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.

Multiple modes of action of hydrophobic amines and their guanidine analogues on ASIC1a.

Hydrophobic monoamines containing only a hydrophobic/aromatic moiety and protonated amino group are a recently described class of acid-sensing ion channel (ASIC) modulators. Intensive studies have revealed a number of active compounds including endogenous amines and pharmacological agents and shown that these compounds potentiate and inhibit ASICs depending on their specific structure and on subunit composition of the target channel. The action of monoamines also depends on the application protocol, membrane voltage, conditioning and activating pH, suggesting complex mechanism(s) of the ligand-receptor interaction. Without understanding of these mechanisms analysis of structure-function relationships and predictive search for new potent and selective drugs are hardly possible. To this end, we investigated the modes of action for a representative series of amine and guanidine derivatives of adamantane and phenylcyclohexyl. The study was performed on transfected Chinese hamster ovary (CHO) cells and rat hippocampal interneurons using whole-cell patch clamp recording. We found that complex picture of monoamine action can be rationalized assuming four modes of action: (1) voltage-dependent pore block, (2) acidic shift of activation, (3) alkaline shift of activation and (4) acidic shift of steady-state desensitization. Structure-activity relationships are discussed in the light of this framework. The experiments on native heteromeric ASICs have shown that some of these mechanisms are shared between them and recombinant ASIC1a, implying that our results could also be relevant for amine action in physiological and pathological conditions.

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.

Blockade of NMDA receptor channels by 9-aminoacridine and its derivatives.

9-Aminoacridine is known as "foot-in-the-door" NMDA receptor channel blocker because its binding prevents channel closure. Structural determinants of this mechanism of block were studied using a series of 9-aminoacridine derivatives. Experiments were performed on native NMDA receptors of hippocampal pyramidal neurons, isolated from rat brain slices. The use-dependence of block and kinetics of recovery from block were used to characterize mechanism of block produced by the compounds. Modifications, which preserve the flat structure of the tricyclic 9-aminoacridine moiety, affect blocking activity and kinetics but not the foot-in-the-door mechanism. On the contrary, disruption of the flat structure changes the mechanism of block to trapping. It is concluded that flat aromatic structure is one of the critical determinants of the action mechanism of 9-aminoacridine.