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. 2011 Jan 13;286(11):9598–9611. doi: 10.1074/jbc.M110.195909

FIGURE 1.

FIGURE 1.

Effect of co-expression of palmitoylated CaV2.2 I-II loop constructs on the subcellular distribution of GFP-tagged CaVβ1b in tsA-201 cells. A, expression of β1b-GFP alone. B, co-expression of β1b-GFP and CaV2.2 I-II loop, palmitoylated on its N terminus (palm CaV2.2 I-II WT). C, co-expression of β1b-GFP and palmitoylated CaV2.2 I-II loop containing the W391A mutation (palm CaV2.2 I-II W391A). In all images (A–C), GFP is shown in green, and nuclear staining (DAPI) is shown in blue. Scale bars, 20 μm. D, representative line scan fluorescence profiles of GFP (green) and DAPI (blue) for cells shown in A–C; for β1b-GFP alone (left), β1b-GFP plus palmitoylated CaV2.2 I-II loop (middle), and β1b-GFP plus palmitoylated CaV2.2 I-II loop containing the W391A mutation (right). Fluorescence was measured along a typical cross-section of a single cell and plotted over distance. It is expressed as arbitrary units (a.u.), determined from images in which all scanning parameters were constant. Lines used for these examples are shown in A-C. The inset schematic shows the palmitoylated construct used and the mechanism for membrane association in B. The palmitoylation motif MTLESIMACCL, shown in blue, was fused to the N terminus of the I-II loop (amino acids 356–483) of Cav2.2. The two Cys residues will be palmitoylated, which should direct the construct to the plasma membrane. The β-binding site on the I-II loop, shown in red, contains a tryptophan residue (Trp391, indicated by the arrow) which is critical for interaction with the β-subunit. E, quantification of fluorescence distribution within a cell. The ratio of fluorescence at the plasma membrane divided by the average fluorescence in the nucleus, in the region indicated by DAPI staining, was calculated for a number of cells for β1b-GFP alone (black bar, n = 11 cells), β1b-GFP plus palmitoylated CaV2.2 I-II loop (white bar, n = 10), and β1b-GFP plus palmitoylated CaV2.2 I-II loop containing the W391A mutation (gray bar, n = 12). Statistical significance of difference between WT and W391A CaV2.2 I-II loop was determined by Student's t test (***, p < 0.001). Error bars, S.E.