Most acid-sensing ion channel (ASIC) subunits are activated by protons, but ASIC2b (a splice variant of ASIC2a) is acid-insensitive. Differences in protonatable residues between the extracellular loop regions of ASIC2a and ASIC2b may explain this difference. Site-directed mutagenesis, combined with immunocytochemistry and whole-cell patch clamp, demonstrated that mutating any one of five ASIC2a sites produces channels that traffic normally to the cell surface membrane but are insensitive to protons. One of the mutants forms functional heteromers with ASIC1a and ASIC2a, demonstrating that ion transport is intact in this mutant. These five sites may be involved in the activation of ASIC2a by protons.