Fig. 7
Using split YFP constructs to measure competition for Slc7a5 between Kv1.2 and Slc3a2. YFP fragments (YFPN residues 1–157, YN; and YFPC residues 158–239, YC) were fused to Kv1.2, Slc3a2, and Slc7a5 as indicated. a Fluorescence-activated cell sorting was performed on split-YFP tagged versions of Kv1.2, Slc7a5, and Slc3a2, with the third protein tagged with LSS-mOrange. All DNA transfections were done at a 1:1:1 ratio. Bars represent the distribution of transfected cells with prominent mOrange fluorescence (upper left quadrant in b), prominent YFP (lower right quadrant), or prominent signals from both fluorophores (upper right quadrant). b Representative data from FACS sorting of cells in the Kv1.2-YFPC + Slc7a5-YFPN + mOrange or +mOrange-Slc3a2 conditions. c Kv1.2-YFPC was expressed with combinations of Slc7a5-YFPN and Slc3a2 as indicated. The data from individual cells are plotted along with the mean ± s.d., n = 7–20. d Conductance-voltage relationships were generated for each condition after current disinhibition with a 30 s hyperpolarization to −120 mV (Kv1.2-YFPC V1/2 = −8.4 ± 7, n = 10; Kv1.2-YFPC + Slc7a5-YFPN V1/2 = −49 ± 2, n = 5; Kv1.2-YFPC + YFPN-Slc3a2 V1/2 = −11 ± 4, n = 11). Individual conductance-voltage relationships for six cells transfected with Kv1.2-YFPC + Slc7a5-YFPN + Slc3a2 are depicted in gray (overall V1/2 = −36 ± 16 mV). Data at each voltage are presented as mean ± s.d., n = 4–11. e Individual conductance-voltage relationships for seven cells transfected with mOrange-Kv1.2 + Slc7a5-YFPC + YFPN-Slc3a2 (transfection ratio 2:1:2)
