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.

Inhibition of Plasma Membrane Na/Ca-Exchanger by KB-R7943 or Lithium Reveals Its Role in Ca-Dependent N-methyl-d-aspartate Receptor Inactivation.

To evaluate the possible role of the plasma membrane Na(+)/Ca(2+)-exchanger (NCX) in regulation of N-methyl-d-aspartate (NMDA) receptors (NMDARs), we studied effects of 2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate (KB-R7943; KBR) and lithium (inhibitors of NCX) on NMDA-elicited whole-cell currents using the patch-clamp technique on rat cortical neurons and human embryonic kidney 293T cells expressing recombinant NMDARs. KBR inhibited NMDAR currents in a voltage-independent manner with similar potency for receptors of GluN1/2A and GluN1/2B subunit compositions that excludes open-channel block and GluN2B-selective inhibition. The inhibition by KBR depended on glycine (Gly) concentration. At 30 µM NMDA, the KBR IC50 values were 5.3 ± 0.1 and 41.2 ± 8.8 µM for 1 and 300 µM Gly, respectively. Simultaneous application of NMDA + KBR in the absence of Gly induced robust inward NMDAR currents that peaked and then rapidly decreased. KBR, therefore, is an agonist (EC50 is 1.18 ± 0.16 µM) of the GluN1 subunit coagonist binding sites. The decrease of NMDA-elicited currents in the presence of KBR was abolished in Ca(2+)-free solution and was not observed in the presence of extracellular Ca(2+) on 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded neurons, suggesting that Ca(2+) affects NMDARs from the cytosol. In agreement, the substitution of Li(+) for extracellular Na(+) caused a considerable decrease of NMDAR currents, which was not observed in the absence of extracellular Ca(2+). Most likely, the accumulation of intracellular Ca(2+) is caused by the inhibition of Ca(2+) extrusion via NCX. Thus, KBR and Li(+) provoke Ca(2+)-dependent receptor inactivation due to the disruption of Ca(2+) extrusion by the NCX. The data reveal the role of NCX in regulation of Ca(2+)-dependent inactivation of NMDARs.

Monoamine NMDA receptor channel blockers inhibit and potentiate native and recombinant proton-gated ion channels.

Acid-sensing ion channels (ASICs) are widely distributed in the peripheral and central nervous system. Although they are involved in many physiological functions, the actual processes that activate ASICs remain unclear. This is particularly true for brain ASICs, which produce only a transient response to a fast drop in pH and cannot mediate sustained current. Therefore, the search for ASIC inhibitors and, especially, potentiators/activators is important. We report that NMDA receptor channel blockers with a comparatively simple structure (9-aminoacridine, memantine, IEM-2117 and IEM-1921) potentiate and/or inhibit ASICs in submillimolar concentrations. The experiments were performed using the patch clamp technique on native ASICs from rat hippocampal interneurons and recombinant ASICs of different subunit compositions expressed in CHO cells. Native ASICs were potentiated by IEM-1921 and IEM-2117, and inhibited by memantine and 9-aminoacridine. Homomeric ASIC1a were inhibited by memantine, IEM-2117 and 9-aminoacridine while IEM-1921 was ineffective. In contrast, homomeric ASIC2a were potentiated by IEM-2117, memantine and IEM-1921, whereas 9-aminoacridine was inactive. The compounds caused a complex effect on ASIC3. 9-aminoacridine and IEM-1921 potentiated the steady-state response of ASIC3 and inhibited the peak component. IEM-2117 not only potentiated ASIC3-mediated currents caused by acidification but also evoked steady-state currents at neutral pH. Our results demonstrate that, depending on the subunit composition, ASICs can be activated or inhibited by simple compounds that possess only amino group and aromatic/hydrophobic moieties. This opens up the possibility to search for new ASIC modulators among a number of endogenous ligands.

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.

Mechanisms of non-steroid anti-inflammatory drugs action on ASICs expressed in hippocampal interneurons.

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 (IC(50) were 622 +/- 34 muM 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 muM 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.

Subunit-dependent cadmium and nickel inhibition of acid-sensing ion channels.

Acid-sensing ion channels (ASIC) are ligand-gated cation channels that are highly expressed in peripheral sensory and central neurons. ASIC are transiently activated by decreases in extracellular pH and are thought to play important roles in sensory perception, neuronal transmission, and excitability, and in the pathology of neurological conditions, such as brain ischemia. We demonstrate here that the heavy metals Ni(2+) and Cd(2+) dose-dependently inhibit ASIC currents in hippocampus CA1 neurons and in Chinese hamster ovary (CHO) cells heterologously expressing these channels. The effects of both Ni(2+) and Cd(2+) were voltage-independent, fast, and reversible. Neither metal affected activation and desensitization kinetics but rather decreased pH-sensitivity. Moreover, distinct ASIC isoforms were differentially inhibited by Ni(2+) and Cd(2+). External application of 1 mM Ni(2+) rapidly inhibited homomeric ASIC1a and heteromeric ASIC1a/2a channels without affecting ASIC1b, 2a, and ASIC3 homomeric channels and ASIC1a/3 and 2a/3 heteromeric channels. In contrast, external Cd(+) (1 mM) inhibited ASIC2a and ASIC3 homomeric channels and ASIC1a/2a, 1a/3, and 2a/3 heteromeric channels but not ASIC1a homomeric channels. The acid-sensing current in isolated rat hippocampus CA1 neurons, thought to be carried primarily by ASIC1a and 1a/2a, was inhibited by 1 mM Ni(2+). The current study identifies ASIC as a novel target for the neurotoxic heavy metals Cd(2+) and Ni(2+).