title: γ-Aminobutyric acid type B (GABAB) receptor expression is needed for inhibition of N-type (Cav2.2) calcium channels by analgesic α-conotoxins.
abstract: α-Conotoxins Vc1.1 and RgIA are small peptides isolated from the venom of marine cone snails. They have effective anti-nociceptive actions in rat models of neuropathic pain. Pharmacological studies in rodent dorsal root ganglion (DRG) show their analgesic effect is mediated by inhibition of N-type (Ca(v)2.2) calcium channels via a pathway involving γ-aminobutyric acid type B (GABA(B)) receptor. However, there is no direct demonstration that functional GABA(B) receptors are needed for inhibition of the Ca(v)2.2 channel by analgesic α-conotoxins. This study examined the effect of the GABA(B) agonist baclofen and α-conotoxins Vc1.1 and RgIA on calcium channel currents after transient knockdown of the GABA(B) receptor using RNA interference. Isolated rat DRG neurons were transfected with small interfering RNAs (siRNA) targeting GABA(B) subunits R1 and R2. Efficient knockdown of GABA(B) receptor expression at mRNA and protein levels was confirmed by quantitative real time PCR (qRT-PCR) and immunocytochemical analysis, respectively. Whole-cell patch clamp recordings conducted 2-4 days after transfection showed that inhibition of N-type calcium channels in response to baclofen, Vc1.1 and RgIA was significantly reduced in GABA(B) receptor knockdown DRG neurons. In contrast, neurons transfected with a scrambled nontargeting siRNA were indistinguishable from untransfected neurons. In the HEK 293 cell heterologous expression system, Vc1.1 and RgIA inhibition of Ca(v)2.2 channels needed functional expression of both human GABA(B) receptor subunits. Together, these results confirm that GABA(B) receptors must be activated for the modulation of N-type (Ca(v)2.2) calcium channels by analgesic α-conotoxins Vc1.1 and RgIA.
authors.affiliation: Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3083, Australia.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Cuny
authors.forename: Andrew
authors.initials: A
authors.lastname: de Faoite
authors.forename: Thuan G
authors.initials: TG
authors.lastname: Huynh
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yasuda
authors.forename: Géza
authors.initials: G
authors.lastname: Berecki
authors.forename: David J
authors.initials: DJ
authors.lastname: Adams
mesh_terms: D000818:Animals; D001418:Baclofen / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020916:Conotoxins / Q000494:pharmacology*; D058786:GABA-B Receptor Agonists / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D017208:Rats, Wistar; D018080:Receptors, GABA-B / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C500545:CACNA1B protein, human; D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D020916:Conotoxins; D058786:GABA-B Receptor Agonists; D021122:Protein Subunits; D018080:Receptors, GABA-B; C510079:conotoxin alpha-RgIA, Conus regius; C507383:alpha-conotoxin Vc1.1; D001418:Baclofen