Focal segmental glomerulosclerosis (FSGS) is a common glomerular disorder causing end-stage renal disease, and hereditary forms of FSGS have been linked to mutations in the Transient Receptor Potential Canonical 6 (TRPC6) gene encoding a nonselective cation channel. We recently found that negative feedback regulation induced by cellular calcium levels, called Ca²⁺-dependent inactivation (CDI), is impaired in FSGS-associated TRPC6 channel mutations within the C-terminal side. Here, we investigated the impact of FSGS mutations in the N-terminal side on CDI of TRPC6 by using electrophysiological experiments. Whole-cell patch-clamp recording confirmed the delayed CDI of TRPC6 currents for cells expressing FSGS-associated mutations (G109S, R175Q, R175W) within the N-terminal ankyrin repeat domains, compared with wild-type TRPC6. Intriguingly, the cells expressing with the early-onset FSGS-associated mutant (R175W, R895L) exhibited severely compared to that of the late-onset mutations (R175Q). These findings suggest that the disruption of CDI is considered to be a fundamental mechanism of genetic FSGS caused by TRPC6 mutations, and evaluation of the CDI in TRPC6 by using whole-cell patch-clamp recording may be a useful and effective approach to estimate the time-to-onset for FSGS.