Mechanistic dissection of a dopamine-gated cation channel from Daphnia reveals key determinants of ligand selectivity and sensitivity

The pentameric ligand-gated ion channels (pLGICs) are a deeply conserved superfamily that includes nicotinic acetylcholine (nAChR), GABAARs, glycine, and serotonin receptors and mediates fast neurotransmission. While vertebrate pLGICs have been extensively characterized, recent discoveries in invertebrates have revealed atypical members with novel ligand specificities, including dopamine-gated ion channels (dop-LGICs) that enable direct ionotropic dopaminergic signaling. Here, we investigate the molecular determinants of catecholamine sensitivity and selectivity in a cation-permeable dopamine-gated receptor from Daphnia magna (Dm-DopC1), which shares high structural homology with vertebrate nAChRs. We find that mutations in loop B (D188N) near-abolish catecholamine responses, while substitutions in loops A (S123P) and C (S227G, E230P) significantly alter sensitivity and ligand selectivity, highlighting the cooperative role of these loops in shaping receptor pharmacology. Complementary face mutations in loops D and E (N92T, A158G) further refined dopamine versus norepinephrine sensitivity. Finally, we also show that this dopamine receptor can be antagonized by both nicotinic and dopaminergic compounds, underscoring its hybrid pharmacological profile. Together, these results reveal how subtle changes in conserved binding motifs can shift receptor specificity from cholinergic to dopaminergic signaling, providing new insights into the evolutionary flexibility of pLGICs and the molecular basis of ionotropic catecholamine transmission in invertebrates.