Identification of a unique Ca²⁺-binding site in rat acid-sensing ion channel 3

Acid-sensing ion channels (ASICs) evolved to sense changes in extracellular acidity with the divalent cation calcium (Ca²⁺) as an allosteric modulator and channel blocker. The channel-blocking activity is most apparent in ASIC3, as removing Ca²⁺ results in channel opening, with the site's location remaining unresolved. Here we show that a ring of rat ASIC3 (rASIC3) glutamates (Glu435), located above the channel gate, modulates proton sensitivity and contributes to the formation of the elusive Ca²⁺ block site. Mutation of this residue to glycine, the equivalent residue in chicken ASIC1, diminished the rASIC3 Ca²⁺ block effect. Atomistic molecular dynamic simulations corroborate the involvement of this acidic residue in forming a high-Affinity Ca²⁺ site atop the channel pore. Furthermore, the reported observations provide clarity for past controversies regarding ASIC channel gating. Our findings enhance understanding of ASIC gating mechanisms and provide structural and energetic insights into this unique calcium-binding site.