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 ϩ 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 2ϩ influx, and calpain activation. This excitotoxic mechanism may be specific to ankyrin G-bound AIS proteins because Nav1.2 channels, but not AIS GABA A 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.

Sensory input drives rapid homeostatic scaling of the axon initial segment in mouse barrel cortex

“…On the other hand, rapid AIS plasticity as reported after significant elevation of neuronal activity in vitro seems to be driven by Ca 2+ signaling-related mechanisms and includes proteolysis in a calpain-dependent manner, which occurs on a much faster time scale of the disassembly of AIS proteins (Benned-Jensen et al, 2016;Evans et al, 2015;Evans et al, 2013;Schafer et al, 2009). Here, we found that rapid AIS plasticity occurs after a brief increase in activity during a behaviorally-relevant context in vivo, with AIS length reduction already 1 h after exposure to EE (Fig.…”

“…Our data so far suggest that AIS increases in length require long periods of deprivation (days to weeks). In contrast, studies applying depolarizing conditions in vitro showed that AIS length changes can be triggered more rapidly, within minutes to hours of increased in vitro network activity (Benned-Jensen et al, 2016;Evans et al, 2015). We next tested whether rapid AIS plasticity could be evoked in vivo in a behaviorally relevant context, by placing young adult wild type mice at P28 (n = 5-6 per group) to an enriched environment (EE) and expose whiskers to a larger range of novel stimuli.…”

“…Depending on the excitation state of the neuronal network both in vitro and in vivo, individual neurons can undergo structural and/or functional AIS plasticity, including modulation of channel density or composition, to adapt to or strengthen the changes in synaptic drive ((Bender et al, 2010;Benned-Jensen et al, 2016;Lezmy et al, 2017;Muir and Kittler, 2014); reviewed in (Jamann et al, 2018;Petersen et al, 2016)). For example, triggering chronic depolarization in neuronal networks in vitro (Evans et al, 2015;Grubb and Burrone, 2010) or using a complete and irrevocable elimination of sensory input in vivo (Kuba et al, 2010) led to significant geometrical and functional AIS changes.…”

Modulation of Kv7 channels and excitability in the brain

“…Under basal conditions, Kv7 channels have very low turnover rate throughout the axon [146]. However, surface expression of Kv7 channels is upregulated after neuronal stimulation [147].…”

Dynamic Gain Analysis Reveals Encoding Deficiencies in Cortical Neurons That Recover from Hypoxia-Induced Spreading Depolarizations

“…Indeed, a structurally intact AIS, with its specialized assembly of voltage-gated channels, cytoskeletal and associated proteins (Rasband, 2010) seems to be prerequisite to its normal function, as even relatively small plastic changes within the AIS (Grubb and Burrone, 2010;Kuba et al, 2010Kuba et al, , 2015Lezmy et al, 2017) elicit significant changes in neuronal excitability. Virtually all these proteins, including ␣and ␤-spectrins, ankyrins, Nav1.2, Nav1.6, and Kv7 channels, are either calpain substrates or subject to calpain-mediated endocytosis (Benned-Jensen et al, 2016). In the AIS, partial proteolysis and loss of anchoring of Na ϩ and K ϩ channels could render these channels nonfunctional and alter their distribution and biophysical properties (Iwata et al, 2004;von Reyn et al, 2012;Brocard et al, 2016).…”