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Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors.
- Source :
-
Biophysical journal [Biophys J] 2016 Jun 07; Vol. 110 (11), pp. 2419-2429. - Publication Year :
- 2016
-
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.<br /> (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Subjects :
- Amantadine pharmacology
Blotting, Western
Genotype
HEK293 Cells
Hemadsorption drug effects
Hemadsorption physiology
Humans
Hydrogen-Ion Concentration
Membrane Potentials drug effects
Membrane Potentials physiology
Recombinant Proteins genetics
Recombinant Proteins metabolism
Rimantadine pharmacology
Transfection
Antiviral Agents pharmacology
Hepacivirus drug effects
Hepacivirus genetics
Patch-Clamp Techniques
Viral Proteins antagonists & inhibitors
Viral Proteins genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1542-0086
- Volume :
- 110
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- Biophysical journal
- Publication Type :
- Academic Journal
- Accession number :
- 27276260
- Full Text :
- https://doi.org/10.1016/j.bpj.2016.04.018