FIGURE 5.

Addition of annexin 12 to the trans side of the bilayer permits imaging of reproducible fluo-3 fluorescence signals. An x-t line scan image of fluo-3 fluorescence during repeated openings of a single RyR2 channel immobilized by addition of 2 μM annexin 12 to the trans side of the bilayer is shown in the top panel. RyR2 Ca²⁺ flux was elicited by three 200-ms voltage steps repeated at 200-ms intervals. Simultaneously recorded single RyR2 Ca²⁺ currents are presented in the middle panel and intensity-time traces derived from the fluorescent images are shown in the bottom panel. The image is 45 μm and 2 s in the horizontal and vertical dimensions, respectively. The reproducibility of the fluo-3 fluorescence during repeated linescans demonstrates the benefit of stabilizing the position of the RyR2 channel in the bilayer.