Functional evaluation of TRPC6 missense variants in cancer patients via molecular docking analysis compared with...

Gain-of-function mutations in the transient receptor potential 6 (TRPC6) channel have been identified as risk factors for doxorubicin (DOX)-induced cardiomyopathy and heart failure. Functional characterization of TRPC6 missense variants is therefore important for cancer patients undergoing anthracycline therapy; however, conventional electrophysiological methods are labor-intensive and time-consuming. Here, we evaluated the functional responses of TRPC6 missense variants to 1-oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC6 agonist, using molecular docking and patch-clamp recording. For the wild type (WT) TRPC6 structure (PDB ID: 6UZ8), OAG exhibited a binding energy of -4.49 kcal/mol and a dissociation constant (Kd) of 0.511 mM. Twenty missense variants were identified from cancer patients, of which 15 had resolvable structures. Among these, nine variants showed increased Kd values and six showed decreased Kd values relative to WT. Patch-clamp recordings demonstrated that WT and mutant channels were inactive at baseline but were activated by 50 μM OAG, except for two loss-of-function variants. Notably, all three variants identified in patients with heart failure exhibited gain-of-function properties in both electrophysiological and in silico analyses. Furthermore, 24-h treatment with 0.5 μM DOX significantly potentiated OAG-induced channel activation in WT and gain-of-function variants, but not in loss-of-function variants. Importantly, our molecular docking and electrophysiological results were strongly correlated, with an 82% concordance rate, exceeding AlphaMissense predictions. These findings indicate that our computational analysis provides a rapid and reliable method for predicting the functional impact of TRPC6 missense variants, which may aid clinical decision-making in cancer patients receiving chemotherapy.