Independent evolution of voltage-independent currents in acoel AMPARs. (A) Main, lower half of the channel pore in Rat-GluA2 AMPARs, subunits shaded in blue, one subunit omitted from foreground, selected amino acid residues labeled in one subunit. TARP-y2 subunits omitted for clarity. From PDB accession no. 5WEO. (A–D) Current traces, example currents at indicated membrane potentials with Ca²⁺-free (red) and 1.8 mM Ca²⁺-containing (blue) extracellular solutions, and graphs, normalized current amplitude (I)—membrane potential (V) relationships for indicated AMPAR subunit combinations (data points mean ± SD, connected by straight lines, n in brackets) for vertebrate deuterostome (A), invertebrate deuterostome (B), protostome (C), and xenacoelomorph (D) AMPARs. Sac-TARP-XP…6113 in B refers to XP_006816113.1, which was used here because currents through Sac-GluA^Q1R + TARP-XP_006822518.1 were small and difficult to work with. Scale bars: x = 10 s; y = 1 µA (A and B) or 0.1 µA (C and D). We co-expressed human TARP-γ2 with Rat-GluA2, as homotetrameric Rat-GluA2^Q1R mutant channels showed no glutamate-gated currents (Fig. S5 G). (E) Exons (dark blue bars) encoding pore-lining residues in selected AMPAR genes from rat and from acoels H. miamia and Symsagittifera roscofensis. White labels indicate exon number within each gene. For the highlighted portion of exon 11 or exon 6, the unedited nucleotide sequence and amino acid sequence are shown above. (F) Acoel AMPAR branch from AMPA gene tree (Fig. S1), including alignment of pore-lining residues (right), with Hofstenia-98014323 orthologues highlighted in light blue. Stars, arrows: gene duplications, mutations, or speciation events referred to in the main text. Gaps in alignment; the Eumecynostomum-c9430 sequence was partial.