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

Abstract

The neuronal cell death-promoting loss of cytoplasmic K+ following injury is mediated by an increase in Kv2.1 potassium channels in the plasma membrane. This phenomenon relies on Kv2.1 binding to syntaxin 1A via 9 amino acids within the channel intrinsically disordered C terminus. Preventing this interaction with a cell and blood-brain barrier-permeant peptide is neuroprotective in an in vivo stroke model. Here a rational approach was applied to define the key molecular interactions between syntaxin and Kv2.1, some of which are shared with mammalian uncoordinated-18 (munc18). Armed with this information, we found a small molecule Kv2.1–syntaxin-binding inhibitor (cpd5) that improves cortical neuron survival by suppressing SNARE-dependent enhancement of Kv2.1-mediated currents following excitotoxic injury. We validated that cpd5 selectively displaces Kv2.1–syntaxin-binding peptides from syntaxin and, at higher concentrations, munc18, but without affecting either synaptic or neuronal intrinsic properties in brain tissue slices at neuroprotective concentrations. Collectively, our findings provide insight into the role of syntaxin in neuronal cell death and validate an important target for neuroprotection.

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

“…Previous preclinical studies from our laboratory have shown efficacious neuroprotection in vivo by targeting the Kv2.1-syntaxin interaction ( fig. S7B), which is the final step in proapoptotic Kv2.1 membrane insertion (9,11). The results in the present study target this process via a completely novel approach, namely, the elimination of the putative insertion sites for cell death trafficking of channels, the Kv2.1 membrane cluster domains.…”

“…TBOA is a glutamate transporter inhibitor that produces a slow excitotoxic injury via prolonged N-methyl-d-aspartate (NMDA) receptor stimulation (26). This induces a pronounced potassium current surge mediated by Kv2.1, culminating in delayed apoptotic cell death, mimicking the excitotoxic conditions present in the ischemic penumbra following stroke (9,11,14,26).…”

The Redox Biology of Excitotoxic Processes: The NMDA Receptor, TOPA Quinone, and the Oxidative Liberation of Intracellular Zinc

“…As glutamate can also generate the production of reactive oxygen species via NMDA receptor activation (Reynolds and Hastings, 1995), by simultaneously measuring calcium and zinc transients in neurons (Figure 10), the Reynolds laboratory provided unequivocal evidence that NMDA receptor stimulation leads to a calcium-dependent production of free radicals, both cytoplasmic and mitochondrial in origin, which, in turn, led to the liberation of intracellular zinc (Dineley et al, 2008). This finding, and subsequent work by us and other groups (Granzotto and Sensi, 2015;Yeh et al, 2017Yeh et al, , 2019, provided a critical link between excitotoxic processes and intracellular liberated zincdependent cell injurious signaling cascades. Zinc translocation from presynaptic, zinc containing terminals to postsynaptic cells via calcium-permeable channels (Sensi et al, 1999a,b) can, under FIGURE 10 | Simultaneous detection of glutamate-induced intracellular calcium and zinc in neurons.…”

“…This has been, admittedly, a very difficult problem, to which large amounts of financial resources and numerous careers have been devoted. Our own recent work points to new therapeutic avenues to pursue, targeting Kv2.1-mediated potassium efflux in cell death processes ( Yeh et al, 2017 , 2019 ; Schulien et al, 2020 ). Although ameliorating excitotoxic injury and clinical disability has been very challenging, a better appreciation of critical mechanisms can only improve the potential to succeed in addressing this important unmet medical need.…”

“…Additional studies revealed that the apoptotic potassium current surge results from a zinc-activated phosphorylation of Kv2.1 by both Src and p38, which in turn, induces a syntaxin-dependent de novo insertion of large number of Kv2.1 channels into the plasma membrane ( Pal et al, 2006 ; Redman et al, 2007 , 2009a ; McCord et al, 2014 ). The characterization of this cell death-enabling pathway led to the generation of a new generation of neuroprotective compounds for the treatment of neurodegenerative disorders in which excitotoxicity has been proposed to play a prominent role, including cerebral ischemia-reperfusion injury ( Yeh et al, 2017 , 2019 ). The efficacy of these agents suggests either that the zinc mobilizing pathway that we have delineated and the excitotoxic pathway are both independently active in ischemic injury, or that these two pathways are intimately linked.…”