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Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors.

ABSTRACT: Hepatitis C is a major worldwide disease and health hazard, affecting ?3% of the world population. The p7 protein of hepatitis C virus (HCV) is an intracellular ion channel and pH regulator that is involved in the viral replication cycle. It is targeted by various classical ion channel blockers. Here, we generated p7 constructs corresponding to HCV genotypes 1a, 2a, 3a, and 4a for recombinant expression in HEK293 cells, and studied p7 channels using patch-clamp recording techniques. The pH50 values for recombinant p7 channels were between 6.0 and 6.5, as expected for proton-activated channels, and current-voltage dependence did not show any differences between genotypes. Inhibition of p7-mediated currents by amantadine, however, exhibited significant, genotype-specific variation. The IC50 values of p7-1a and p7-4a were 0.7 ± 0.1 nM and 3.2 ± 1.2 nM, whereas p7-2a and p7-3a had 50- to 1000-fold lower sensitivity, with IC50 values of 2402 ± 334 nM and 344 ± 64 nM, respectively. The IC50 values for rimantadine were low across all genotypes, ranging from 0.7 ± 0.1 nM, 1.6 ± 0.6 nM, and 3.0 ± 0.8 nM for p7-1a, p7-3a, and p7-4a, respectively, to 24 ± 4 nM for p7-2a. Results from patch-clamp recordings agreed well with cellular assays of p7 activity, namely, measurements of intracellular pH and hemadsorption assays, which confirmed the much reduced amantadine sensitivity of genotypes 2a and 3a. Thus, our results establish patch-clamp studies of recombinant viroporins as a valid analytical tool that can provide quantitative information about viroporin channel properties, complementing established techniques.

SUBMITTER: Breitinger U

PROVIDER: S-EPMC4906147 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors.

Breitinger Ulrike U Farag Noha S NS Ali Nourhan K M NKM Breitinger Hans-Georg A HA

Biophysical journal 20160601 11

Hepatitis C is a major worldwide disease and health hazard, affecting ∼3% of the world population. The p7 protein of hepatitis C virus (HCV) is an intracellular ion channel and pH regulator that is involved in the viral replication cycle. It is targeted by various classical ion channel blockers. Here, we generated p7 constructs corresponding to HCV genotypes 1a, 2a, 3a, and 4a for recombinant expression in HEK293 cells, and studied p7 channels using patch-clamp recording techniques. The pH50 val ...[more]

PMID: 27276260

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Project description:Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca(2+) on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K(+) (E K), indicating K(+) selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at -80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na(+) and K(+) selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na(+) influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca(2+) revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca(2+) activated SV channels, vacuolar Ca(2+) inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca(2+) was also determined. These channels were active even without Ca(2+) (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca(2+) on the cytoplasmic side. Apart from monovalent cations (K(+) and Na(+)), SV channels were permeable to divalent cations (Ca(2+) and Mg(2+)). Both monovalent and divalent cations passed through the channels in the same direction-from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.

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Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors.

Publications

Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors.

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