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.