FIGURE 3.

KCa2.3 recycles to the plasma membrane in HEK cells. A, representative IB demonstrating that plasma membrane KCa2.3 is rapidly endocytosed to a steady-state level in HEK cells. Plasma membrane proteins were biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (see “Experimental Procedures”) after which the cells were incubated at 37 °C for the times indicated. Initial plasma membrane expression was determined by omitting the 37 °C incubation step (cell surface, 1st lane). The remaining cell surface biotin was stripped (MESNA), after which the endocytosed, biotinylated protein was pulled down using streptavidin-agarose, and the proteins were separated by SDS-PAGE and blotted for KCa2.3. The efficiency of stripping was determined by subjecting cells to MESNA in the absence of a 37 °C endocytosis step (strip control). The 3rd to 6th lanes demonstrate rapid endocytosis of KCa2.3 to a steady-state level. Similar results were observed in three separate experiments. B, representative IB demonstrating that KCa2.3 recycles back to the plasma membrane following endocytosis. Plasma membrane proteins were biotinylated and allowed to endocytose for 30 min at 37 °C as in A, after which the remaining cell surface biotin was stripped. As shown in the 1st lane, KCa2.3 was endocytosed as in A. The cells were returned to 37 °C for various periods of time as indicated, after which the cells were subject to a second round of biotin stripping from the cell surface. If KCa2.3 returns to the plasma membrane, the biotin associated with the channel will be stripped resulting in a decreasing signal upon IB, and this is what is observed. C, experiment in B was repeated three times, and the resulting IB was digitized, and band intensities for the various time points were determined as a percent change from time 0. The decrease in protein was fit to an exponential decay function with a time constant of 4.7 ± 0.3 min (n = 3).