Identification of the modulatory Ca²⁺-binding sites of acid-sensing ion channel 1a

Acid-sensing ion channels (ASICs) are neuronal Na⁺-permeable ion channels activated by extracellular acidification. ASICs are involved in learning, fear sensing, pain sensation and neurodegeneration. Increasing the extracellular Ca²⁺ concentration decreases the H⁺ sensitivity of ASIC1a, suggesting a competition for binding sites between H⁺ and Ca²⁺ ions. Here, we predicted candidate residues for Ca²⁺ binding on ASIC1a, based on available structural information and our molecular dynamics simulations. With functional measurements, we identified several residues in cavities previously associated with pH-dependent gating, whose mutation reduced the modulation by extracellular Ca²⁺ of the ASIC1a pH dependence of activation and desensitization. This occurred likely owing to a disruption of Ca²⁺ binding. Our results link one of the two predicted Ca²⁺-binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg²⁺ regulates ASICs in a similar way as does Ca²⁺. We show that Mg²⁺ shares some of the binding sites with Ca²⁺. Finally, we provide evidence that some of the ASIC1a Ca²⁺-binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca²⁺ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.