Abstract
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 Ca(2+) concentration decreases the H(+) sensitivity of ASIC1a, suggesting a competition for binding sites between H(+) and Ca(2+) ions. Here, we predicted candidate residues for Ca(2+) 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 Ca(2+) of the ASIC1a pH dependence of activation and desensitization. This occurred likely owing to a disruption of Ca(2+) binding. Our results link one of the two predicted Ca(2+)-binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg(2+) regulates ASICs in a similar way as does Ca(2+). We show that Mg(2+) shares some of the binding sites with Ca(2+). Finally, we provide evidence that some of the ASIC1a Ca(2+)-binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca(2+) affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.