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

摘要

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 Gαi/o subunits. However, the identification of the specific Gα 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 Gα subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended Gα 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 Gα protein was examined by RT-PCR experiments. Fluorescence imaging showed that Gαi1, Gαi3, and Gαo were expressed in SG neurons. The transfection of Gαi1-specific siRNA resulted in a significant decrease in Noc-mediated Ca²⁺ current inhibition, while silencing of either Gαi3 or Gαo was without effect. Taken together, these results suggest that in SG neurons Gαi1 subunits selectively couple NOP receptors to N-type Ca²⁺ channels.