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. 2016 Dec 19;114(1):E37–E46. doi: 10.1073/pnas.1612263114

Fig. 6.

Fig. 6.

PIP2 depletion and Gq/11-coupled receptor activation lead to dynamic NHERF1 dissociation and a conformational change of the TRPC5 C terminus. (A–N) Dynamic intermolecular FRET measurements with dual-emission photometry. (A–E) FRET measurements between N-terminally Cerulean-tagged NHERF1 (Cerulean–NHERF1) and (A) C-terminally eYFP-tagged TRPC5 (TRPC5–YFP), (B) C-terminally eYFP-tagged TRPC5–T972D (TRPC5–T972D–YFP), (C) C-terminally eYFP-tagged TRPC5–T972A (TRPC5–T972A–YFP), and (D) C-terminally eYFP-tagged TRPC6 (TRPC6–YFP). (A) Representative FRET measurement showing normalized fluorescence traces of Cerulean (cyan) and eYFP (yellow) on excitation of 430 nm (Left). (A–D) Representative traces of the FRET signal. Applications of 20 µM wortmannin (Wort) and of 100 µM carbachol (CCh) are indicated. (E) Summary of changes of FRET signal amplitudes induced by wortmannin (cyan bars) or CCh (dark blue bars) in both orders of application. Numbers indicate the numbers of measured cells from at least four independent experiments. Significant differences compared with wild-type TRPC5–eYFP-expressing cells (mean ± SEM, two-tailed, unpaired t test; **P < 0.01, ***P < 0.001, black asterisks). Significant differences compared with TRPC5–T972D–eYFP-expressing cells (mean ± SEM, two-tailed, unpaired t test; **P < 0.01, ***P < 0.001, gray asterisk). (F–N) FRET measurements between C-terminally eYFP- and eCFP-tagged TRPC5 (F–I) and between C-terminally eYFP- and eCFP-tagged TRPC5–T972A (J–M). (F and J) Representative FRET measurement showing normalized fluorescence traces of eCFP (cyan) and eYFP (yellow) on excitation of 430 nm (Left). (F–M) Representative traces of the FRET signal. Applications of 20 µM wortmannin (Wort), of 100 µM ATP, and of 100 µM OAG are indicated. (N) Summaries of changes of FRET signal amplitudes induced by wortmannin (cyan bars), ATP (violet bars), OAG (blue bars), and OAG and wortmannin together (blue hatched bars). Numbers indicate the numbers of measured cells from at least four independent experiments. Significant differences between wortmannin- and wortmannin plus OAG-induced FRET amplitudes (mean ± SEM, two-tailed, unpaired t test; **P < 0.01, ***P < 0.001, black asterisks).