Fig. 4: Identification of the hyperforin binding site at TRPC6 channels.

A The topology model of human TRPC6 (hTRPC6) shows α-helices in cylinders and dashed lines describe region with not sufficient density in CryoEM structure PDB: 6uz8. Potential hyperforin bindings site is marked with a red star. B Sketch demonstrating that amino acids LLKL were mutated in hTRPC6 into the respective amino acids IMRI of hTRPC3 to block hyperforin-mediated TRPC6 activation. In a second step, the amino acids IMRI in hTRPC3 were mutated into the corresponding amino acids LLKL of hTRPC3 to induce a hyperforin-sensitive hTRPC3 channel. hTRPC6 (black), TRPC6mut = IMRI^TRPC6mut (red), hTRPC3 (gray), TRPC3mut = LLKL^TRPC3mut. C Single-cell Ca²⁺ imaging was conducted in HEK293 cells transiently expressing pcDNA3.1 plasmid vector with DNA coding only for eYFP (ctl, white), hTRPC6 (black), hTRPC6mut (red), hTRPC3 (gray), or hTRPC3mut (blue) all expressed as C-terminal eYFP fusion proteins. Cells were stimulated with the solvent DMSO (0.1%), OAG (100 µM) or hyperforin (10 µM) and intracellular Ca²⁺ alterations were detected using fura-2 AM (n = 7–9 ± SEM, cells were selected according to their eYFP fluorescence and their OAG sensitivity; Statistical significance was analyzed by ANOVA with post hoc Dunnett’s test ***p < 0.001) C Whole-cell currents were recorded from HEK293 cells transiently expressing eYFP (ctl, white), hTRPC6 (black), hTRPC6mut (red), hTRPC3 (gray), or hTRPC3mut (blue) all expressed as C-terminal eYFP fusion proteins. Mean current density are depicted at +100 and −100 mV after application of hyperforin (10 µM). Currents were normalized to the basic currents before compound application were subtracted (n = 3 ± SEM¸ Statistical significance was analyzed by ANOVA with post hoc Dunnett’s test ***p < 0.001). D Representative time traces were monitored in HEK293 ctl cells (dashed line), hTRPC6-expressing HEK293 cells (black) or hTRPC6mut (red) stimulated with OAG (100 µM) 60 s after starting the experiment and after 300 s hyperforin (10 µM) was applied. E Representative time traces were monitored in HEK293 ctl cells (dashed line), hTRPC3-expressing HEK293 cells (gray) or hTRPC3mut (blue) stimulated with OAG (100 µM) 60 s after starting the experiment and after 300 s hyperforin (10 µM) was applied. F Whole-cell currents recorded from HEK293 ctl cells (dashed line), hTRPC6-expressing HEK293 cells (black) or hTRPC6mut (red). Application of hyperforin (10 µM) resulted in an increase in outward and inward current in hTRPC6 expressing cells. This effect is lost in TRPC6mut expressing cells. G Whole-cell currents recorded from HEK293 ctl cells (dashed line), hTRPC3-expressing HEK293 cells (gray) or hTRPC3mut (blue). Application of hyperforin (10 µM) showed no effect in ctl and hTRPC3 expressing cells but resulted in an increase in outward and inward current in hTRPC3mut expressing cells. H The hyperforin binding site LLKL at human hTRPC6 differs in the last amino acid from rat and mouse TRPC6 LLKF. To test if this amino acid interferes with hyperforin binding to TRPC6, we compared hTRPC6 with hTRPC6 LLKF. Single-cell calcium imaging was conducted in HEK239 cells transiently expressing hTRPC6 or hTRPC6 LLKF. Cells were stimulated with hyperforin (10 µM) and Fura-2-AM 340/380 nm ratio changes were analyzed and afterward converted into intracellular Ca²⁺ in nM. No significant differences were observed (n = 3 ± SEM, cells were selected according to their eYFP fluorescence; statistical significance was calculated using unpaired t-test, not significant 0.0576).