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An HSV vector system for selection of ligand-gated ion channel modulators

Nature Methods volume 4, pages 733–739 (2007)Cite this article

Abstract

Pathological alterations of ion channel activity result from changes in modulatory mechanisms governing receptor biology. Here we describe a conditional herpes simplex virus (HSV) replication−based strategy to discover channel modulators whereby inhibition of agonist-induced channel activation by a vector-expressed modulatory gene product prevents ion flux, osmotic shock and cell death. Inhibition of channel activity, in this case, the rat vanilloid (Trpv1 or the glycine receptor (GlyRα1), can allow selection of escape vector plaques containing the 'captured' modulatory gene for subsequent identification and functional analysis. We validated this prediction using mixed infections of a wild-type Trpv1 expression vector vTTHR and a nonfunctional 'poreless' Trpv1 subunit−expressing vector, vHP, wherein vHP was highly selected from a large background of vTTHR viruses in the presence of the Trpv1 agonist, capsaicin. The approach should be useful for probing large libraries of vector-expressed cDNAs for the presence of ion channel modulators.

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Figure 1: HSV vector constructs and protein expression profiles.
Figure 2: Whole-cell patch-clamp recordings of vTT-expressed Trpv1 activation by capsaicin.
Figure 3: Inhibition of vTT replication by Trpv1 agonists.
Figure 4: Rescue of vTT replication by Trpv1 by known antagonists.
Figure 5: Construction and characterization of vectors vTTHR and vHP.
Figure 6: Selection for vHP in coinfection experiments.

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Acknowledgements

We thank D. Fink for useful discussions and D. Julius for providing Trpv1 cDNA. Human GLRA1 cDNA was obtained from M.C. NDT9515223 was a gift from Neurogen Corp. Supported by grants from the US National Institutes of Health (NIH) National Institute of Diabetes and Digestive and Kidney Diseases 2P01 DK04493512A1 (J.C.G.); P01 DK044935-11 (J.C.G.); NIH National Cancer Institute 1R01 CA119298-01 (J.C.G.); NIH National Institute of Neurological Disorders and Stroke 5R01 NS44323-04 (J.C.G.); NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases 5U54 AR050733-02 (J.C.G.); NIH National Heart, Lung and Blood Institute 2U01 HL066949-06 (J.C.G.), NIH R01 DK49430 (W.C.D.) and NIH R01 DK54171 (P.A.F.).

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Authors and Affiliations

  1. Department of Molecular Genetics and Biochemistry, 200 Lothrop Street, Biomedical Science Tower, University of Pittsburgh, Pittsburgh, 15261, Pennsylvania, USA

    Rahul Srinivasan, Shaohua Huang, Suchita Chaudhry, Michael Cascio, Darren Wolfe & Joseph C Glorioso

  2. Department of Pharmacology, 200 Lothrop Street, Biomedical Science Tower, University of Pittsburgh, Pittsburgh, 15261, Pennsylvania, USA

    Adrian Sculptoreanu, Peter A Friedman & William C de Groat

  3. Department of Pathology, 200 Lothrop Street, Biomedical Science Tower, University of Pittsburgh, Pittsburgh, 15261, Pennsylvania, USA

    David Krisky

Authors
  1. Rahul Srinivasan
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  2. Shaohua Huang
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  4. Adrian Sculptoreanu
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  7. Peter A Friedman
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  9. Darren Wolfe
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  10. Joseph C Glorioso
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Contributions

R.S. and J.C.G. wrote the manuscript. R.S. engineered the vTTHR and vHP vectors and either performed or was directly involved in all the experiments. S.H. and D.K. engineered vHG and vTT viral vectors. S.C. performed experiments for poreless Trpv1. A.S. performed electrophysiological recordings. M.C. provided the GLRA1 cDNA and advised R.S. on glycine selection experiments. P.A.F. performed calcium influx studies. W.C.D. advised R.S. and A.S.; J.C.G. and D.W. advised R.S.

Corresponding author

Correspondence to Joseph C Glorioso.

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The authors declare no competing financial interests.

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Supplementary Figures 1–5, Supplementary Methods (PDF 1416 kb)

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Srinivasan, R., Huang, S., Chaudhry, S. et al. An HSV vector system for selection of ligand-gated ion channel modulators. Nat Methods 4, 733–739 (2007). https://doi.org/10.1038/nmeth1077

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