Lee, H. C.

ABSTRACT Gain-of-function mutations in the transient receptor potential 6 (TRPC6) channel have recently been recognized as risk factors for both doxorubicin (DOX)-induced cardiomyopathy. Functional evaluation of TRPC6 missense variants is therefore important for cancer patients undergoing anthracycline treatment. However, traditional electrophysiological methods are labor-intensive and time-consuming. In this study, we compared 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 techniques. 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 TRPC6 missense variants were identified from cancer patients in the Mayo Clinic database. Of these, fifteen variants had resolvable structures, nine of which displayed increased Kd values and six decreased Kd values compared to WT in molecular docking analysis. Patch clamp recordings revealed that TRPC6 WT and mutant channels were inactive at baseline but were activated upon 50 μM OAG stimulation, except two loss-of-function variants. Moreover, a 24-h treatment with 0.5 μM DOX significantly enhanced OAG-induced channel activation. All three variants identified in patients with heart failure demonstrated gain-of-function properties in both electrophysiological measurements and in-silico predictions. Importantly, the results obtained from molecular docking and patch clamp recordings were strongly correlated, showing an 82% concordance, higher than the predictions from AlphaMissense. 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.

We have previously shown that NAD+ inhibits renal Na(+)-Pi symport; however, the biochemical mechanism of NAD+ in this action is not clarified. We now propose that NAD+ acts indirectly by first being converted to cyclic ADP-ribose (cADPR), a potent stimulator of intracellular Ca2+ mobilization. In permeabilized opossum kidney (OK) cells, a cell line often employed as a model for study of proximal tubular epithelial transport, cADPR is synthesized from beta-NAD+ in a substrate concentration (0.01-1 mM) and time-dependent manner. That cADPR was generated from beta-NAD+ by OK cells was verified by coelution with authentic cADPR on anion exchange high-performance liquid chromatography and by homologous desensitization of the Ca2+ release bioassay to authentic cADPR. cADPR synthesized by permeabilized OK cells was not influenced by the addition of parathyroid hormone. The OK cell also contains the enzyme activity necessary to catalyze catabolism of cADPR. Identification of these two key enzyme activities of cADPR metabolism in OK cells is consistent with a possible role of cADPR in regulation of the Na(+)-Pi symporter by NAD+ in response to metabolic stimuli. d