Rapid Internalization of the Oncogenic K+ Channel KV10.1

PLOS ONE, Oct 2011

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.

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Rapid Internalization of the Oncogenic K+ Channel KV10.1

Citation: Kohl T, Lo rinczi E, Pardo LA, Stu hmer W ( + Rapid Internalization of the Oncogenic K Channel KV10.1 Tobias Kohl 0 1 Eva Lo rinczi 0 1 Luis A. Pardo 0 1 Walter Stu hmer 0 1 Bernard Attali, Sackler Medical School, Tel Aviv University, Israel 0 Current address: Department of Cardiology & Pneumology, Translational Cardiology, Georg August University Medical Center , Go ttingen , Germany 1 1 Max-Planck-Institute of Experimental Medicine, Department of Molecular Biology of Neuronal Signals , Go ttingen, Germany , 2 DFG Research Center for Molecular Physiology of the Brain (CMPB) , Go ttingen , Germany 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 a-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. - Funding: This work was support by the Max Planck Society and the DFG Research Center for Molecular Physiology of the Brain (CMBP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Protein turnover within cells plays a key role in maintaining cellular homeostasis and plasticity. Here we report an analysis of the mechanisms controlling the surface expression and turnover of the oncogenic voltage-gated K+ channel KV10.1. KV10.1 (Eag1) is a voltage-gated, delayed rectifier K+ channel from the Ether-a`-go-go (KCNH) gene family [1,2]. It is mainly found in distinct neuronal tissues at both the mRNA and protein level [3,4,5]. Yet KV10.1 is overexpressed in a wide range of solid tumors [4]. In this context KV10.1 is emerging as a prognostic marker for poor outcome and as a drug-target for KV10.1-positive tumors [4,6,7,8]. The precise mechanism how KV10.1 promotes proliferation of cancer cells is still under debate, although it is known that it includes both permeation-dependent and -independent components. Notably, non-conducting signaling functions might rely on ion channel conformation [9,10,11]. Ectopic expression of KV10.1 at the cell surface has been proven relevant for tumor biology, since a KV10.1-specific blocking antibody reduces proliferation in a variety of cancer cell types expressing KV10.1 both in vitro and in vivo [12]. Furthermore, experimental evidence supports a role for KV10.1-mediated currents in facilitating cell-cycle progression: progression through the early G1 phase of the cell cycle is promoted by membrane hyperpolarization [13], and KV10.1 -mediated K+ efflux could contribute to this hyperpolarization, a model that is also confirmed by the anti-proliferative effects of KV10.1 channel blockers [10,12,14,15,16,17]. KV10.1 was also shown to be a cell-cycle regulated channel: KV10.1 currents are down-regulated at the G2-M transition, upon cell differentiation and also within cells arrested in G0/G1 [14,18,19]. It is likely that these events are regulated both at the level of channel activity and surface expression. In neurons, the surface-expression of endogenous KV10.1 is tightly controlled. No currents mediated by endogenous KV10.1 in neuronal tissue have been published to date in spite of the fact that in Drosophila, eag modulates K+ currents and synaptic function [20,21,22,23,24]. We recently identified a small KV10.1 surface population localizing preferentially to presynaptic membranes in rat hippocampal neurons, while large intracellular pools of KV10.1 can be readily detected in permeabilized cells [25,26]. KV10.1 channels activate at subthreshold potentials and show progressively slower activation kinetics at hyperpolarized prepulse potentials, a feature reminiscent of the Cole-Moore shift described on squid axon channels [27,28]. These properti (...truncated)


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Tobias Kohl, Eva Lörinczi, Luis A. Pardo, Walter Stühmer. Rapid Internalization of the Oncogenic K+ Channel KV10.1, PLOS ONE, 2011, Volume 6, Issue 10, DOI: 10.1371/journal.pone.0026329