M1 and M2 sequence logos and overview of nonsense suppression approach. (A) Sequence logo of M1 and M2 based on mammalian ASIC an ENaC sequences (see Figs. S2 and S3). Residues are numbered according to position in mASIC1a. The height of each residue is proportional to its frequency at this position. Here, we have employed a numbering system for M1 in which equivalent residues from ENaCs and ASICs are referred to with the same number. The conserved W in M1 is designated the 0′ position. (B) Experimental approach used to determine ion selectivity of mASIC1a constructs. cRNA encoding WT or mutant mASIC1a was injected into Xenopus oocytes, and reversal potentials with extracellular Na⁺, K⁺, Li⁺, and Cs⁺ were determined using a 200-ms voltage ramp from −60 to +60 mV during the peak current. Currents during the voltage ramps at pH 7.4 (blue) were subtracted from currents during activating pH (red). (C) Incorporation of the ncAA naphthalene was achieved via the nonsense suppression method in Xenopus oocytes. The suppressor tRNA, THG73, lacking its terminal CA dinucleotide was enzymatically ligated to the ncAA naphthalene attached to a CA dinucleotide using an RNA ligase. The tRNA carrying naphthalene was injected into Xenopus oocytes together with mASIC1a mRNA containing an amber stop codon at the site of interest.