Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification is lacking due to the sensitivity of this structure to conventional manipulations. Here, we explored the basis for ion selectivity by incorporating unnatural amino acids into the channel, engineering channel stoichiometry and performing free energy simulations. We observed no preference for sodium at the “GAS belt” in the central constriction. Instead, we identified a band of glutamate and aspartate side chains at the lower end of the pore that enables preferential sodium conduction.DOI: http://dx.doi.org/10.7554/eLife.24630.001

Acid-sensing ion channels (ASICs) are proton-gated ion channels broadly expressed in the vertebrate nervous system, converting decreased extracellular pH into excitatory sodium current. ASICs were previously thought to be a vertebrate-specific branch of the DEG/ENaC family, a broadly conserved but functionally diverse family of channels. Here, we provide phylogenetic and experimental evidence that ASICs are conserved throughout deuterostome animals, showing that ASICs evolved over 600 million years ago. We also provide evidence of ASIC expression in the central nervous system of the tunicate, Furthermore, by comparing broadly related ASICs, we identify key molecular determinants of proton sensitivity and establish that proton sensitivity of the ASIC4 isoform was lost in the mammalian lineage. Taken together, these results suggest that contributions of ASICs to neuronal function may also be conserved broadly in numerous animal phyla.

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin Psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition remain unknown. Here we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveal that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications.

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Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that contribute to fast synaptic transmission. Pharmacological inhibition of ASIC1a has been shown to reduce neurotoxicity and infarct volumes during stroke. The cysteine knot toxin Psalmotoxin-1 (PcTx1) is one of the most potent and selective inhibitors of ASIC1a. PcTx1 binds at the subunit interface, but both the stoichiometric requirements and the dynamics of the conformational consequences of the ion channel-peptide interaction remain unknown. Here, we use a combination of electrophysiology, voltage-clamp fluorometry and subunit concatenation to decipher the mechanism of PcTx1 inhibition. We observe a long-lived PcTx1-induced conformational change in the ASIC1a extracellular domain that is destabilized by the F350L mutation at the PcTx1 binding site. Concatemeric channel constructs show that two WT ASIC1a subunits are sufficient for WT-like current inhibition, while the presence of a single mutated subunit is enough to destabilize the PcTx1-induced conformation. Our results therefore demonstrate a divergence between the functional effects of PcTx1 on the pore and its conformational consequences in the extracellular domain. It further highlights how engineering of ion channels enables precise control over individual subunits for pharmacological and conformational assessment to determine the mechanism of ion channel-ligand interactions.