Figure 2. F− transport and selectivity in Fluc proteins.
(A) Liposome flux assays for Fluc Ec2 and Bpe (∼10 pmol protein/mg lipid). Anion efflux from liposomes containing KF+ KCl, 150 mM each, was initiated by addition of 1 µM Vln (filled triangle). Anions trapped in protein-free liposomes were released with 50 mM β-OG (open triangles). Anion appearance in the external solution was monitored (F− red, Cl− black traces), and signals normalized to final levels. (B) Timecourse of insertion of Fluc-Ec2 reconstituted liposomes (5 µg/mg) into a planar bilayer under salt-gradient conditions (300 mM NaF/30 mM NaF) at −100 mV. Arrow indicates addition of 0.5-µl liposomes. Dashed line is zero-current level. (C) Macroscopic I–V relations under ionic conditions indicated. Inset: Current responses to voltage pulses from −90 mV to +90 mV in 20-mV increments under salt-gradient conditions as in (B). Reversal potentials from 4–5 separate bilayers were 53 ± 1 mV and 119 ± 3 mV for salt-gradient and bi-ionic conditions, respectively.