Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes

Gomes, Fernanda Ramos; Romaniello, Vincenzo; Sánchez, Araceli; Weber, Claudia; Narayanan, Pratibha; Psol, Maryna; Pardo, Luis A.

doi:10.1074/jbc.m115.668749

Cited by 15 publications

(14 citation statements)

References 63 publications

(76 reference statements)

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“…Similarly, abolishing the ability of Kv10.1 to conduct ions through mutagenesis is not enough to completely inhibit its tumor-promoting activity [96]. Furthermore, a non-conducting Kv10.1 splice variant (E65, found in cancer cell lines), activates CDK1 when injected into Xenopus oocytes [97]. The depletion of Kv10.1 would, thus decrease both the full length and the spliced forms of the channel and prevent E65-mediated CDK1 activation.…”

Section: Cancers 2020 12 X For Peer Reviewmentioning

confidence: 99%

Kv10.1 Regulates Microtubule Dynamics during Mitosis

Movsisyan

Pardo

2020

Cancers

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Kv10.1 (potassium voltage-gated channel subfamily H member 1, known as EAG1 or Ether-à-go-go 1), is a voltage-gated potassium channel, prevailingly expressed in the central nervous system. The aberrant expression of Kv10.1 is detected in over 70% of all human tumor tissues and correlates with poorer prognosis. In peripheral tissues, Kv10.1 is expressed almost exclusively during the G2/M phase of the cell cycle and regulates its progression—downregulation of Kv10.1 extends the duration of the G2/M phase both in cancer and healthy cells. Here, using biochemical and imaging techniques, such as live-cell measurements of microtubule growth and of cytosolic calcium, we elucidate the mechanisms of Kv10.1-mediated regulation at the G2/M phase. We show that Kv10.1 has a dual effect on mitotic microtubule dynamics. Through the functional interaction with ORAI1 (calcium release-activated calcium channel protein 1), it modulates cytosolic calcium oscillations, thereby changing microtubule behavior. The inhibition of either Kv10.1 or ORAI1 stabilizes the microtubules. In contrast, the knockdown of Kv10.1 increases the dynamicity of mitotic microtubules, resulting in a stronger spindle assembly checkpoint, greater mitotic spindle angle, and a decrease in lagging chromosomes. Understanding of Kv10.1-mediated modulation of the microtubule architecture will help to comprehend how cancer tissue benefits from the presence of Kv10.1, and thereby increase the efficacy and safety of Kv10.1-directed therapeutic strategies.

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Section: Cancers 2020 12 X For Peer Reviewmentioning

confidence: 99%

Kv10.1 Regulates Microtubule Dynamics during Mitosis

Movsisyan

Pardo

2020

Cancers

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“…The C-terminal mutation should block all CaM-dependent regulation, since inhibition has been suggested to depend on CaM bridging the N-and C-termini (Whicher and MacKinnon 2016). Mutant and WT channels were expressed at comparable levels (Figure 1B), though there can be variability in molecular weight that is likely due to differences in glycosylation (see Fig 3B and Ramos Gomes et al 2015). GFP was co-expressed to identify transfected cells.…”

Section: Resultsmentioning

confidence: 99%

Regulation of Eag by ca²⁺/calmodulin controls presynaptic excitability inDrosophila

Bronk

Kuklin

Gorur-Shandilya

et al. 2017

Preprint

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Drosophila ether-à-go-go (eag) is the founding member of a large family of voltage-gated K+ channels, the KCNH family, which includes Kv10, 11 and 12, (Ganetzky et al. 1999). Concurrent binding of calcium/calmodulin (Ca2+/CaM) to N-and C-terminal sites inhibits mammalian EAG1 channels at sub-micromolar Ca2+ concentrations (Schonherr et al. 2000), likely by causing pore constriction (Whicher and MacKinnon 2016). Although the Drosophila EAG channel was believed to be Ca2+-insensitive (Schonherr et al. 2000), both the N-and C-terminal sites are conserved. Here we show that Drosophila EAG is inhibited by high Ca2+ concentrations that are only present at plasma membrane Ca2+ channel microdomains. To test the role of this regulation in vivo, we engineered mutations that block CaM-binding to the major C-terminal site of the endogenous eag locus, disrupting Ca2+-dependent inhibition. eag CaMBD mutants have reduced evoked release from larval motor neuron presynaptic terminals and show decreased Ca2+ influx in stimulated adult projection neuron presynaptic terminals, consistent with an increase in K+ conductance. These results are predicted by a conductance-based multi-compartment model of the presynaptic terminal in which some fraction of EAG is localized to the Ca2+ channel microdomains that control neurotransmitter release. The reduction of release in the larval neuromuscular junction drives a compensatory increase in motor neuron somatic excitability. This misregulation of synaptic and somatic excitability has consequences for systems-level processes and leads to defects in associative memory formation in adults.New and NoteworthyRegulation of excitability is critical to tuning the nervous system for complex behaviors. We demonstrate here that the EAG family of voltage-gated K+ channels exhibit conserved gating by Ca2+/CaM. Disruption of this inhibition in Drosophila results in decreased evoked neurotransmitter release due to truncated Ca2+ influx in presynaptic terminals. In adults, disrupted Ca2+ dynamics cripples memory formation. These data demonstrate that the biophysical details of channels have important implications for cell function and behavior.

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“…Here we present a systematic characterisation of the monoclonal antibody mAb62, targeting the K v 10.1 potassium channel in vivo in a nude mouse subcutaneous tumour model as a potential tool for cancer targeting. This particular antibody specifically recognises the full-length K v 10.1, but not its shorter splice variants, which do not display the corresponding epitope (Ramos Gomes et al 2015 ). By NIRF imaging we showed a selective binding of the NIR fluorescently labelled mAb62 to K v 10.1-expressing MDA-MB-435S tumours in vivo, with a peak at 2 days post injection, which could be blocked by an excess of unlabelled mAb62.…”

Section: Discussionmentioning

confidence: 99%

“…The monoclonal mouse antibody mAb62 (IgG κ2b) generated in our group (Gomez-Varela et al 2007 ; Ramos Gomes et al 2015 ) was labelled with Cy5.5 (Cy5.5 maleimide monoreactive dye; Amersham/GE Healthcare Life Sciences) using a standard protocol, resulting in mAb62-Cy5.5 (dye/protein ratio of ~0.7).…”

Section: Methodsmentioning

confidence: 99%

In vivo imaging of tumour xenografts with an antibody targeting the potassium channel Kv10.1

et al. 2016

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The Kv10.1 (Eag1) voltage-gated potassium channel represents a promising molecular target for novel cancer therapies or diagnostic purposes. Physiologically, it is only expressed in the brain, but it was found overexpressed in more than 70 % of tumours of diverse origin. Furthermore, as a plasma membrane protein, it is easily accessible to extracellular interventions. In this study we analysed the feasibility of the anti-Kv10.1 monoclonal antibody mAb62 to target tumour cells in vitro and in vivo and to deliver therapeutics to the tumour. Using time-domain near infrared fluorescence (NIRF) imaging in a subcutaneous MDA-MB-435S tumour model in nude mice, we showed that mAb62-Cy5.5 specifically accumulates at the tumour for at least 1 week in vivo with a maximum intensity at 48 h. Blocking experiments with an excess of unlabelled mAb62 and application of the free Cy5.5 fluorophore demonstrate specific binding to the tumour. Ex vivo NIRF imaging of whole tumours as well as NIRF imaging and microscopy of tumour slices confirmed the accumulation of the mAb62-Cy5.5 in tumours but not in brain tissue. Moreover, mAb62 was conjugated to the prodrug-activating enzyme β-D-galactosidase (β-gal; mAb62-β-gal). The β-gal activity of the mAb62-β-gal conjugate was analysed in vitro on Kv10.1-expressing MDA-MB-435S cells in comparison to control AsPC-1 cells. We show that the mAb62-β-gal conjugate possesses high β-gal activity when bound to Kv10.1-expressing MDA-MB-435S cells. Moreover, using the β-gal activatable NIRF probe DDAOG, we detected mAb62-β-gal activity in vivo over the tumour area. In summary, we could show that the anti-Kv10.1 antibody is a promising tool for the development of novel concepts of targeted cancer therapy.

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Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes

Cited by 15 publications

References 63 publications

Kv10.1 Regulates Microtubule Dynamics during Mitosis

Kv10.1 Regulates Microtubule Dynamics during Mitosis

Regulation of Eag by ca²⁺/calmodulin controls presynaptic excitability inDrosophila

In vivo imaging of tumour xenografts with an antibody targeting the potassium channel Kv10.1

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Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes

Cited by 15 publications

References 63 publications

Kv10.1 Regulates Microtubule Dynamics during Mitosis

Kv10.1 Regulates Microtubule Dynamics during Mitosis

Regulation of Eag by ca2+/calmodulin controls presynaptic excitability inDrosophila

In vivo imaging of tumour xenografts with an antibody targeting the potassium channel Kv10.1

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Regulation of Eag by ca²⁺/calmodulin controls presynaptic excitability inDrosophila