Disruption of K V 2.1 somato-dendritic clusters prevents the apoptogenic increase of potassium currents

Abstract

As the predominant mediator of the delayed rectifier current, KV2.1 is an important regulator of neuronal excitability. KV2.1, however, also plays a well-established role in apoptotic cell death. Apoptogenic stimuli induce syntaxin-dependent trafficking of KV2.1, resulting in an augmented delayed rectifier current that acts as a conduit for K+ efflux required for pro-apoptotic protease/nuclease activation. Recent evidence suggests that KV2.1 somato-dendritic clusters regulate the formation of endoplasmic reticulum–plasma membrane junctions that function as scaffolding sites for plasma membrane trafficking of ion channels, including KV2.1. However, it is unknown whether KV2.1 somato-dendritic clusters are required for apoptogenic trafficking of KV2.1. By overexpression of a protein derived from the C-terminus of the cognate channel KV2.2 (KV2.2CT), we induced calcineurin-independent disruption of KV2.1 somato-dendritic clusters in rat cortical neurons, without altering the electrophysiological properties of the channel. We observed that KV2.2CT-expressing neurons are less susceptible to oxidative stress-induced cell death. Critically, expression of KV2.2CT effectively blocked the increased current density of the delayed rectifier current associated with oxidative injury, supporting a vital role of KV2.1-somato-dendritic clusters in apoptogenic increases in KV2.1-mediated currents.

Defining the Kv2.1–syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant

“…S1F). This finding is consistent with the previously observed lack of declustering properties of Kv2.1 C1a overexpression in cortical neurons (36), strongly suggesting that inhibiting the Kv2.1-syntaxin interaction subdues the translocation of new Kv2.1 channels to the membrane.…”

“…Recently, Kv2.1 was found to associate with the endoplasmic reticulum proteins VAPA and VAPB to form distinct channel clusters at contact points between the plasma membrane and the endoplasmic reticulum (52,53). These clusters have been shown to be necessary for trafficking of the death-promoting population of Kv2.1 (36), suggesting that the interaction between Kv2.1 and the VAP proteins may be involved in cell death processes. With the discovery of cpd5 as a first-in-class neuroprotective agent, we demonstrate a translational workflow that uses structural modeling to guide the resolution of protein-protein interactions involving poorly characterized disordered domains, leading to both mechanistic insights and identification of an effective drug.…”

“…Mechanistically, dispersal of somatodendritic Kv2.1 clusters has also been identified as a viable approach for preventing the increase of death-inducing K + currents (36). To rule out the possibility that cpd5 is providing neuroprotection by declustering Kv2.1, we transfected neurons with Kv2.1-eGFP and evaluated their clustering status after 24 h exactly as described previously (36). We found no significant effect of 24 h of 10 μM cpd5 treatment on Kv2.1 clusters (SI Appendix, Fig.…”

Molecular Neuroprotection Induced by Zinc-Dependent Expression of Hepatitis C–Derived Protein NS5A Targeting Kv2.1 Potassium Channels

“…Electrophysiological Recordings. Whole-cell voltage-clamp currents from rat cortical neurons were measured with an Axopatch 200B amplifier and pClamp software (Molecular Devices, San Jose, CA) using 3-to 5-MV electrodes and a 10-kHz low-pass filter (Justice et al, 2017). The extracellular solution contained 2 mM MgCl 2 , 2.5 mM KCl, 115 mM NaCl, 10 mM HEPES, 10 mM D-glucose, 1 mM CaCl 2 , and 0.25 mM tetrodotoxin, pH 7.2.…”

Targeted disruption of Kv2.1-VAPA association provides neuroprotection against ischemic stroke in mice by declustering Kv2.1 channels

“…Two separate membrane populations of Kv2.1 exist in neurons: (i) freely dispersed conducting channels, which mediate canonical delayed rectifier potassium currents that regulate neuronal excitability (4,5,12), and (ii) electrically silent, micrometer-sized somatodendritic channel clusters (12). This second, much larger population of clustered Kv2.1 channels forms endoplasmic reticulum-PM (ER-PM) junctions that function as scaffolding sites for ion channel trafficking to the membrane (13), facilitating surface delivery of new proapoptotic Kv2.1 channels (14,15). Critically, overexpression of the C terminus of the cognate channel Kv2.2 (Kv2.2 CT) can disrupt Kv2.1 clusters without altering the biophysical properties of existing active channels (14,16) and, importantly, block proapoptotic potassium channel current enhancement, resulting in neuroprotection following oxidative insult (14).…”