The funnel-web spider venom derived single knot peptide Hc3a modulates acid-sensing ion channel 1a desensitisation

[Display omitted] Acid-sensing ion channel 1a (ASIC1a) is a proton-gated channel involved in synaptic transmission, pain signalling, and several ischemia-associated pathological conditions. The spider venom-derived peptides PcTx1 and Hi1a are two of the most potent ASIC1a inhibitors known and have been instrumental in furthering our understanding of the structure, function, and biological roles of ASICs. To date, homologous spider peptides with different pharmacological profiles at ASIC1a have yet to be discovered. Here we report the characterisation of Hc3a, a single inhibitor cystine knot peptide from the Australian funnel-web spider Hadronyche cerberea with sequence similarity to PcTx1. We show that Hc3a has complex pharmacology and binds different ASIC1a conformational states (closed, open, and desensitised) with different affinities, with the most prominent effect on desensitisation. Hc3a slows the desensitisation kinetics of proton-activated ASIC1a currents across multiple application pHs, and when bound directly to ASIC1a in the desensitised conformation promotes current inhibition. The solution structure of Hc3a was solved, and the peptide-channel interaction examined via mutagenesis studies to highlight how small differences in sequence between Hc3a and PcTx1 can lead to peptides with distinct pharmacology. The discovery of Hc3a expands the pharmacological diversity of spider venom peptides targeting ASIC1a and adds to the toolbox of compounds to study the intricacies of ASIC1 gating.