FIGURE 2.

K_ATP channels undergo multiple rounds of endocytosis and recycling. A, schematic of secondary antibody capture assay (see “Experimental Procedures”). HEK293 cells stably expressing Kir6.2-HA and SUR1 were incubated with rat anti-HA antibodies for 2 h at 37 °C to allow labeling and subsequent endocytosis of cell surface channels. Following blocking of surface-bound antibody with unlabeled secondary antibody, a subsequent incubation at 37 °C was performed in the presence of extracellular Alexafluor⁴⁸⁸-conjugated anti-rat antibodies (green) to label channels returning to the cell surface. The cells were then fixed and permeabilized, and nonrecycled channels were stained with Cy3-conjugated anti-rat antibodies (red). B, representative images of time course of channel recycling assayed as outlined by the schematic in A. The scale bar equals 10 μm. C, time course of channel recycling determined by quantitative chemiluminescence assay (see “Experimental Procedures”). The data points represent the means, and the error bars indicate S.E. (n = 3). The increase in recycling was expressed relative to surface channels present at time 0.