Fig. 4
Single-channel patch-clamp experiments were conducted on TRPC3 in HEK293 cells (purple) and on reconstituted TRPC3 extracted from Expi293F cells using DDDG (blue) or DDM (orange). (A) Representative single-channel currents showing 200 ms of GSK-induced TRPC3 activity. TRPC3 was either expressed in HEK293 cells (purple) or purified from Exppi293F cells using DDDG (blue) or DDM (orange) and reconstituted into DPhPC bilayers. GSK was applied at 10 µM for adherent HEK293 cells and 20 µM for reconstituted TRPC3. Currents were recorded at + 80 mV. Channel states are indicated as closed (c), sublevels (s), or open (o). (B) Unitary currents at the open (o) level in response to repeated GSK applications of (10 µM for adherent HEK293 cells and 20 µM for reconstituted TRPC3) at a membrane potential of + 80 mV. Data are presented as means ± SEM. Statistical significance was determined using a two-tailed multiple t-test for normally distributed data and the Wilcoxon test for non-normally distributed data. Non-significant differences are denoted as “ns.” C–E) Fraction of events corresponding to the closed (panel C), sublevel (panel D), and open (Panel E) states, derived from Gaussian fitting of single-channel recordings from HEK293 cells expressing TRPC3 (purple), reconstituted TRPC3 purified with DDDG (blue) or with DDM (orange). GSK was applied at 10 µM for HEK293 cells and 20 µM for reconstituted TRPC3 at a membrane potential of + 80 mV. Data are presented as means ± SEM. Statistical significance was assessed using a two-tailed multiple t-test or the Wilcoxon test, with differences considered significant at * p < 0.05, ** p < 0.01, and *** p < 0.001. Non-significant differences are indicated as “ns”. We demonstrated that purified TRPC3 remains functional and its activity is comparable to TRPC3 overexpressed in HEK293 cells. Please note: Uncropped representative traces from Fig. 4A are shown in the Supplementary Information including the statistical analysis of the fraction of events at multiple levels (SI Figure S12).
