Fig. 6: De novo mutations in patients with epilepsy affecting the ligand-binding pocket.

a Plot of known (orange) and undescribed (pink) disease-causing variants in patients with epilepsy and neurodevelopmental disorders onto the structure. Only one subunit is shown in cartoon mode and side chains for the residues at the gain-of-function mutant position are shown as sticks. Gain-of-function mutants refer to the numbering in the human TRPM3a2 isoform. b Close-up view of a). The identified mutations, I999S and F1150S, contribute to the ligand-binding pocket. Close-up view on I999 and F1150 (right). A π-π stacking between F1150 and F885 would be abrogated in the F1150S mutant. The I999S mutation could form a hydrogen bond between its serine side chain hydroxyl group and the main chain carbonyl oxygen of L996, potentially affecting the stability of the S4 helix by inducing a kink. c Representative jRCAMP1b fluorescence assays showing the effect of primidone in cells expressing I999S and F1150S, either alone or in a 1-to-1 ratio with WT TRPM3. d Concentration dependence of the inhibitory effect of primidone on the PS response for the conditions shown in (c). Each data point represents the mean ± SEM from 4 biological replicates (individual wells). The dotted line indicates the concentration-response curve for WT TRPM3. e Concentration dependence of the effect of primidone on the basal fluorescence for the conditions shown in (c). Each data point represents the mean ± SEM from 4 biological replicates (individual wells). f Representative traces showing the effects of other ligands (isosakuranetin, (R)-CIM0216 and ononetin) on I999S and F1150S. Note the severely affected inhibition by isosakuranetin and ononetin, the insensitivity of F1150S for CIM0216, and the activation of F1150S by isosakuranetin. Concentration-response curves for these ligands are shown in Supplementary Fig. 20. Source data are provided as a Source Data file.