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This Article Full Text Full Text (PDF) All Versions of this Article: 100/3/1420    most recent 90405.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Margas, W. Articles by Ruiz-Velasco, V. PubMed PubMed Citation Articles by Margas, W. Articles by Ruiz-Velasco, V.

Coupling Specificity of NOP Opioid Receptors to Pertussis-Toxin-Sensitive G Proteins in Adult Rat Stellate Ganglion Neurons Using Small Interference RNA

Department of Anesthesiology, Penn State University College of Medicine, Hershey, Pennsylvania

Submitted 26 March 2008; accepted in final form 15 June 2008

The opioid receptor-like 1 (NOP or ORL1) receptor is a G-protein-coupled receptor the endogenous ligand of which is the heptadecapeptide, nociceptin (Noc). NOP receptors are known to modulate pain processing at spinal, supraspinal, and peripheral levels. Previous work has demonstrated that NOP receptors inhibit N-type Ca²⁺ channel currents in rat sympathetic stellate ganglion (SG) neurons via pertussis toxin (PTX)-sensitive G_i/o subunits. However, the identification of the specific G subunit that mediates the Ca²⁺ current modulation is unknown. The purpose of the present study was to examine coupling specificity of Noc-activated NOP receptors to N-type Ca²⁺ channels in SG neurons. Small interference RNA (siRNA) transfection was employed to block the expression of PTX-sensitive G subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended G subunit. Evaluation of cell surface protein expression and Ca²⁺ channel modulation were assessed by immunofluorescence staining and electrophysiological recordings, respectively. Furthermore, the presence of mRNA of the intended siRNA target G protein was examined by RT-PCR experiments. Fluorescence imaging showed that G_i1, G_i3, and G_o were expressed in SG neurons. The transfection of G_i1-specific siRNA resulted in a significant decrease in Noc-mediated Ca²⁺ current inhibition, while silencing of either G_i3 or G_o was without effect. Taken together, these results suggest that in SG neurons G_i1 subunits selectively couple NOP receptors to N-type Ca²⁺ channels.