A duplicated RIC-3 binding motif at the MA–M4 transition mirrors the effects observed for the MX-segment RIC-3 site. (a) Duplicated RIC-3 binding motif, DWLRxxxVLDR, depicted on the cartoon structure of 5-HT3A transmembrane and intracellular domains: two amino acid clusters (cyan) consisting of Asp, Trp, Leu, Arg (DWLR) and Val, Leu, Asp, Arg (VLDR), respectively, are present in both MX-helix and straddling the MA–M4 helices. (b) Triple Ala substitution at residues W447, R449, and L454 of the MAM4 peptide reduces the RIC-3 band intensity to background levels. (c) Serotonin-induced current amplitudes for AAA substitutions normalized to WT currents show significantly reduced amplitudes as compared to WT 5-HT3A receptor; biological replicates n = 21 for WT, n = 8 for MX-AAA, n = 16 for MAM4-AAA. (d) Remaining 5-HT current for each construct in the presence of RIC-3. AAA substitutions lead to less decreased currents with RIC-3 co-expression as compared to WT; biological replicates n = 14 for WT, n = 15 for MX-AAA, n = 21 for MAM4-AAA. Statistical significance in c and d was determined vs. WT using ordinary one-way ANOVA with Tukey’s post-hoc test. Significance is indicated as **** P = 0.000004 (c: MX-AAA vs. MAM4-AAA), **** P = 0.000000000017930 (c: MAM4-AAA vs. WT), ** P = 0.001628 (d: MX-AAA vs. WT), ** P = 0.005000 (d: MAM4-AAA vs. WT). Source data are available for this figure: SourceData F5.