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γ-Aminobutyric Acid Type B (GABAB) Receptor Expression Is Needed for Inhibition of N-type (Cav2.2) Calcium Channels by Analgesic α-Conotoxins

2012; Elsevier BV; Volume: 287; Issue: 28 Linguagem: Inglês

10.1074/jbc.m112.342998

1083-351X

Hartmut Cuny, Andrew de Faoite, Thuan G. Huynh, Takahiro Yasuda, Géza Berecki, David J. Adams,

Pain Mechanisms and Treatments

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 (Cav2.2) calcium channels via a pathway involving γ-aminobutyric acid type B (GABAB) receptor. However, there is no direct demonstration that functional GABAB receptors are needed for inhibition of the Cav2.2 channel by analgesic α-conotoxins. This study examined the effect of the GABAB agonist baclofen and α-conotoxins Vc1.1 and RgIA on calcium channel currents after transient knockdown of the GABAB receptor using RNA interference. Isolated rat DRG neurons were transfected with small interfering RNAs (siRNA) targeting GABAB subunits R1 and R2. Efficient knockdown of GABAB 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 GABAB 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 Cav2.2 channels needed functional expression of both human GABAB receptor subunits. Together, these results confirm that GABAB receptors must be activated for the modulation of N-type (Cav2.2) calcium channels by analgesic α-conotoxins Vc1.1 and RgIA.Background: A class of analgesic α-conotoxins potently inhibits N-type calcium channels.Results: The activity of α-conotoxins Vc1.1 and RgIA was reduced following knockdown of GABAB receptor expression in sensory neurons and could be reconstituted in HEK 293 cells expressing human GABAB receptors and Cav2.2.Conclusion: GABAB receptors are needed for inhibition of Cav2.2 by Vc1.1 and RgIA.Significance: These analgesic α-conotoxins activate human GABAB receptors. 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 (Cav2.2) calcium channels via a pathway involving γ-aminobutyric acid type B (GABAB) receptor. However, there is no direct demonstration that functional GABAB receptors are needed for inhibition of the Cav2.2 channel by analgesic α-conotoxins. This study examined the effect of the GABAB agonist baclofen and α-conotoxins Vc1.1 and RgIA on calcium channel currents after transient knockdown of the GABAB receptor using RNA interference. Isolated rat DRG neurons were transfected with small interfering RNAs (siRNA) targeting GABAB subunits R1 and R2. Efficient knockdown of GABAB 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 GABAB 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 Cav2.2 channels needed functional expression of both human GABAB receptor subunits. Together, these results confirm that GABAB receptors must be activated for the modulation of N-type (Cav2.2) calcium channels by analgesic α-conotoxins Vc1.1 and RgIA. Background: A class of analgesic α-conotoxins potently inhibits N-type calcium channels. Results: The activity of α-conotoxins Vc1.1 and RgIA was reduced following knockdown of GABAB receptor expression in sensory neurons and could be reconstituted in HEK 293 cells expressing human GABAB receptors and Cav2.2. Conclusion: GABAB receptors are needed for inhibition of Cav2.2 by Vc1.1 and RgIA. Significance: These analgesic α-conotoxins activate human GABAB receptors.