Rapid Internalization of the Oncogenic K+ Channel KV10.1

KV10.1 is a mammalian brain voltage-gated potassium channel whose ectopic expression outside of the brain has been proven relevant for tumor biology. Promotion of cancer cell proliferation by KV10.1 depends largely on ion flow, but some oncogenic properties remain in the absence of ion permeation. Additionally, KV10.1 surface populations are small compared to large intracellular pools. Control of protein turnover within cells is key to both cellular plasticity and homeostasis, and therefore we set out to analyze how endocytic trafficking participates in controlling KV10.1 intracellular distribution and life cycle. To follow plasma membrane KV10.1 selectively, we generated a modified channel of displaying an extracellular affinity tag for surface labeling by α-bungarotoxin. This modification only minimally affected KV10.1 electrophysiological properties. Using a combination of microscopy and biochemistry techniques, we show that KV10.1 is constitutively internalized involving at least two distinct pathways of endocytosis and mainly sorted to lysosomes. This occurs at a relatively fast rate. Simultaneously, recycling seems to contribute to maintain basal KV10.1 surface levels. Brief KV10.1 surface half-life and rapid lysosomal targeting is a relevant factor to be taken into account for potential drug delivery and targeting strategies directed against KV10.1 on tumor cells

Clathrin-mediated endocytosis is modestly involved in the internalization of K_V10.1.

<p>A) Complexes of K_V10.1-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 K_V10.1-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 K_V10.1-BBS surface-expression. C) K_V10.1 is internalized by fluid phase uptake. Complexes of K_V10.1-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.</p

Labeling of K_V10.1-BBS with fluorescent BTX conjugates specifically labels the cell membrane.

<p>A) Incubation of cells with BTX-Alexa594 results in membrane stains (top row, right)) in Hek cells transfected with K_V10.1-BBS. This labeling is blocked by preincubation of cells with unlabeled BTX (center). No labeling is detectable in cells expressing wild type K_V10.1 (left). Transfected cells can be identified based on expression of GFP from the pTracer plasmid (bottom). GFP signals do not correlate to K_V10.1-BBS expression levels. B) Double-labeling K_V10.1: Cells expressing the fusion protein K_V10.1-BBS-Venus were labeled with BTX-Alexa647 to distinguish the membrane versus internal population of K_V10.1.</p

Endocytosis of K_V10.1 is constitutive and shows saturation after 45 minutes.

<p>A) left) Internalized K_V10.1-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 K_V10.1-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. (<i>From top to bottom: a:</i> pyruvate-carboxylase, b: propionyl-CoA carboxylase, c: methycrotonyl-CoA carboxylase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026329#pone.0026329-Hollinshead1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026329#pone.0026329-Ruggiero1" target="_blank">[47]</a>). Right: Acid washing at pH3 removes surface-labels while washing at pH5 does not. B) The endocytosis rate of K_V10.1 was measured by determining the cellular uptake of BTX-biotin via K_V10.1-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 K_V10.1. Whole-cell signals (lane 4) were determined after omitting acid washing.</p

K_V10.1 in the plasma membrane is rapidly transported to punctuate endosomal stuctures.

<p>A) Formation of vesicular structures proceeds immediately after surface -labeling of K_V10.1-BBS with BTX-Alexa594 during 5 minutes at 30°C (left to right). Confocal laser scans were performed B) after surface-labeling K_V10.1-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).</p

K_V10.1-BBS with or without bound BTX-conjugates shows electrophysiological behavior similar to K_V10.1.

<p>K_V10.1-BBS with or without bound BTX-conjugates shows electrophysiological behavior similar to Kv10.1. A) The K_V10.1-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. K_V10.1-BBS (filled circles, n = 11) activates at more negative potentials compared to K_V10.1 (open circles, n = 28). B) K_V10.1 and K_V10.1-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. K_V10.1-BBS (filled circles, n = 11) is characterized by shorter rise time (faster activation) as compared to K_V10.1 (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 K_V10.1-BBS cells did not change upon binding of BTX-Alexa488 to the BBS-site.</p

K_V10.1 is internalized and sorted to lysosomes for degradation.

<p>A) Internalized complexes of K_V10.1-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 K_V10.1-BBS molecules by the lysosome inhibitor chloroquine (CQN) was detected in western blots (row 1): internalization of K_V10.1-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 K_V10.1-BBS molecules surface labels were removed labeling on ice. Endogenously biotinylated carboxylases were detected to normalize signals for pull-down efficiencies.</p

The K_V10.1 life cycle includes recycling of internalized channels to the plasma membrane.

<p>A) Internalized K_V10.1-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- K_V10.1-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.</p

Calmodulin regulates human ether à go-go 1 (hEAG1) potassium channels through interactions of the eag-domain with the cyclic nucleotide binding homology domain

The ether à go-go family of voltage-gated potassium channels is structurally distinct. The N-terminus contains an eag domain (eagD) that contains a Per-Arnt-Sim (PAS) domain that is preceded by a conserved sequence of 25-27 amino acids known as the PAS-cap. The C-terminus contains a region with homology to cyclic nucleotide binding domains (cNBHD), which is directly linked to the channel pore. The human EAG1 (hEAG1) channel is remarkably sensitive to inhibition by intracellular calcium (Ca²⁺ᵢ) through binding of Ca²⁺-calmodulin to three sites adjacent to the eagD and cNBHD. Here, we show that the eagD and cNBHD interact to modulate Ca²⁺-calmodulin as well as voltage-dependent gating. Sustained elevation of Ca²⁺ᵢ resulted in an initial profound inhibition of hEAG1 currents, which was followed by a phase when current amplitudes partially recovered, but activation gating was slowed and shifted to depolarized potentials. Deletion of either the eagD or cNBHD abolished the inhibition by Ca²⁺ᵢ. However, deletion of just the PAS-cap resulted in a >15-fold potentiation in response to elevated Ca²⁺ᵢ. Mutations of residues at the interface between the eagD and cNBHD have been linked to human cancer. E600 on the cNBHD, when substituted with residues with a larger volume, resulted in hEAG1 currents that were profoundly potentiated by Ca²⁺ᵢ in a manner similar to the ΔPAS-cap mutant. These findings provide the first evidence that eagD and cNBHD interactions are regulating Ca²⁺-dependent gating and indicate that the binding of the PAS-cap with the cNBHD is required for the closure of the channels upon CaM binding