Fig. 2

Localization of Kv1.3 channels after sequential mutation of the acidic ER export motif. The Kv1.3-eGFP and each of the mutated Kv1.3-eGFP proteins (see methods) were transiently expressed in BSC40 cells and the fluorescence was observed using confocal microscopy (a-h) in the presence of the membranous ER resident protein Sec61β and the Golgi resident protein Golgin97. The Kv1.3-eGFP co-localization with these organelle specific resident proteins and the resulting fluorescent trafficking profile (a) is similar in appearance with the Kv1.3-eGFP E443A (b), Kv1.3-eGFP E445A (c), Kv1.3-eGFP E443A-E445A (e), and Kv1.3-eGFP E445A-E447A (f) profiles indicating that there is no significant ER retention of any of these mutated proteins. A noticeable difference in the co-localization of Kv1.3-eGFP and Sec61β is observed in the Kv1.3-eGFP E447A (d), Kv1.3-eGFP E443A-E445A-E447A (g), and Kv1.3-eGFP ∆C (h) trafficking profiles where there is either a modest (d and g) or severe (h) retention within the ER network (d, g, and h; white arrows). Line scans of each fluorescent channel are shown. The white arrow in the merged image represents the placement and direction of the line scan. Scale bar = 10 μm