Analgesic conotoxins: block and G protein-coupled receptor modulation of N-type (Ca(V) 2.2) calcium channels

Abstract

Conotoxins (conopeptides) are small disulfide bonded peptides from the venom of marine cone snails. These peptides target a wide variety of membrane receptors, ion channels and transporters, and have enormous potential for a range of pharmaceutical applications. Structurally related ω-conotoxins bind directly to and selectively inhibit neuronal (N)-type voltage-gated calcium channels (VGCCs) of nociceptive primary afferent neurones. Among these, ω-conotoxin MVIIA (Prialt) is approved by the Food and Drug Administration (FDA) as an alternative intrathecal analgesic for the management of chronic intractable pain, particularly in patients refractory to opioids. A series of newly discovered ω-conotoxins from Conus catus, including CVID-F, are potent and selective antagonists of N-type VGCCs. In spinal cord slices, these peptides reversibly inhibit excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurones, and in the rat partial sciatic nerve ligation model of neuropathic pain, significantly reduce allodynic behaviour. Another family of conotoxins, the α-conotoxins, are competitive antagonists of mammalian nicotinic acetylcholine receptors (nAChRs). α-Conotoxins Vc1.1 and RgIA possess two disulfide bonds and are currently in development as a treatment for neuropathic pain. It was initially proposed that the primary target of these peptides is the α9α10 neuronal nAChR. Surprisingly, however, α-conotoxins Vc1.1, RgIA and PeIA more potently inhibit N-type VGCC currents via a GABA(B) GPCR mechanism in rat sensory neurones. This inhibition is largely voltage-independent and involves complex intracellular signalling. Understanding the molecular mechanisms of conotoxin action will lead to new ways to regulate VGCC block and modulation in normal and diseased states of the nervous system.

Figure 1

Block of recombinant N-type (Ca_v2.2) VGCCs by ω-conotoxins in Xenopus oocytes. (A) Three-dimensional structure of ω-conotoxin CVID highlighting the β-bridge and sheets (red arrows), the turns (blue arrows) and the location of disulfide bridges (ball-and-stick) [adapted from Lewis et al. (2000) ]. (B) Representative normalized Ba²⁺ current traces (I_Ba) obtained before and after (arrowhead) application of 100 nM ω-conotoxin CVIE, CVIF or CVIB. Inset: voltage protocol. (C) Concentration–response curves for the normalized peak I_Ba. (D) Recovery from block by ω-conotoxin CVIE (100 nM) is voltage-dependent. I_Ba traces were evoked by 0.1 Hz, 200 ms step depolarization to 0 mV from the indicated holding potential. (E) Recovery from CVIE block depends on the β subunit: channels with the ‘non-inactivating’β_2a auxiliary subunit show full recovery, whereas channels with β₃ exhibit weak recovery. Scale bars represent 1 µA and 100 ms. Bottom: reversibility of block after bath application of 100 nM ω-conotoxin CVIE, CVIF, CVIB or GVIA seen with α_1B-b/α₂δ1/β_2a or α_1B-b/α₂δ1/β₃ VGCCs. Data marked by ‘Ref’ are from Mould et al. (2004) and represent recovery from block by 1 nM GVIA. Data in B, C, D and E are modified with permission from Berecki et al. (2010).

Figure 2

α-Conotoxins inhibit N-type (Ca_V2.2) VGCCs via the GABA_B receptor. (A) Three-dimensional structure of α-conotoxin Vc1.1. Disulfide bonds are shown in ball-and-stick representation and the N and C termini are marked adapted from Clark et al. (2006). (B) Superimposed traces of depolarization-activated whole-cell I_Ba recorded using 2 mM Ba²⁺ as the charge carrier, elicited in the absence (a) and presence (b) of 100 nM Vc1.1. Inset: voltage protocol. (C) Concentration–response curve for inhibition of I_Ba in DRG neurones by Vc1.1; IC₅₀= 1.7 nM. B and C are adapted from Callaghan et al. (2008), with permission. (D) The GABA_B receptor mediates Vc1.1 inhibition of N-type (Ca_V2.2) VGCC currents in transiently transfected HEK-293 cells. Time course of peak I_Ba evoked with 0.1 Hz, 250 ms depolarizing test pulses from −80 to 10 mV and recorded using 20 mM Ba²⁺ as the charge carrier; upward deflections represent current inhibition. Baclofen (50 µM) or Vc1.1 (100 nM) does not affect I_Ba in cells co-transfected only with cDNAs of α_1-B, α₂δ₁ and β₁ VGCC subunits (n= 3). Inset: representative I_Ba traces are shown at the times indicated by lowercase letters. In cells co-transfected with (Ca_V2.2) VGCC subunits and GABA_B1,B2 subunits, Vc1.1 and baclofen (n= 4) (E) inhibit I_Ba. Scale bars in D and E represent 500 pA and 50 ms (D and E; G. Berecki and D.J. Adams, unpubl. obs.).

Figure 3

GABA_B receptor activation by analgesic α-conotoxins. The highly conserved structural scaffold of the α-conotoxins consists of two disulfide bonds that stabilize a central helical region. GABA_B receptor is a heterodimer with two almost identical subunits that are both required for a functional receptor. The GABA_B1 subunit is involved in ligand binding and the GABA_B2 subunit interacts with the G-protein. The natural ligand of the receptor, GABA, binds to a cleft within the large N-terminal ‘Venus fly-trap (VFT)’ domain of the GABA_B1 subunit, triggering a conformational change in the receptor that facilitates interaction with the G-protein by the GABA_B2 subunit. GPCR activation leads to dissociation of Gα from Gβγ subunits. A single GPCR can couple to either one or more families of Gα proteins, which activate several downstream effectors (Tedford and Zamponi, 2006). Upon ligand binding, Gβγ subunits function as a dimer to interact with many signalling molecules and with ion channels. The schematically shown pore-forming α_1-B subunit of the N-type (Ca_v2.2) VGCC consists of IV homologous domains linked by cytoplasmic loops (referred to as I-II, II-III and III-IV linkers) and cytoplasmic N- and C-terminal regions.