An HSV vector system for selection of ligand-gated ion channel modulators.
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 (GlyRalpha1), 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.
Srinivasan
Rahul
R
Department of Molecular Genetics and Biochemistry, 200 Lothrop Street, Biomedical Science Tower, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Huang
Shaohua
S
Chaudhry
Suchita
S
Sculptoreanu
Adrian
A
Krisky
David
D
Cascio
Michael
M
Friedman
Peter A
PA
de Groat
William C
WC
Wolfe
Darren
D
Glorioso
Joseph C
JC
D000818:Animals; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics*; D008024:Ligands; D051381:Rats; D018009:Receptors, Glycine / Q000235:genetics*; D050916:TRPV Cation Channels / Q000235:genetics*; D014779:Virus Replication
D008024:Ligands; D018009:Receptors, Glycine; D050916:TRPV Cation Channels; C493922:Trpv1 protein, rat