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

The voltage-gated K channel Kv2.1 has been intimately linked with neuronal apoptosis. After ischemic, oxidative, or inflammatory insults, Kv2.

Disruption of K2.1 somato-dendritic clusters prevents the apoptogenic increase of potassium currents.

As the predominant mediator of the delayed rectifier current, K2.1 is an important regulator of neuronal excitability. K2.

Regulation of Pro-Apoptotic Phosphorylation of Kv2.1 K+ Channels.

Caspase activity during apoptosis is inhibited by physiological concentrations of intracellular K+. To enable apoptosis in injured cortical and hippocampal neurons, cellular loss of this cation is facilitated by the insertion of Kv2.1 K+ channels into the plasma membrane via a Zn2+/CaMKII/SNARE-dependent process.

Syntaxin-binding domain of Kv2.1 is essential for the expression of apoptotic K+ currents.

Intracellular signalling cascades triggered by oxidative injury can lead to upregulation of Kv2.1 K(+) channels at the plasma membrane of dying neurons. Membrane incorporation of new channels is necessary for enhanced K(+) efflux and a consequent reduction of intracellular K(+) that facilitates apoptosis.

Regulation of neuronal proapoptotic potassium currents by the hepatitis C virus nonstructural protein 5A.

Apoptosis-enabling neuronal potassium efflux is mediated by an enhancement of K+ currents. In cortical neurons, increased currents are triggered by dual phosphorylation of Kv2.1 by Src and p38 at channel residues Y124 and S800.

Regulation of apoptotic potassium currents by coordinated zinc-dependent signalling.

Oxidant-liberated intracellular Zn(2+) regulates neuronal apoptosis via an exocytotic membrane insertion of Kv2.1-encoded ion channels, resulting in an enhancement of voltage-gated K(+) currents and a loss of intracellular K(+) that is necessary for caspase-mediated proteolysis. In the present study we show that an N-terminal tyrosine of Kv2.

Protein kinase C regulation of neuronal zinc signaling mediates survival during preconditioning.

Sub-lethal activation of cell death processes initiate pro-survival signaling cascades. As intracellular Zn(2+) liberation mediates neuronal death pathways, we tested whether a sub-lethal increase in free Zn(2+) could also trigger neuroprotection. Neuronal free Zn(2+) transiently increased following preconditioning, and was both necessary and sufficient for conferring excitotoxic tolerance.

Assessment of cell viability in primary neuronal cultures.

This unit contains five protocols for assaying cell viability in vitro using primary neuronal cultures, including a novel method for use with transfected neurons. Three of the assays are based on the principle that cell death cascades alter membrane permeability. The lactate dehydrogenase (LDH) release assay measures the amount of the cytoplasmic enzyme released into the bathing medium, while the trypan blue and propidium iodide assays measure the ability of cells to exclude dye from their cytoplasm.

Microglia induce neurotoxicity via intraneuronal Zn(2+) release and a K(+) current surge.

Microglial cells are critical components of the injurious cascade in a large number of neurodegenerative diseases. However, the precise molecular mechanisms by which microglia mediate neuronal cell death have not been fully delineated. We report here that reactive species released from activated microglia induce the liberation of Zn(2+) from intracellular stores in cultured cortical neurons, with a subsequent enhancement in neuronal voltage-gated K(+) currents, two events that have been intimately linked to apoptosis.

Apoptotic surge of potassium currents is mediated by p38 phosphorylation of Kv2.1.

Kv2.1, the primary delayed rectifying potassium channel in neurons, is extensively regulated by phosphorylation. Previous reports have described Kv2.

Mediation of neuronal apoptosis by Kv2.1-encoded potassium channels.

Cellular K+ efflux is a requisite event in the unfolding of apoptosis programs across many types of cells and death-inducing stimuli; however, the molecular identities of the ion channels mediating this key event have remained undefined. Here, we show that Kv2.1-encoded K+ channels are responsible for the expression of apoptosis in cortical neurons in vitro.

Caspase 3 activation is essential for neuroprotection in preconditioning.

Sublethal insults can induce tolerance to subsequent stressors in neurons. As cell death activators such as ROS generation and decreased ATP can initiate tolerance, we tested whether other cellular elements normally associated with neuronal injury could add to this process. In an in vivo model of ischemic tolerance, we were surprised to observe widespread caspase 3 cleavage, without cell death, in preconditioned tissue.