Coupling specificity of NOP opioid receptors to pertussis-toxin-sensitive Galpha proteins in adult rat stellate ganglion neurons using small interference RNA.
Coupling specificity of NOP opioid receptors to pertussis-toxin-sensitive Galpha proteins in adult rat stellate ganglion neurons using small interference RNA.
复制标题
使用小干扰 RNA 将 NOP 阿片受体与成年大鼠星状神经节神经元中百日咳毒素敏感 Gα 蛋白的特异性偶联。
DOI：
10.1152/jn.90405.2008
复制
发表时间：
2008
影响因子：
2.5
通讯作者：
Ruiz-Velasco,Victor
中科院分区：
文献类型：
--
作者：
Margas,Wojciech;Sedeek,Khaled;Ruiz-Velasco,Victor
阿片受体样1（NOP或ORL 1）受体是一种G蛋白偶联受体，其内源性配体是十七肽，痛敏肽（Noc）。已知NOP受体调节脊髓、脊髓上和外周水平的疼痛处理。以前的研究表明，NOP受体通过百日咳毒素（PTX）敏感的Gαi/o亚基抑制大鼠交感星状神经节（SG）神经元N型钙通道电流。然而，介导Ca 2+电流调节的特异性Gα亚基的鉴定是未知的。本研究的目的是研究在SG神经元的N-型钙通道的耦合特异性的Noc激活的NOP受体。采用小干扰RNA（siRNA）转染技术阻断PTX敏感性Gα亚基的表达。RT-PCR结果显示siRNA特异性地降低了Gα亚基的表达。分别通过免疫荧光染色和电生理记录评估细胞表面蛋白表达和Ca 2+通道调节。此外，通过RT-PCR实验检测预期siRNA靶Gα蛋白的mRNA的存在。荧光成像显示SG神经元表达Gαi1、Gαi3和Gα。Gαi1特异性siRNA的转染导致Noc介导的Ca 2+电流抑制显著降低，而Gα i3或Gα o的沉默则没有影响。这些结果表明，在SG神经元中，Gα i1亚基选择性地将NOP受体与N型Ca ~（2+）通道偶联。
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/osubunits. 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 Gαowere 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 Gαi3or Gαowas without effect. Taken together, these results suggest that in SG neurons Gαi1subunits selectively couple NOP receptors to N-type Ca2+channels.