desensitization curve to more alkaline pH values. The reason for this shift was slowing down the recovery from desensitization of ASICs. Thus, diclofenac may serve as a neuroprotective agent during pathological conditions associated with acidification.