Pharmacological inhibition of focal segmental glomerulosclerosis-related, gain of function mutants of TRPC6 channels by semi-synthetic derivatives of larixol

BACKGROUND AND PURPOSE Gain of functionmutations in TRPC6 channels can cause autosomal dominant forms of focal segmental glomerulosclerosis (FSGS). Validated inhibitors of TRPC6 channels that are biologically active on FSGS-related TRPC6 mutants are eagerly sought. EXPERIMENTAL APPROACH We synthesized new TRPC6-inhibiting modulators from larixol, a resiniferous constituent of Larix decidua, and tested the potency and selectivity in cell lines stably expressing various TRPC channel isoforms. Channel activation was followed by Ca2+ influx analyses and electrophysiological recordings. The most promising compound larixyl carbamate (LC) was tested on native TRPC6 channels and TRPC6 constructs carrying FSGS-related point mutations. KEY RESULTS LC exhibited an about 30-fold preference for TRPC6 over TRPC3 channels and a fivefold preference for TRPC6 over TRPC7 channels. Six FSGS-related TRPC6 mutants, including the highly active M132T and R175Q variants, were strongly inhibited by 1 mu M LC. Surprisingly, no TRPC6-related Ca2+ signals were detectable in primary murine podocytes, or in acutely isolated glomeruli. in these preparations. Quantitative PCR revealed a 20-fold to 50-fold lower abundance of TRPC6 transcripts in rat or mouse podocytes, compared with pulmonary artery smooth muscle cells from the same species. Accordingly, electrophysiological recordings demonstrated that DAG-induced currents in murine podocytes are very small, but sensitive to LC. CONCLUSIONS AND IMPLICATIONS In spite of their low abundance in native podocytes, native TRPC6 channels are targetable using larixol-derived TRPC6 inhibitors. As observed with wild-type TRPC6 channels, FSGS-related TRPC6 mutants were sensitive to the newly developed inhibitors, paving the way for experimental therapies.