Ala substitutions within the MX-segment reduce modulation of functional 5-HT _3A surface expression by RIC-3. (a) View of 5-HT_3A channel (PDB ID 6W1J; Basak et al., 2020) from the intracellular side, with residues W347, R349, and L353 in blue (left), and alignment of the MX-segments with Ala substitutions as indicated (right). (b) Serotonin-induced current amplitudes of all constructs normalized to the current amplitude of WT 5-HT_3A receptor. The triple Ala substitution leads to significantly reduced current amplitudes; biological replicates n = 21 for WT, n = 7 for W347A, n = 7 for R349A, n = 6 for L353A, n = 8 for MX-AAA. (c) Current amplitudes for each construct co-expressed with RIC-3 normalized to the amplitudes of each channel expressed alone, respectively; biological replicates n = 14 for WT, n = 15 for W347A, n = 12 for R349A, n = 10 for L353A, n = 15 for MX-AAA. Statistical significance in b and c was determined vs. WT using ordinary one-way ANOVA with Tukey’s post-hoc test. Significance is indicated: * P = 0.0485, ** P = 0.001628, *** P = 0.000321, **** P = 0.00005 (c: L353A vs. WT), **** P = 0.000000000018616 (b: MX-AAA vs. WT).