Structure-Activity Studies Reveal the Molecular Basis for GABAB-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1.

TL;DR: Findings suggest that selectively targeting GABABR-mediated HVA calcium channel inhibition by α-conotoxins could be effective for the treatment of chronic visceral pain.

Abstract: α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABAB receptors (GABABR). Therefore, it is unclear whether activation of GABABR or inhibition of α9α10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which α-conotoxins provide analgesia, we synthesized a suite of Vc1.1 analogues where all residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine (A), lysine (K), and aspartic acid (D). Our results show that the amino acids in the first loop play an important role in binding of the peptide to the receptor, whereas those in the second...

Summary

Introduction

• Structure-Activity Studies Reveal the Molecular Basis for GABA(B)-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1.
• Structure-activity studies reveal the molecular basis for GABAB-receptor mediated inhibition of high voltage-activated calcium channels by α-conotoxin Vc1.1.

Conus victoriae

• Testing for antagonist activity against a range of nAChR subtypes revealed that Vc1.1 was a selective inhibitor of the α9α10 nAChR subtype10, 11.
• Therefore, deliniating the precise molecular mechanisms responsible for their analgesic effects in vivo is important.
• To develop these conotoxins as potential analgesics it is important to identify the key peptide residues for the interaction with both the α9α10 nAChR and the GABABR.
• A comprehensive structure-activity study of Vc1.1 at the α9α10 nAChR, where an alanine, lysine and aspartic acid scan was performed, revealed that key regions in Vc1.1 for activity were residues Asp5-Asp7 and Asp11-Ile15.26.
• To further clarify the roles of the α9α10 nAChR and HVA calcium channel inhibition on the analgesic effects of this sub-family of α-conotoxins, the authors undertook a systematic study of the structure-activity relationships of Vc1.1 inhibition of HVA calcium channel currents.

Results and Discussion

• Activity of Vc1.1 analogues on HVA calcium channels current in DRG neurons: Representative traces of HVA calcium channel inhibition in mice DRG neurons by Vc1.1 analogues equipotent to Vc1.1 and less effective than Vc1.1 are shown in Figure S2A.
• Representative traces of IBa inhibition in mice DRG neurons by Vc1.1 analogues equipotent and less potent to Vc1.1 are shown in Figure S2B.
• Both peptides were characterised by NMR spectroscopy and Hα chemical shift analysis indicated that the structures of Vc1.1[D11A,E14A] and cVc1.1[D11A,E14A] were consistent with the native sequences (Figure S3, Table S1).
• Therefore, the authors sought to investigate the antinociceptive effects of cVc1.1[D11A,E14A], which is selective only for the GABABR in a mouse model of CVH.

Conclusion

• A subset of α-conotoxins, including Vc1.1 and RgIA, are thought to produce their pain relieving effects by inhibiting either nicotinic acetylcholine receptors or HVA calcium channels via GABABR activation in mammalian sensory neurons.
• By demonstrating that an α-conotoxin analogue selective for GABABR mediating HVA calcium channel inhibition is analgesic in a mouse model of chronic visceral pain, this study provides compelling evidence for the role of this analgesic mechanism.
• This finding opens novel perspectives in the pharmacological intervention of chronic pain management and the potential use of GABABR-targeting α-conotoxins as treatments for chronic pain conditions.

Full text

University of Wollongong
Research Online
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
Structure-Activity Studies Reveal the Molecular
Basis for GABA(B)-Receptor Mediated Inhibition
of High Voltage-Activated Calcium Channels by α-
Conotoxin Vc1.1
Mahsa Sadeghi
University of Wollongong;@/23567B=D32B/B
Bodil B. Carstens
University of Queensland
Brid P. Callaghan
University of Wollongong, University of Queensland
James T. Daniel
University of Queensland
Han Shen Tae
University of Wollongong6@A/3B=D32B/B
See next page for additional authors
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Structure-Activity Studies Reveal the Molecular Basis for GABA(B)-
Receptor Mediated Inhibition of High Voltage-Activated Calcium
Channels by α-Conotoxin Vc1.1
Abstract
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Structure-activity studies reveal the molecular basis for GABA
B
-receptor
mediated inhibition of high voltage-activated calcium channels by α
αα
α-conotoxin
Vc1.1.
Mahsa Sadeghi
1
, Bodil B. Carstens
2
, Brid P. Callaghan
3
, James T. Daniel
4
, Han-Shen
Tae
1
, Tracey O’Donnell
5,6
, Joel Castro
5,6
, Stuart M. Brierley
5,6
, David J. Adams
1
,
David J. Craik
2
and Richard J. Clark
4*
.
1
Illawarra Health and Medical Research Institute (IHMRI), University of
Wollongong, Wollongong, NSW 2522, Australia.
2
The University of Queensland, Institute for Molecular Bioscience, Brisbane, Qld
4072, Australia.
3
Department of Anatomy & Neuroscience, University of Melbourne, Parkville,
Victoria 3010, Australia
4
The University of Queensland, School of Biomedical Sciences, Brisbane, Qld 4072,
Australia.
5
Visceral Pain Research Group, Human Physiology, Centre for Neuroscience, College
of Medicine and Public Health, Flinders University, Bedford Park, South Australia,
5042, Australia.
6
Centre for Nutrition and Gastrointestinal Diseases, Discipline of Medicine,
University of Adelaide, South Australian Health and Medical Research Institute
(SAHMRI), North Terrace, Adelaide, Southern Australia 5000, Australia.
Address correspondence to: Dr. Richard J Clark, The University of Queensland,
School of Biomedical Sciences, Brisbane, QLD, 4072, Australia. Phone: +61 7 3365
1527. Fax: +61 7 3365 1766. Email: richard.clark@uq.edu.au
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Abbreviated title: Molecular basis of calcium channel inhibition by Vc1.1
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Abstract
α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are
characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-
conotoxins with distinct selectivity for nAChR subtypes have been identified as potent
analgesics in animal models of chronic pain. However, a number of α-conotoxins have
been shown to inhibit N-type
calcium channel currents in rodent dissociated dorsal root
ganglion (DRG) neurons via activation of G protein-coupled GABA
B
receptors
(GABA
B
R). Therefore it is unclear whether activation of GABA
B
R or inhibition of
α9α10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which α-
conotoxins provide analgesia, we synthesised a suite of Vc1.1 analogues where all
residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine
(A), lysine (K) and aspartic acid (D). Our results show that the amino acids in the first
loop play an important role in binding of the peptide to the receptor whereas those in the
second loop play an important role for the selectivity of the peptide for the GABA
B
R over
α9α10 nAChRs.
We designed a cVc1.1 analogue that is >8000-fold selective for
GABA
B
R-mediated inhibition of high voltage-activated (HVA) calcium channels over
α9α10 nAChRs and show that it is analgesic in a mouse model of chronic visceral
hypersensitivity (CVH). cVc1.1[D11A,E14A] caused dose-dependent inhibition of
colonic nociceptors with greater efficacy in ex vivo CVH colonic nociceptors relative
to healthy colonic nociceptors. These findings suggest that selectively targeting
GABA
B
R-mediated HVA calcium channel inhibition by α-conotoxins could be
effective for the treatment of chronic visceral pain.
Keywords: conotoxin, calcium channels, GPCRs, GABA
B
receptor, chronic pain,
analgesia
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Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Structure-activity studies reveal the molecular basis for gaba(b)-receptor mediated inhibition of high voltage-activated calcium channels by î±-conotoxin vc1.1" ?

To investigate the mechanisms by which α-conotoxins provide analgesia, the authors synthesized a suite of Vc1. The authors designed a cVc1. 1 analogue that is > 8000-fold selective for GABABR-mediated inhibition of high voltage-activated ( HVA ) calcium channels over α9α10 nAChRs and show that it is analgesic in a mouse model of chronic visceral hypersensitivity ( CVH ). This journal article is available at Research Online: https: //ro. uow. edu. au/ihmri/1263 These findings suggest that selectively targeting GABABR-mediated HVA calcium channel inhibition by α-conotoxins could be effective for the treatment of chronic visceral pain. 

Q2. What have the authors stated for future works in "Structure-activity studies reveal the molecular basis for gaba(b)-receptor mediated inhibition of high voltage-activated calcium channels by î±-conotoxin vc1.1" ?

1. 18 The importance of both loops for α9α10 nAChR inhibition by α-conotoxins is further supported by the structure-activity studies on RgIA, which have shown that mutations in both loops can have significant impact on potency. 27, 30 Therefore, the difference in the importance of the loop sequences on biological activity suggests that introducing a combination of mutations into loop 2 of Vc1. 21 These results suggest that GABABR-dependant HVA calcium channel inhibition underlies the analgesic effects of Vc1. Therefore, as predicted from the mutational Page 13 of 44 ACS Paragon Plus Environment ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 data, introduction of the two alanine mutations caused a significant reduction in potency, which was further reduced by introduction of the cyclic backbone. 

Q3. What is the effect of alanine substitution on HVA calcium channel currents in mice?

alanine substitution at other residues resulted in loss of inhibitory activity on HVA calcium channels in mice DRG neurons. 

Q4. What is the mechanism of analgesic action of RgIA analogues?

RgIA analogues selective for only α9α10 nAChR inhibition have been shown to be effective in animal models of chemotherapy-induced neuropathic pain. 

Q5. What is the effect of alanine substitution on the HVA calcium channel current?

In cells showing HVA calcium channel current (IBa) inhibition in response to α-conotoxin exposure, subsequent application of baclofen (50 µM) suppressed IBa further depending on the tested analogues. 

Q6. What is the effect of cVc1.1 on colonic nociceptors?

Whilst both cVc1.1 and cVc1.1[D11A,E14A] cause greater inhibition of colonic nociceptors in CVH states compared with healthy states, they evoke equivalent inhibition in CVH states, again implicating GABABR activation in this anti-nociceptive action. 

Q7. What is the role of the -conotoxins in analgesia?

Although theα9α10 nAChR subtype was originally proposed to be the primary analgesic target ofVc1.1 and RgIA, several observations have cast doubt on its importance for α-conotoxin analgesia instead supporting a more prominent role of HVA calcium channel inhibition via GABABR activation. 

Q8. What is the mechanism of inhibition of HVA calcium channel currents?

inhibiting HVA calcium channel currents either directly with selective antagonists or indirectly via GPCR modulation causes analgesia in animals and humans8. 

Q9. How many times did cVc1.1 inhibit 910 nACh?

Vc1.1[D11A,E14A] and cVc1.1[D11A, E14A] were 3,700-fold and 8,800-fold more selective for HVA calcium channel inhibition over inhibition of α9α10 nAChRs, respectively. 

Q10. What is the role of the peptides in the analgesic effects of V?

Given that Vc1.1 and RgIA have been shown to inhibit both α9α10 nAChRs and GABABR-mediated HVA calcium currents, the challenge has been to determine the contribution of these two mechanisms to the analgesic effects of these peptides. 

Q11. What is the effect of GABABR on the nociceptive action of c?

The authors have previously shown an increased expression of CaV2.2 within colon-innervating DRG neurons from CVH mice,21 which is likely to explain the enhanced anti-nociceptive actions of both cVc1.1 and cVc1.1[D11A,E14A] following GABABR activation. 

Q12. What is the role of the cyclised loops in the inhibition of HVA calcium channels?

these individual Vc1.1 mutations substantially reduced inhibition of α9α10 nAChRs26 and modelling of the interaction between Vc1.1 and α9α10 predicted that Asp11 and Glu14 form key salt bridge interactions with arginine residues on the α9α10 nAChR.27 Furthermore, during their previous development of an orally active, backbone cyclised analogue of Vc1.1, the authors observed a five-fold decrease and 12-fold increase in potency for α9α10 nAChR and HVA calcium channel inhibition, respectively, when Vc1.1 was cyclised.31 

Q13. What is the peptide that inhibits HVA calcium channel currents?

it was subsequently shown that Vc1.1 and the α9α10 nAChR-selective αconotoxin RgIA12 were both able to also potently inhibit HVA calcium channel currents in rat dorsal root ganglion (DRG) neurons and that this inhibition was dependent on GABABR activation 13-15. 

Q14. What is the effect of cVc1.1 on nociceptors?

cVc1.1[D11A,E14A] caused dose-dependent inhibition of ex vivo colonic nociceptors from healthy (Figure 5A) and CVH (Figure 5B) mice. 

Q15. What is the significance of the cVc1.1 inhibitor?

This substantial selectivity for HVA calcium channel inhibition suggests that cVc1.1[D11A, E14A] is an excellent molecule for investigating the role of this pathway in the analgesic action of Vc1.1. 

Q16. What is the recent study showing that a subset of -conotoxins?

a subset of α-conotoxins have been shown to inhibit high voltage-activated(HVA) calcium channel currents via activation of the GABAB receptor (GABABR) (Figure 1B) . 

Q17. What is the effect of alanine substitution on the HVA calcium channel current in mice?

Representative traces of HVA calcium channel inhibition in mice DRG neurons by Vc1.1 analogues equipotent to Vc1.1 and less effective than Vc1.1 are shown in Figure S2A. 

Q18. What is the effect of aspartic acid substitution on IBa inhibition in DRG neurons?

Although the estimated IC50 for these analogues (13.1, 6.8, 23.1 and 170.7 nM, respectively) are similar to that of Vc1.1 (2.5 nM), the maximum inhibition of IBa at 100 nM concentration of these peptides is greater than those with Vc1.1, highlighting that the efficacy of these peptides is greater on inhibiting IBa in DRG neurons.