Fig. 6: Characterization of the simplified hyperforin-derivative Hyp13.

Chemical structure of hyperforin (A) and Hyp13 (B). The phloroglucinol core structure is highlighted in red. (C) Concentration dependent effect of Hyp13 in HEK293 cells expressing hTRPC6 channels in whole cell patch clamp experminents. D Hyp13 also interacts to the LLKL binding motif at TRPC6. 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 hyperforin (10 µM) or Hyp13 (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). E 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. F 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. G Whole-cell currents recorded from HEK293 ctl cells (dashed line), hTRPC6-expressing HEK293 cells (black) or hTRPC6mut (red). Application of Hyp13 (10 µM) resulted in an increase in outward and inward current in hTRPC6 expressing cells. This effect is lost in TRPC6mut expressing cells. H Whole-cell currents recorded from HEK293 ctl cells (dashed line), hTRPC3-expressing HEK293 cells (gray) or hTRPC3mut (blue). Application of Hyp13 (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.