Delayed inactivation of TRPC6 as a determinative characteristic of FSGS-associated variants

Transient receptor potential canonical 6 (TRPC6) is a receptor-operated nonspecific cation channel. To date, more than 30 TRPC6 variants have been reported to focal segmental glomerulosclerosis (FSGS), which can present from infancy to adulthood and is characterized by proteinuria and often nephrotic syndrome leading to kidney failure. These variants may exhibit gain-of-function (e.g. K874X) or loss-of-function (e.g. L395A, G757D) phenotypes, making the role of TRPC6 in FSGS controversial. Here, we characterized Ca²⁺-dependent inactivation (CDI) of TRPC6 after the receptor activation and found that >85% of TRPC6 variants exhibit delayed CDI. Thus, prolonged TRPC6 channel opening due to impaired inactivation may be a common feature of FSGS-associated variants. This effect was confirmed in immortalized mouse podocytes (MPC-5) in which the coiled-coil (CC) domain was deleted from the channel (C6_ΔCC). Podocytes expressing C6_ΔCC exhibited delayed CDI and increased basal Ca²⁺ levels as well as disruption of the F-actin cytoskeleton. Moreover, transcriptomic data from C6_ΔCC-expressing podocytes showed weak expression of the podocyte markers Synpo and Magi2. These results indicate that CDI of TRPC6 is critical for maintaining proper podocyte function. Notably, we observed a correlation between the magnitude of the prolongation of TRPC6 channel activity and the age diagnosed with FSGS. Our findings thus demonstrate that delayed inactivation due to lack of CDI is a determinative characteristic of FSGS-associated TRPC6 variants, affecting both the structure and the function of glomerular podocytes.