Figure 4.

Fluorescence lifetime imaging microscopy (FLIM) images of the cells cotransfected with enhanced cyan fluorescent protein (ECFP)-chloride channel 1 (ClC-1) and epithelial sodium channel (ENaC) subunits with C-terminal mutations. The cells were cotransfected with ECFP-ClC-1 and αβγ-ENaC. One subunit in αβγ-ENaC complex was truncated at the C-terminus and N-terminally tagged with enhanced yellow fluorescent protein (EYFP) (A). The lifetime images are shown in pseudocolors (nanoseconds). The apparent mean lifetime for each image is shown at the bottom of each image. Note: each image has separate lifetime range (ns). B: apparent FRET efficiencies, E, in the cells cotransfected with ECFP-ClC-1 and αβγ-ENaC. One subunit in αβγ-ENaC complex was truncated at the C-terminus and N-terminally tagged with EYFP; EYFP-α-ΔC (4), EYFP-β-ΔC (5), or EYFP-γ-ΔC (6). The ENaC subunit with a point at the C-terminus was C-terminally tagged with EYFP; α-Y644A-EYFP (7), β-Y620A-EYFP (8), or γ-Y627A-EYFP (9). ECFP-EYFP fusion (1) served as a positive control while separately coexpressed ECFP and EYFP (2), and ECFP-ClC-1 expressed alone served as a negative controls. *Statistically significant changes (Student’s t test) compared with negative controls (2 or 3). These experiments were repeated at least five times with similar results.