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.
机构:
Oregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Baconguis, Isabelle
Bohlen, Christopher J.
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Univ Calif San Francisco, Dept Physiol, San Francisco, CA 94158 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Bohlen, Christopher J.
Goehring, April
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Oregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Goehring, April
Julius, David
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Univ Calif San Francisco, Dept Physiol, San Francisco, CA 94158 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Julius, David
Gouaux, Eric
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Oregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Oregon Hlth & Sci Univ, Howard Hughes Med Inst, Portland, OR 97239 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
机构:
Oregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Baconguis, Isabelle
Gouaux, Eric
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机构:
Oregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Oregon Hlth & Sci Univ, Howard Hughes Med Inst, Portland, OR 97239 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
机构:
Oregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Baconguis, Isabelle
Bohlen, Christopher J.
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h-index: 0
机构:
Univ Calif San Francisco, Dept Physiol, San Francisco, CA 94158 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Bohlen, Christopher J.
Goehring, April
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h-index: 0
机构:
Oregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Goehring, April
Julius, David
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h-index: 0
机构:
Univ Calif San Francisco, Dept Physiol, San Francisco, CA 94158 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Julius, David
Gouaux, Eric
论文数: 0引用数: 0
h-index: 0
机构:
Oregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Oregon Hlth & Sci Univ, Howard Hughes Med Inst, Portland, OR 97239 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
机构:
Oregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Baconguis, Isabelle
Gouaux, Eric
论文数: 0引用数: 0
h-index: 0
机构:
Oregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA
Oregon Hlth & Sci Univ, Howard Hughes Med Inst, Portland, OR 97239 USAOregon Hlth & Sci Univ, Vollum Inst, Portland, OR 97239 USA