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

Abstract

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. It was recently shown that a K+ current surge is also present in hepatocytes undergoing apoptosis, and that the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) could inhibit Kv2.1-mediated currents and block cell death. Here, we show that NS5A1b (from HCV genotype 1b) expression in rat neurons depresses delayed rectifier potassium currents, limits the magnitude of the K+ current surge following exposure to activated microglia, and is neuroprotective. In a non-neuronal recombinant expression system, cells expressing Kv2.1 mutated at residue Y124, but not S800 mutants, are insensitive to NS5A1b-mediated current inhibition. Accordingly, NS5A1b coexpression prevents phosphorylation of wild-type Kv2.1 by Src at Y124, but is unable to inhibit p38 phosphorylation of the channel at S800. The actions of the viral protein are genotype-selective, as NS5A1a does not depress neuronal potassium currents nor inhibit Src phosphorylation of Kv2.1. Our results indicate that NS5A1b limits K+ currents following injury, leading to increased neuronal viability. NS5A1b may thus serve as a model for a new generation of neuroprotective agents.

Figure 1.

NS5A interferes with K⁺ channel function and is neuroprotective. A, Top, Whole-cell K⁺ currents in rat cortical neurons in culture, evoked by 10 mV incremental steps (−80 to +50 mV). Neurons were transfected with vector (VEC) or NS5A (genotype 1b) and exposed to vehicle or AMG. Scales are 1 nA and 5 ms. Bottom, Mean (± SEM; n = 7–20 cells per group) current densities (at +10 mV) from cells such as those shown above. ANOVA/Bonferroni, all p < 0.001; ***VEC versus NS5A, ^†VEC versus VEC/AMG, ^‡NS5A versus NS5A/AMG and VEC/AMG versus NS5A/AMG. B, Resting membrane voltage (Vm) and input resistance were measured under current clamp (R_in) and threshold synaptic conductance (Thresh-g_syn) was measured under dynamic clamp in untransfected (UT) and vector-expressing cells (VEC) or NS5A1b-transfected neurons (mean ± SEM; n = 5–8 cells per group). Scales are 20 mV and 500 ms. (C) Repetitive firing was recorded for VEC and NS5A1b transfected neurons using 1 s depolarizing current steps of increased amplitudes. Top, Traces are representative of neurons in each group at three depolarizing currents (n = 8). Bottom, Frequency-current relationships were fit to nonlinear regressions and compared using an F test. NS5A1b transfected neurons have a significantly higher repetitive firing frequency as a function of current when compared with VEC transfected neurons (p < 0.01). D, Rat cortical neurons expressing NS5A1b, but not empty vector (VEC), are protected from exposure to AMG. Shown are mean (±SD) luciferase values (counts per second, CPS) of a representative experiment, performed in quadruplicate; *p < 0.05, ANOVA/Dunnet. Inset, mean ± SEM of viability for a total of 7 independent experiments (each performed in quadruplicate), expressed as a percentage of control; **p < 0.01, paired t test.

Figure 2.

NS5A1b inhibits Src, but not p38, phosphorylation of Kv2.1. A, Top, Whole-cell currents (−80 to +30 mV step) in CHO cells expressing Kv2.1, Kv2.1(S800E), or Kv2.1 (Y124F) together with either VEC or NS5A1b (marked by a +). Scales are 5 nA and 5 ms. Bottom, Average K⁺ current suppression by NS5A1b coexpression, expressed as a percentage of the average currents present in vector-expressing cells; n = 9–19 cells per group (42 cells total); *p < 0.05, significantly different from the other two groups, ANOVA/Dunnet. B, CHO cells were cotransfected with Kv2.1(S800A), Kv2.1, p38, NS5A1b or vector. Kv2.1 immunoprecipitates were run by SDS-PAGE and probed with a phospho-specific antibody recognizing phosphorylated residue S800. Top, Representative immunoblot; Bottom, summary and quantification of results in three independent experiments, normalized to total Kv2.1 protein; **p < 0.01, ANOVA/Dunnet versus Kv2.1 control (third bar). C, CHO cells were cotransfected with Kv2.1, Src, NS5A1b or vector. Kv2.1 immunoprecipitates were run by SDS-PAGE and probed with a phosphotyrosine antibody. Top, Representative immunoblot; Bottom, summary and quantification of results in three independent experiments; **p < 0.01, ANOVA/Bonferroni versus Src/vector group (second vs third bar).

Figure 3.

Genotype 1a NS5A does not block K⁺ currents in neurons, or prevents SRC phosphorylation of Kv2.1. A, Whole-cell K⁺ currents in rat cortical neurons in culture, evoked by 10 mV incremental steps (−80 to +50 mV). Neurons were transfected with vector (VEC), NS5A1a, or NS5A1a(PA2) plasmids (Top). Scales are 3 nA and 25 ms. Bottom, Mean (±SEM; n = 14–16 cells per group; 44 total cells) current densities (at +10 mV) from neurons transfected with empty vector, NS5A (genotype 1a) or a mutant (PA2) of NS5A1a. B, Experiments similar to those shown in Figure 2C, illustrating the lack of inhibition of Src phosphorylation of Kv2.1 channels in CHO cells expressing NS5A1b (n = 3); **p < 0.01, ANOVA/Dunnet; all groups are significantly different from Kv2.1+vector group (first bar).