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. 2014 Jan 9;164(3):1415–1429. doi: 10.1104/pp.113.229757

Figure 1.

Figure 1.

The C-linker domain is important for PM expression and channel functionality of the Shaker K+ channel KAT2 from Arabidopsis. A, Analyses of the native KAT2 subunit after transient expression in tobacco protoplasts and X. laevis oocytes. A1, The native KAT2 was studied by subcellular localization of GFP fusions in tobacco protoplasts and current recordings in X. laevis oocytes. Shown from left to right are a schematic representation of the C-linker sequence of the KAT2 subunit; a representative confocal microscopy image of GFP and FM4-64 fluorescence in a protoplast expressing the channel-GFP construct, together with the bright-field image of the corresponding protoplast (bar = 10 µm; chlorophyll autofluorescence in chloroplasts [red] is also seen in the confocal images); GFP (green) and FM4-64 (red) fluorescence intensity profiles across the protoplast membrane (and a pocket of cytoplasm; the white arrow on the confocal image marks the position of the analyzed section crossing the PM and pockets of cytoplasm [Ext. and Cyt. indicate external and cytosolic sides, respectively]); and representative current traces recorded in oocytes expressing the channel construct (without GFP tag) in 100 mm K+ bath solution. Applied activation membrane voltages ranged from +40 to −170 mV (increments of 15 mV; holding potential, 0 mV; deactivation potential, −40 mV). A2, Further analyses were performed on oocytes expressing KAT2-GFP. Shown from left to right are linear Z stacks obtained at the upper pole of the oocyte (green signals correspond to GFP fluorescence and red signals correspond to FM4-64 fluorescence); GFP (green) and FM4-64 (red) fluorescence intensity profiles across the oocyte membrane (the white arrow on the confocal image marks the position of the analyzed section crossing the PM and the cytoplasm; bar = 10 µm); and protein gel blot analysis of PMs and internal membranes (IM) from Shaker-GFP-injected oocytes probed with a monoclonal antibody to GFP. Note the presence of monomers (108 kD) and dimers (216 kD) in the PM fraction. B, Alignment of the C-linker sequences of Shaker subunits KAT2 and AtKC1 from Arabidopsis. Identical residues are displayed with a gray background. C, Left, amino acid sequences of the C-linker upstream junction with the channel sixth transmembrane segment (S6) and downstream junction with the CNBD in the KAT2*AtKC1-C-linker chimera. Blue and black colors refer to KAT2 and AtKC1 sequences, respectively. Right, pictogram of the KAT2*AtKC1-C-linker chimera structure in which the sequences from KAT2 (blue) and AtKC1 (black) are indicated. D to F, Surface expression and activity at the PM of the chimeric channels (obtained by sequence swapping between KAT2 and AtKC1): KAT2*AtKC1-C-linker chimera (D), KAT2*AtKC1(302-354) (E), and KAT2*AtKC1(355-389) (F), investigated by the expression of Shaker-GFP fusions in tobacco mesophyll protoplasts and Shaker subunits (without GFP tag) in X. laevis oocytes as described in A1. A white arrow on the confocal image marks the position of the analyzed section crossing the PM and pockets of cytoplasm. Bars = 10 μm.