Fig. 1
Overexpression of TRPC3 in adherent HEK293 (A), Expi293F (B), and K. phaffii cells (C), and electrophysiology of TRPC3-YFP-3xFLAG in adherent HEK293 cells by whole-cell analysis (D–F). A–C) In all cell types, TRPC3 is prominently expressed in the PM. Overexpression of TRPC3 in HEK293 (A) and Expi293F cells (B) was examined by fluorescence microscopy, where the YFP tag was excited at 495 nm, and emission was detected at 530 nm. TRPC3 is highlighted in yellow localizing in the PM. C) TRPC3 is enriched in microsomal fractions of K. phaffii. A CBS7435 his4Δ strain and an otherwise isogenic strain expressing TRPC3-3F6H driven by the strong AOX1 promoter were induced with methanol. Different fractions (total cell lysates (TCL)), as well as cytosolic and microsomal fractions) were prepared, separated by SDS–PAGE, and analyzed by immunoblotting with anti-FLAG antibody (TLCWT = total cell lysate of K. phaffi without TRPC3 (negative controle), TLC = total cell lysate of K. phaffi expressing TRPC3). D-F) Whole-cell patch clamp analysis was conducted in adherent HEK293 cells activated with 10 µM GSK. The TRPC3–YFP–3 x FLAG construct (orange, n = 9), TRPC3–YFP construct (blue, n = 11), and TRPC3–3xFLAG construct (green, n = 10) were compared. No significant variances in TRPC3 channel activity were observed across the constructs. D) Statistical evaluation of maximum current densities of the TRPC3 channels at − 90/70 mV with mean ± SEM are displayed. Statistical analysis was performed using a two-tailed Student’s t-test for normally distributed data and a Wilcoxon test for non-normally distributed data, with significance denoted as follows: ns = non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001. E) Representative time courses of current activation by 10 µM GSK in TRPC3 channels are shown. F) Representative I–V curves of activated TRPC3 channels with 10 µM GSK are displayed. Please note: Uncropped fluorescent images of TRPC3-YFP-3xFLAG over expression in adherent HEK293 and Expi293F cells are provided in the Supplementary Information (see Figure S1).
