FIGURE 11.

Small N-terminal motif is required for the proper endocytosis and recycling of KCa2.3 in HEK and HMEC-1 cells. BLAP-tagged full-length KCa2.3 (A) or the N-terminal deletions, ΔLeu⁸³–Asp¹³⁵ (B), ΔMet¹⁸⁶–Leu²³⁵ (C), ΔMet¹⁸⁶–Glu²⁰⁵ (D), ΔGly²⁰⁶–Pro²¹⁷ (E), ΔPro²¹⁸–Leu²³⁵ (F), and ΔGln²⁰⁷–Gln²¹⁰ (G), were transiently transfected into HEK cells, and the plasma membrane channel was labeled with streptavidin-Alexa555 and localized by IF at either time 0 or 3 h. Each of these deletions was localized to the plasma membrane at time 0 h (left panels). After 3 h at 37 °C (right panels), the deletion ΔLeu⁸³–Asp¹³⁵ (B) was correctly localized at the plasma membrane, whereas the ΔMet¹⁸⁶–Leu²³⁵ deletion (C) was completely endocytosed. Further deletions within this domain demonstrate that the amino acids Met¹⁸⁶–Glu²⁰⁵ (D) and Pro²¹⁸–Leu²³⁵ (F) localize normally at 0 and 3 h, whereas Gly²⁰⁶–Pro²¹⁷ (E) as well as the smaller deletion ΔGln²⁰⁷–Gln²¹⁰ (G) are mislocalized to intracellular endosomes. For these studies, single confocal sections are shown. H and I, to quantify the time course for plasma membrane, ΔGly²⁰⁶–Pro²¹⁷ (H) and ΔGln²⁰⁷–Gln²¹⁰ (I) degradation plasma membrane proteins were biotinylated followed by streptavidin pulldown and blotted for KCa2.3 at the times indicated. J, blots were quantified by densitometry and fit to an exponential decay function with time constants of 6.7 h (n = 2) for ΔGly²⁰⁶–Pro²¹⁷ (dashed line, open triangles) and 8.1 h (n = 2) for ΔGln²⁰⁷–Gln²¹⁰ (solid line, filled circles). To confirm a similar function for the N-terminal domain of KCa2.3 in endothelial cells, we transiently transfected HMEC-1 cells with either BLAP-tagged KCa2.3 (K) or ΔGly²⁰⁶–Pro²¹⁷ (L) and evaluated localization as above. Each of these channels is correctly localized to the plasma membrane at time 0 h. However, after 3 h at 37 °C, KCa2.3 is almost exclusively localized to the plasma membrane, whereas ΔGly²⁰⁶–Pro²¹⁷ (L) has been completely endocytosed. For these studies, projection images from multiple z-sections are shown. Nuclei are labeled with DAPI (blue).