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.

Highlights

  • A class of analgesic ␣-conotoxins potently inhibits N-type calcium channels

  • Knockdown of GABAB Receptor mRNA Expression in dorsal root ganglion (DRG) Neurons—To effectively knockdown the expression of functional GABAB receptors, we tested small interfering RNAs (siRNA) targeting either the R1 or R2 subunit in isolated DRG neurons. quantitative real time PCR (qRT-PCR) showed that following 24 – 48 h post-transfection, the mRNA levels of R1 in cells transfected with an R1-specific siRNA significantly decreased to 32.6 Ϯ 3.5% (n ϭ 4) of control cells transfected with a scrambled, nontargeting siRNA (p Ͻ 0.0001)

  • This study demonstrates that functional GABAB receptors are needed to observe the analgesic ␣-conotoxins Vc1.1 and RgIA decrease N-type voltage-gated calcium channels (VGCC) peak current amplitude

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Summary

Background

A class of analgesic ␣-conotoxins potently inhibits N-type calcium channels. Results: The activity of ␣-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. 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 ␣-conotoxins Vc1.1 and RgIA. GABAB Receptor Needed for ␣-Conotoxin Inhibition of Cav2.2 does not interact directly with voltage-gated calcium channels (VGCC) but instead via a voltage-independent G protein-coupled GABAB receptor-mediated mechanism [8, 15] These findings indicate that several different membrane receptors, including the GABAB receptor, may contribute to the pain-relieving activity of a class of ␣-conotoxins. We investigated Cav2.2 channel modulation by Vc1.1 in HEK 293 cells stably expressing Cav2.2 channels and transient transfection with both subunits of the human GABAB receptor These approaches showed that GABAB receptor expression is necessary for the observed inhibition of N-type VGCCs by the ␣-conotoxins Vc1.1 and RgIA. A preliminary report of some of these results has been presented in abstract form [41]

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