Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition.

Christian B Borg,Stephanie A Heusser,Janne M Colding,Stephan A Pless

doi:10.7554/elife.73384

Abstract

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 reveals 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.

Highlights

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels expressed throughout the central and peripheral nervous system (Gründer 2020)
In line with previous findings, 30 nM of PcTx1 led to potent alkaline shifts in both the activation curve and the steady-state desensitization (SSD) of ASIC1a WT expressed in Xenopus laevis oocytes (Escoubas, De Weille et al 2000, Chen, Kalbacher et al 2005)
We introduced a cysteine residue into the mouse ASIC1a (mASIC1a) extracellular domain (ECD) at amino acid position 80 or 105 (Figure 1A), as labelling of these positions with the environmentally sensitive

Summary

Introduction

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels expressed throughout the central and peripheral nervous system (Gründer 2020). ASICs exhibit a rich pharmacology with complex molecular details Modulators can affect both ASIC activation and steady-state desensitization (SSD)—a state reached at sub-activating proton concentrations that reduces subsequent activation—and metabotropic downstream signaling (Kellenberger and Schild 2015, Wang, Wang et al 2015, Wang, Liu et al 2020). Psalmotoxin-1 (PcTx1) is a 40-residue cysteine knot peptide toxin extracted from tarantula venom (Escoubas, De Weille et al 2000). It acts as a potent gating modifier on most ASIC1a-containing channels (Chen, Kalbacher et al 2005, Chen, Kalbacher et al 2006) and shifts activation and SSD curves to more alkaline values, thereby effectively inhibiting the channel in the low nanomolar range at neutral pH (Cristofori-Armstrong and Rash 2017). Structural studies have shown that PcTx1 binds to the subunit interfaces of cASIC1, forming contact points with the primary and complementary side of the acidic pocket (Dawson, Benz et al 2012, Baconguis, Bohlen et al 2014)

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Conclusion