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. Transient expression of two different dominant-negative forms of this subunit in neurons completely eliminated the enhancement of K+ currents that normally accompanies the cell death process. Importantly, neurons deficient in functional Kv2.1-encoded K+ channels were protected from oxidant and staurosporine-induced apoptosis. Finally, Chinese hamster ovary cells, which do not express endogenous voltage-gated K+ channels, became substantially more sensitive to apoptosis after transient expression of wild-type Kv2.1. These results suggest that Kv2.1-encoded K+ channels are necessary for the apoptotic signaling cascade in mammalian cortical neurons in culture and are sufficient for increasing the susceptibility to apoptogens in a nonexcitable cell.

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. To dissect the preconditioning pathways activating caspases, and the mechanisms by which these proteases are held in check, we developed an in vitro model of excitotoxic tolerance. In this model, antioxidants and caspase inhibitors blocked ischemia-induced protection against N-methyl-D-aspartate toxicity. Moreover, agents that blocked preconditioning also attenuated induction of HSP 70; transient overexpression of a constitutive form of this protein prevented HSP 70 upregulation and blocked tolerance. We outline a neuroprotective pathway where events normally associated with apoptotic cell death are critical for cell survival. P rior exposure to sublethal challenges can render neuronal tissue less vulnerable to severe insults (1). Preconditioning models share several key features, including limited window of efficacy, requirement for protein synthesis, involvement of ATP sensitive K ϩ (K ATP ) channels, and heat-shock protein (HSP) induction (2, 3). However, the underlying mechanisms mediating neuroprotection remain undefined. The up-regulation of prosurvival elements within preconditioned cells seems to depend upon activation of pathways typically associated with degeneration. For example, generation of reactive oxygen species (ROS) is critical for induction of tolerance in cardomyocytes (4, 5) and neurons (6-9). Metabolic dysfunction also contributes to preconditioning, as decline in ATP͞ADP ratios leads to mitochondrial K ATP channel opening (8) and ROS production (10). In fact, neuronal preconditioning is attenuated with K ATP antagonists (2), and K ATP activators are neuroprotective (11).Although ROS and energetic dysfunction contribute to preconditioning, little is known about how far these pathways progress before being halted, or the mechanism by which they are blocked. Here, we investigated the extent of activation of cell death pathways during ischemic preconditioning (IP) in vivo as well as the mechanism by which activation of these pathways results in tolerance in vitro. We propose a new model of IP in which neuroprotection depends upon activation of factors typically associated with neurodegeneration. Materials and MethodsFocal IP, Immunohistochemistry, and Immunoblotting. Transient MCAO was performed on spontaneously hypertensive rats (12). Immunohistochemistry for activated caspase 3 was performed as described (12). At various times after preconditioning, tissue was harvested, and proteins were run by SDS͞PAGE (13). For details, see Supporting Materials and Methods, which is published as supporting information on the PNAS web site, www.pnas.org.Tissue Culture and in Vitro Precondit...

Kv2.1, the primary delayed rectifying potassium channel in neurons, is extensively regulated by phosphorylation. Previous reports have described Kv2.1 phosphorylation events affecting channel gating and the impact of this process on cellular excitability. Kv2.1, however, also provides the critical exit route for potassium ions during neuronal apoptosis via p38 MAPK-dependent membrane insertion, resulting in a pronounced enhancement of K ؉ currents. Here, electrophysiological and viability studies using Kv2.1 channel mutants identify a p38 phosphorylation site at Ser-800 (S800) that is required for Kv2.1 membrane insertion, K ؉ current surge, and cell death. In addition, a phospho-specific antibody for S800 detects a p38-dependent increase in Kv2.1 phosphorylation in apoptotic neurons and reveals phosphorylation of S800 in immunopurified channels incubated with active p38. Consequently, phosphorylation of Kv2.1 residue S800 by p38 leads to trafficking and membrane insertion during apoptosis, and remarkably, the absence of S800 phosphorylation is sufficient to prevent completion of the cell death program.apoptosis ͉ ion channel ͉ MAPK

Abstract:The membrane-permeant oxidizing agent 2,2Ј-dithiodipyridine (DTDP) can induce Zn 2ϩ release from metalloproteins in cell-free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neurons, detected by the presence of both DNA laddering and asymmetric chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad-spectrum cysteine protease inhibitor butoxy-carbonylaspartate-fluoromethylketone. N,N,NЈ,NЈ-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and other cell-permeant metal chelators also effectively blocked DTDP-induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK-801, by the intracellular calcium release antagonist dantrolene, or by high concentrations of ryanodine. DTDP generated increases in fluorescence signals in cultured neurons loaded with the zinc-selective dye Newport Green. The fluorescence signals following DTDP treatment also increased in fura-2-and magfura-2-loaded neurons. These responses were completely reversed by TPEN, consistent with a DTDP-mediated increase in intracellular free Zn 2ϩ concentrations. Our studies suggest that under conditions of oxidative stress, Zn 2ϩ released from intracellular stores may contribute to the initiation of neuronal apoptosis.