Mammalian ASIC channels of the DEG/ENaC superfamily are gated by extracellular protons and function to mediate touch and pain sensitivity, learning and memory, and fear conditioning. The recently solved crystal structure of chicken ASIC1a and preliminary functional studies suggested that a highly negatively charged pocket in the extracellular domain of the channel might be the primary proton binding domain. However, more recent extensive mutagenesis analysis paints a more complex mechanism of channel gating, involving binding of protons at sites immediately after the first transmembrane domain (TM1) and displacement of inhibitory Ca2+ ions from the acidic pocket in the extracellular domain and from another Ca2+ binding site at the mouth of the pore. We recently identified and functionally characterized Caenorhabditis elegans ACD-1, the first acidinactivated DEG/ENaC channel. ACD-1 is expressed in C. elegans amphid glia and functions with neuronal DEG/ENaC channel DEG-1 to mediate acid avoidance and chemotaxis to the amino acid lysine.The post-TM1 residues that were proposed to bind protons in ASIC1a are not conserved in ACD-1, but some of the amino acids constituting the acidic pocket are. However, ACD-1 proton sensitivity is completely independent from extracellular Ca2+, and protons appear to bind the channel in a less cooperative manner. We thus wondered if residues in the acidic pocket might contribute to ACD-1 acid sensitivity. We show here that while ACD-1 sensitivity to extracellular protons is influenced by mutations in the acidic pocket, other sites are likely to participate.We also report that one histidine at the base of the thumb and residues in the channel pore influence proton inhibition in a voltage-independent manner, suggesting that they affect the coupling of proton binding with the gating rather than proton binding itself. We conclude that ACD-1 inhibition by protons is likely mediated by binding of proton ions tomultiple sites throughout the extracellular domain of the channel. Our data also support a model inwhich residues in the acidic pocket contribute to determining the channel state perhaps by changing the strength of the interaction between adjacent thumb and finger domains.
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