Figure 1.
KV10.1-BBS is a tagged version of KV10.1.
KV10.1-BBS is a voltage-gated ion channel which contains an extracellular loop with the α-bungarotoxin-binding site (BBS) that can bind α-bungarotoxin-conjugates (BTX-XX).
Figure 2.
Labeling of KV10.1-BBS with fluorescent BTX conjugates specifically labels the cell membrane.
A) Incubation of cells with BTX-Alexa594 results in membrane stains (top row, right)) in Hek cells transfected with KV10.1-BBS. This labeling is blocked by preincubation of cells with unlabeled BTX (center). No labeling is detectable in cells expressing wild type KV10.1 (left). Transfected cells can be identified based on expression of GFP from the pTracer plasmid (bottom). GFP signals do not correlate to KV10.1-BBS expression levels. B) Double-labeling KV10.1: Cells expressing the fusion protein KV10.1-BBS-Venus were labeled with BTX-Alexa647 to distinguish the membrane versus internal population of KV10.1.
Figure 3.
KV10.1-BBS with or without bound BTX-conjugates shows electrophysiological behavior similar to KV10.1.
KV10.1-BBS with or without bound BTX-conjugates shows electrophysiological behavior similar to Kv10.1. A) The KV10.1-BBS current-voltage relationship is shifted to more negative values. Whole cell currents were triggered by stepping from a holding potential of −100 mV to test potentials (−100 mV to +80 mV) for 500 ms. Current amplitudes at the end of test pulses were normalized to amplitudes recorded at +80 mV (I/Imax) and plotted against the applied membrane potential. KV10.1-BBS (filled circles, n = 11) activates at more negative potentials compared to KV10.1 (open circles, n = 28). B) KV10.1 and KV10.1-BBS channel activation depends on the holding potential. Current traces were measured upon application of 500 ms depolarization to +40 mV after conditioning pulses (5000 ms) at potentials ranging from −120 mV to −70 mV in 10 mV increments. (C) The rise time of activation from 20 to 80% of maximal current was plotted against the holding potential. KV10.1-BBS (filled circles, n = 11) is characterized by shorter rise time (faster activation) as compared to KV10.1 (open circles, n = 28). D) Representative traces of Kv10.1-BBS mediated currents in unlabeled (left; scale bars, 1 nA, 50 ms) and labeled (right; scale bars, 0.25 nA, 50 ms) cells. No changes in kinetics were observed. E) Current density of KV10.1-BBS cells did not change upon binding of BTX-Alexa488 to the BBS-site.
Figure 4.
KV10.1 in the plasma membrane is rapidly transported to punctuate endosomal stuctures.
A) Formation of vesicular structures proceeds immediately after surface -labeling of KV10.1-BBS with BTX-Alexa594 during 5 minutes at 30°C (left to right). Confocal laser scans were performed B) after surface-labeling KV10.1-BBS with BTX-Alexa488 and C) after 30 minutes of incubation at 30°C. Intracellular vesicles were identified in xy-sections and xz-projections at positions indicated by arrows (green and gray: BTX-Alexa488, red: membrane stain with FM 4–64). More endosomal structures appear in the plane of the basal membrane than 3 µm above (C, top and bottom row, respectively).
Figure 5.
Endocytosis of KV10.1 is constitutive and shows saturation after 45 minutes.
A) left) Internalized KV10.1-BBS molecules were detected in western blots (row 1, lane 3 & 4) and correspond up to ∼20% of initially labeled surface-channels (lane 1). Intracellular KV10.1-BBS molecules were discriminated from surface molecules by removing surface-labels using acid wash before harvest and pull-down. Endogenous biotinylated carboxylases were detected with streptavidin-peroxidase in western blots to correct for slight variations in pull-down and blotting efficiency. (From top to bottom: a: pyruvate-carboxylase, b: propionyl-CoA carboxylase, c: methycrotonyl-CoA carboxylase [46], [47]). Right: Acid washing at pH3 removes surface-labels while washing at pH5 does not. B) The endocytosis rate of KV10.1 was measured by determining the cellular uptake of BTX-biotin via KV10.1-BBS surface-molecules. The relative amount of internalized BTX-biotin was plotted over time (error bars: SD; top) and starts to saturate after 45 minutes. To generate this data, internalized BTX-biotin was blotted on membranes and detected with streptavidin-peroxidase (bottom, representative blot of duplicates). Ratios were determined as ‘intracellular signal/(whole-cell signal – intracellular signal)’ and corrected for unspecific uptake of BTX-biotin in cells expressing KV10.1. Whole-cell signals (lane 4) were determined after omitting acid washing.
Figure 6.
Clathrin-mediated endocytosis is modestly involved in the internalization of KV10.1.
A) Complexes of KV10.1-BBS with BTX-Alexa594 (red) colocalize with clathrin-GFP (green) in punctuate structures (magnified insets, middle) compatible with endosomes. Line profile plots (bottom) through vesicles (white arrows) represent three classes of labeling. The colocalization map (right panel) highlights punctae with a high vs. low degree of colocalization. B) Surface-expression of KV10.1-BBS is altered in cells that overexpress components of the clathrin-dependent endocytic machinery. Surface channels were labeled with BTX-biotin, isolated and immune-detected in western blots. Overexpression of the dominant-negative dynamin-K44A led to slight increases in surface expression, while overexpressing AP-180 slightly decreased KV10.1-BBS surface-expression. C) KV10.1 is internalized by fluid phase uptake. Complexes of KV10.1-BBS with BTX-Alexa594 colocalize with dextran-rhodamine, a marker for fluid phase uptake, after 3 minutes of chase reaction. Corresponding ROIs from the merged dual-color image (center) and the intensity correlation image (right) highlight structures with colocalization.
Figure 7.
KV10.1 is internalized and sorted to lysosomes for degradation.
A) Internalized complexes of KV10.1-BBS with BTX-Alexa488 (green) colocalize with the lysosome stain ‘lysotracker red’ (red). The presented image was recorded close to the plane of the basal membrane (left). Line profile plots through highlighted punctuate structures distinguish dual- or single-color labeling and shows that lysotracker red did not show signals in the green detection channel (right) and consequently no photoconversion. The intensity correlation image (center) maps punctae with a high degree of colocalization. B) A rescue of internalized KV10.1-BBS molecules by the lysosome inhibitor chloroquine (CQN) was detected in western blots (row 1): internalization of KV10.1-BBBS complexed to BTX-biotin is shown for ± CQN during 90 minutes in lane 1 & 2 and for 240 minutes in lanes 3 & 4, respectively. For isolation of internalized KV10.1-BBS molecules surface labels were removed labeling on ice. Endogenously biotinylated carboxylases were detected to normalize signals for pull-down efficiencies.
Figure 8.
The KV10.1 life cycle includes recycling of internalized channels to the plasma membrane.
A) Internalized KV10.1-BBS channels complexed to BTX-biotin recycle back to the plasma membrane and were detected with streptavidin-Alexa594. Before, BTX-biotin surface-labels had been removed by acid wash. Thereafter incubation at permissive temperatures (30°C) lead to more pronounced membrane signals than at non-permissive temperatures (4°C) (right column versus center, respectively) GFP is expressed from pTracer- KV10.1-BBS plasmids as a marker of transfection (second row). Identical exposure times and look up tables were used. Membrane-label intensity was quantified and normalized to membrane-signals before acid wash B) A reduction of intracellular BTX-biotin due to recycling and degradation was detected in western blots (scheme). Intracellular BTX-biotin levels decreased by ∼60% during 30 minutes of incubation at 30°C (lanes 3 & 4) compared to 4°C (lanes 1 and 2). A second acid wash lead to another decrease of BTX-biotin levels by ∼30% presumably by removing recycled BTX-biotin molecules from the cell surface.