Targeting a Potassium Channel/Syntaxin Interaction Ameliorates Cell Death in Ischemic Stroke

Abstract

The voltage-gated K ϩ channel Kv2.1 has been intimately linked with neuronal apoptosis. After ischemic, oxidative, or inflammatory insults, Kv2.1 mediates a pronounced, delayed enhancement of K ϩ efflux, generating an optimal intracellular environment for caspase and nuclease activity, key components of programmed cell death. This apoptosis-enabling mechanism is initiated via Zn 2ϩ -dependent dual phosphorylation of Kv2.1, increasing the interaction between the channel's intracellular C-terminus domain and the SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein syntaxin 1A. Subsequently, an upregulation of de novo channel insertion into the plasma membrane leads to the critical enhancement of K ϩ efflux in damaged neurons. Here, we investigated whether a strategy designed to interfere with the cell death-facilitating properties of Kv2.1, specifically its interaction with syntaxin 1A, could lead to neuroprotection following ischemic injury in vivo. The minimal syntaxin 1A-binding sequence of Kv2.1 C terminus (C1aB) was first identified via a far-Western peptide screen and used to create a protherapeutic product by conjugating C1aB to a cell-penetrating domain. The resulting peptide (TAT-C1aB) suppressed enhanced whole-cell K ϩ currents produced by a mutated form of Kv2.1 mimicking apoptosis in a mammalian expression system, and protected cortical neurons from slow excitotoxic injury in vitro, without influencing NMDA-induced intracellular calcium responses. Importantly, intraperitoneal administration of TAT-C1aB in mice following transient middle cerebral artery occlusion significantly reduced ischemic stroke damage and improved neurological outcome. These results provide strong evidence that targeting the proapoptotic function of Kv2.1 is an effective and highly promising neuroprotective strategy.

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

“…1 A and B show both a representative snapshot and the conformational ensembles entailed by the C1aB K6W7 sequence motif for 4 different peptides. Two of the strongest syntaxin-binding peptides (24), pep-22 and pep-28, consistently show a fully solvent-exposed W7 residue with the preceding K6 in the opposite trans direction. In contrast, the 2 weakest syntaxinbinding peptides, pep-20 and pep-21, show increasing occlusion of W7 by interactions with the K6 side chain on deletion of either the K8W9 residues or just the W9 from the corresponding C1aB sequence.…”

“…Syntaxin binding to the C1a region of Kv2.1 is largely restricted to within a 9-aa sequence of the channel that we previously defined as C1aB (H 1 LSPNKWKW 9 ). We had observed a rapid dropoff in syntaxin binding when C-terminal C1aB residues were removed in a large avidity panel of Kv2.1 C1a-derived 15-mer peptides (24), alerting us to the possibility that the essential interactions of the syntaxin-binding domain could be further refined. We thus carried out molecular dynamics simulations of Kv2.1-derived peptides containing partial or complete C1aB sequences to analyze the stability of these peptides.…”

“…acute neuronal ischemic injury, limiting infarct damage and improving neurologic function in vivo (24).…”

Potassium channel dysfunction in neurons and astrocytes in Huntington's disease

“…The dysfunction of potassium channels can induce numerous CNS disorders, including HD . With a tremendous functional relevance in both physiological and pathological brain, potassium channels have become an important drug target in clinical therapies . Here we summarize recent progress on understanding the role of potassium channels in the pathology of HD obtained from various mouse models.…”

“…10,[14][15][16] With a tremendous functional relevance in both physiological and pathological brain, potassium channels have become an important drug target in clinical therapies. 12,[17][18][19][20] Here we summarize recent progress on understanding the role of potassium channels in the pathology of HD obtained from various mouse models. With a thorough examination of the dysfunction of potassium channels in HD, new insights for the development of therapeutic strategies for the treatment of this devastating neurodegenerative disease could be gained.…”