Journal: The Journal of Biological Chemistry
Article Title: Free fatty acids inhibit an ion-coupled membrane transporter by dissipating the ion gradient
doi: 10.1016/j.jbc.2024.107955
Figure Lengend Snippet: AA dissipates cation gradients in single-transporter assays. A , time-dependent E FRET population contour plots of the labeled ccPEB1a-Y198F encapsulated in the R276S/M395R-Cys Glt Ph proteoliposomes during sequential changes of the external buffer. Before imaging, proteoliposomes were loaded with KCl and immobilized in the microscope chamber. Vesicles were perfused with KCl buffer with 20 μM AA, and the first movie was taken after 10 min incubation (K + /AA). NaCl buffer with 1 μM L-Asp and 20 μM AA was injected at 4 s during the next recording, marked by a red arrow (Na + /Asp/AA). The imaging chamber was washed with 0.5 ml of KCl buffer and imaged in the same buffer (K + ). Finally, NaCl buffer with 1 μM L-Asp was injected while recording (Na + /Asp). B , the experiment was performed as in A , except the proteoliposomes were preloaded with NaCl buffer and perfused with solutions as shown. During the wash step, the proteoliposomes were first perfused with KCl buffer with 20 μM AA, incubated for 10 min, and washed with an excess KCl buffer. C , the experiment was performed as in B, except Na + /Asp perfusion was omitted and Na + buffers were used during the wash steps. Schematic representations of the proteoliposomes, emphasizing the ion content and expected transport activity, are shown above the panels. Blue and pink fonts denote K + and Na + ions. L-Asp and AA are presented as a purple and red circle , respectively. The number of trajectories analyzed is shown on the panels ( N ). AA, arachidonic acid.
Article Snippet: All imaging experiments were performed on a previously described home-built prism-based TIRF microscope constructed around a Nikon Eclipse Ti inverted microscope body at 25 °C ( ).
Techniques: Labeling, Imaging, Microscopy, Incubation, Injection, Activity Assay
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