Critical role of Casein kinase 2 in hepatitis C NS5A-mediated inhibition of Kv2.1 K(+) channel function.

Inhibiting injury-induced increases in outward K(+) currents is sufficient to block cell death in cortical neuronal injury models. It is now known that apoptosis is facilitated in hepatocytes by the same K(+) channel as in cortical neurons, namely, the delayed rectifier K(+) channel Kv2.1. The hepatitis C virus (HCV) protein NS5A prevents the apoptosis-enabling loss of intracellular potassium by inhibiting Kv2.1 function and thus blocking hepatocyte cell death. Critically, neurons expressing NS5A1b (from HCV genotype 1b), but not NS5A1a, can be protected from lethal injurious stimuli via a block of Kv2.1-mediated potassium currents. Here, we identify a key component unique to NS5A1b, which is necessary for restricting Kv2.1 currents and establishing neuroprotection. By comparing the sequence differences between NS5A1b and 1a we identify putative casein kinase 2 (CK2) phosphorylation regions unique to the 1b genotype. We show that selective inhibition of CK2 in cortical neurons results in loss of NS5A1b's ability to depress outward potassium currents, and, surprisingly, potentiates currents in non-NS5A-expressing cells. As such, our results suggest that NS5A1b-mediated inhibition of Kv2.1 function is critically dependent on its phosphorylation status at genotypic-specific CK2-directed residues. Importantly, inhibiting NS5A viral replicative function with the novel HCV drug Ledipasvir does not impair the ability of this protein to block Kv2.1 function. This suggests that the modulation of NS5A function by CK2 may be a component of HCV unique to the regulation of apoptosis.

Cyclin e1 regulates Kv2.1 channel phosphorylation and localization in neuronal ischemia.

Kv2.1 is a major delayed rectifying K(+) channel normally localized to highly phosphorylated somatodendritic clusters in neurons. Excitatory stimuli induce calcineurin-dependent dephosphorylation and dispersal of Kv2.1 clusters, with a concomitant hyperpolarizing shift in the channel's activation kinetics. We showed previously that sublethal ischemia, which renders neurons transiently resistant to excitotoxic cell death, can also induce Zn(2+)-dependent changes in Kv2.1 localization and activation kinetics, suggesting that activity-dependent modifications of Kv2.1 may contribute to cellular adaptive responses to injury. Recently, cyclin-dependent kinase 5 (Cdk5) was shown to phosphorylate Kv2.1, with pharmacological Cdk5 inhibition being sufficient to decluster channels. In another study, cyclin E1 was found to restrict neuronal Cdk5 kinase activity. We show here that cyclin E1 regulates Kv2.1 cellular localization via inhibition of Cdk5 activity. Expression of cyclin E1 in human embryonic kidney cells prevents Cdk5-mediated phosphorylation of Kv2.1, and cyclin E1 overexpression in rat cortical neurons triggers dispersal of Kv2.1 channel clusters. Sublethal ischemia in neurons induces calcineurin-dependent upregulation of cyclin E1 protein expression and cyclin E1-dependent Kv2.1 channel declustering. Importantly, overexpression of cyclin E1 in neurons is sufficient to reduce excitotoxic cell death. These results support a novel role for neuronal cyclin E1 in regulating the phosphorylation status and localization of Kv2.1 channels, a likely component of signaling cascades leading to ischemic preconditioning.