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

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 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.

Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions

“…Consistent with previous work (40), we show that the Alexa Fluor 488 C 5 maleimide-labeled K105C mutant responded to pH 5.6 activation with an inward current that acutely desensitizes within the 20 s application frame, accompanied by an upward deflection of the fluorescent signal (Figure 2B, left). The fluorescent signal typically displayed a transient peak that settled into a plateau, likely reflecting channel opening and desensitization, respectively (40). During six consecutive activations with pH 5.6, the K105C mutant showed minor tachyphylaxis with near-identical fluorescence signals (Figure 2C, left).…”

“…Figure S3C, Suppl. Table T1), as reported earlier (41). On the other hand, SSD of the V80C/N414K mutant showed only a shallow pH dependence and was not fully resolved within the pH range tested (Figure 3D, and E, right, Suppl.…”

“…Consecutive activations with pH 5.6 evoked strong tachyphylaxis in V80C/N414K, but not in V80C, while the fluorescence response remained stable across six consecutive activations for both mutants (Figure 3B, right, and 3C). In V80C, the changes in fluorescence appeared in a similar pH range as SSD, while activation happened at lower pH (Figure 3D and E S3A), as reported earlier (40). On the other hand, the SSD of the V80C/N414K mutant showed only a shallow pH dependence and was not fully resolved within the pH range tested (Figure 3D, and E, right, Suppl.…”

“…To attach the fluorophore, we replaced residue K105 with a cysteine (K105C, Figure 2A) and labeled it with Alexa Fluor 488 C 5 maleimide. VCF readouts from this position have shown to act as a proxy of ECD conformational changes during mASIC1a gating and peptide binding (35, 39, 40). Consistent with previous work (40), we show that the Alexa Fluor 488 C 5 maleimide-labeled K105C mutant responded to pH 5.6 activation with an inward current that acutely desensitizes within the 20 s application frame, accompanied by an upward deflection of the fluorescent signal (Figure 2B, left).…”

“…VCF readouts from this position have shown to act as a proxy of ECD conformational changes during mASIC1a gating and peptide binding (35, 39, 40). Consistent with previous work (40), we show that the Alexa Fluor 488 C 5 maleimide-labeled K105C mutant responded to pH 5.6 activation with an inward current that acutely desensitizes within the 20 s application frame, accompanied by an upward deflection of the fluorescent signal (Figure 2B, left). The fluorescent signal typically displayed a transient peak that settled into a plateau, likely reflecting channel opening and desensitization, respectively (40).…”