title: Live Imaging of Kv7.2/7.3 Cell Surface Dynamics at the Axon Initial Segment: High Steady-State Stability and Calpain-Dependent Excitotoxic Downregulation Revealed.
abstract: The voltage-gated K(+) channels Kv7.2 and Kv7.3 are located at the axon initial segment (AIS) and exert strong control over action potential generation. Therefore, changes in their localization or cell surface numbers are likely to influence neuronal signaling. However, nothing is known about the cell surface dynamics of Kv7.2/7.3 at steady state or during short-term neuronal stimulation. This is primarily attributable to their membrane topology, which hampers extracellular epitope tagging. Here we circumvent this limitation by fusing an extra phluorin-tagged helix to the N terminus of human Kv7.3. This seven transmembrane chimera, named super ecliptic phluorin (SEP)-TAC-7.3, functions and traffics as a wild-type (WT) channel. We expressed SEP-TAC-7.3 in dissociated rat hippocampal neurons to examine the lateral mobility, surface numbers, and localization of AIS Kv7.2/7.3 heteromers using live imaging. We discovered that they are extraordinarily stable and exhibit a very low surface mobility both during steady state and neuronal stimulation. In the latter case, we also found that neither localization nor cell surface numbers were changed. However, at high glutamate loads, we observed a rapid irreversible endocytosis of Kv7.2/7.3, which required the activation of NR2B-containing NMDA receptors, Ca(2+) influx, and calpain activation. This excitotoxic mechanism may be specific to ankyrin G-bound AIS proteins because Nav1.2 channels, but not AIS GABAA receptors, were also endocytosed. In conclusion, we have, for the first time, characterized the cell surface dynamics of a full-length Kv7 channel using a novel chimeric strategy. This approach is likely also applicable to other Kv channels and thus of value for the additional characterization of this ion channel subfamily.
  
  
  CONCLUSIONS
  The voltage-gated K(+) channels Kv7.2 and Kv7.3 exert strong control over action potential generation, but little is known about their cell surface dynamics. Using a novel phluorin-based approach, we here show that these channels are highly stable at steady state and different types of neuronal stimulation. However, at high glutamate loads, they undergo a rapid calpain-dependent endocytosis that likely represents an early response during excitotoxic states.
authors.affiliation: Ionchannel Group, Department of Biomedical Sciences, Faculty of Health Sciences, and tau@sund.ku.dk.
authors.forename: Tau
authors.initials: T
authors.lastname: Benned-Jensen
authors.affiliation: Neuronal Signaling Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Rasmus Kordt
authors.initials: RK
authors.lastname: Christensen
authors.affiliation: Ionchannel Group, Department of Biomedical Sciences, Faculty of Health Sciences, and.
authors.forename: Federico
authors.initials: F
authors.lastname: Denti
authors.affiliation: Neuronal Signaling Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Perrier
authors.affiliation: Ionchannel Group, Department of Biomedical Sciences, Faculty of Health Sciences, and.
authors.forename: Hanne Borger
authors.initials: HB
authors.lastname: Rasmussen
authors.affiliation: Ionchannel Group, Department of Biomedical Sciences, Faculty of Health Sciences, and.
authors.forename: Søren-Peter
authors.initials: SP
authors.lastname: Olesen
mesh_terms: D000818:Animals; D017487:Ankyrins / Q000235:genetics; D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure; D020013:Calcium Signaling / Q000235:genetics; D002154:Calpain / Q000378:metabolism*; D002678:Chimera / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics*; D005260:Female; D006801:Humans; D051658:KCNQ2 Potassium Channel / Q000378:metabolism* / Q000648:ultrastructure; D051659:KCNQ3 Potassium Channel / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism* / Q000648:ultrastructure; D018408:Patch-Clamp Techniques; D011247:Pregnancy; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D011963:Receptors, GABA-A / Q000235:genetics; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics
chemical_list: C490614:Ank3 protein, rat; D017487:Ankyrins; D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; C114401:Kcnq2 protein, mouse; C496917:Kcnq3 protein, mouse; C121001:NR2B NMDA receptor; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D011963:Receptors, GABA-A; D016194:Receptors, N-Methyl-D-Aspartate; D002154:Calpain
keywords: FRAP; Kv7 channels; excitotoxicity; glutamate; live imaging; phluorin