Molecular determinants conferring the stoichiometric-dependent activity of α-conotoxins at the human α9α10 nicotinic acetylcholine receptor subtype

TLDR: The molecular determinants of α-conotoxins Vc1.1, RgIA#, and PeIA inhibition at hypothetical stoichiometries of the human α9α10 nAChR are investigated to suggest that only Vc 1.1 exhibits stoichiometric-dependent inhibition at the α9 α10 n AChR.

Abstract: α9α10 nicotinic acetylcholine receptors (nAChRs) putatively exist at different stoichiometries. We systematically investigated the molecular determinants of α-conotoxins Vc1.1, RgIA#, and PeIA inhibition at hypothetical stoichiometries of the human α9α10 nAChR. Our results suggest that only Vc1.1 exhibits stoichiometric-dependent inhibition at the α9α10 nAChR. The hydrogen bond between N154 of α9 and D11 of Vc1.1 at the α9(+)-α9(−) interface is responsible for the stoichiometric-dependent potency of Vc1.1.

Figures

Figure 4. Activity of the α-conotoxins at human α9α10 and α9[N154G]α10 nAChRs expressed from varied α9:α10 mRNA ratios. Bar graphs of relative ACh-evoked current amplitude mediated by hα9α10 and hα9[N154G]α10 nAChRs in the presence of (A) 1 μM Vc1.1, (B) 300 nM RgIA#, and (C) 30 nM PeIA. Whole-cell hα9α10 nAChR-mediated currents at 1:3 and 3:1 ratios were activated by 50 μM and 30 μM ACh, respectively, and hα9[N154G]α10 nAChR-mediated currents at 1:3 and 3:1 ratios were activated by 20 μM and 30 μM ACh, respectively (close to their respective EC50 values). ** p < 0.0001

Figure 2: Sequence alignment and structures of the α-conotoxins. (A) Sequence alignment of the α-conotoxin Vc1.1, PeIA, RgIA# and their analogues. There are two disulphide bonds (black lines) linking CI and CIII, and CII and CIV, respectively. The DPR motif is highlighted using the grey background. Vc1.1 and PeIA possess a 4/7 disulphide framework, and RgIA has a 4/3 disulphide framework. * represents the amide group of the C-termini. (B) and (D) NMR structures of Vc1.1 (PDB code: 2H8S) and RgIA (PDB code: 2JUT), and (C) crystal structure of PeIA (PDB code: 5JME).

Table 1. Inhibition of h910 and h9[N154]10 expressed from α9/α9[N154G]:α10 mRNA injection ratios of 1:3 and 3:1 by Vc1.1 and analogues.

Full text

University of Wollongong
Research Online
33)=)88)-)3:0)5,-,1+)3"-9-)8+059:1:;:- )+;3:?6.#+1-5+--,1+15-)5,-)3:0

Molecular Determinants Conferring the
Stoichiometric-Dependent Activity of α-
Conotoxins at the Human α9α10 Nicotinic
Acetylcholine Receptor Subtype
Rilei Yu
Qingdao National Laboratory for Marine Science and Technology, Ocean University of China
Han-Shen Tae
University of Wollongong
Nargis Tabassum
Ocean University of China, Qingdao National Laboratory for Marine Science and Technology
Juan Shi
Ocean University of China, Qingdao National Laboratory for Marine Science and Technology
Tao Jiang
Qingdao National Laboratory for Marine Science and Technology, Ocean University of China
See next page for additional authors
"-9-)8+0 5315-19:0-67-5)++-99159:1:;:165)38-7691:68?.68:0-%51<-891:?6.'63365/65/68.;8:0-815.684):165+65:)+::0-% '1*8)8?
8-9-)8+07;*9;6=-,;);
!;*31+):165-:)139
(;"$)-$)*)99;4#011)5/$,)4963-+;3)8-:-8415)5:965.-8815/:0-#:61+0164-:81+
-7-5,-5:+:1<1:?6.@656:6>159)::0-;4)5@@1+6:151++-:?3+06315-"-+-7:68#;*:?7-6;85)36.-,1+15)30-419:8?


Molecular Determinants Conferring the Stoichiometric-Dependent
Activity of α-Conotoxins at the Human α9α10 Nicotinic Acetylcholine
Receptor Subtype
Abstract
@@51+6:151+)+-:?3+06315-8-+-7:68950"97;:):1<-3?->19:):,1B-8-5:9:61+0164-:81-9'-
9?9:-4):1+)33?15<-9:1/):-,:0-463-+;3)8,-:-8415)5:96.@+656:6>159&+"/)5,!-1501*1:165):
0?76:0-:1+)39:61+0164-:81-96.:0-0;4)5@@50" ;88-9;3:99;//-9::0):653?&+->01*1:9
9:61+0164-:81+,-7-5,-5:1501*1:165)::0-@@50"C-0?,86/-5*65,*-:=--56.@)5,
6.&+)::0-@@A15:-8.)+-198-976591*3-.68:0-9:61+0164-:81+,-7-5,-5:76:-5+?6.&+
Disciplines
-,1+15-)5,-)3:0#+1-5+-9
Publication Details
(;"$)-$)*)99;4#011)5/$,)4963-+;3)8-:-8415)5:965.-8815/:0-
#:61+0164-:81+-7-5,-5:+:1<1:?6.@656:6>159)::0-;4)5@@1+6:151++-:?3+06315-"-+-7:68
#;*:?7-6;85)36.-,1+15)30-419:8?
Authors
"13-1(;)5#0-5$)-)8/19$)*)99;4;)5#01$)61)5/)5,)<1,,)49
C1926;85)3)8:1+3-19)<)13)*3-):"-9-)8+0 5315- 0D7986;6=-,;);10481

1
Molecular determinants conferring the stoichiometric-dependent activity of
α-conotoxins at the human α9α10 nAChR subtype
Rilei Yu
1,3†*
, Han-Shen Tae
2†
, Nargis Tabassum
1,3
, Juan Shi
1,3
, Tao Jiang
1,3
and David J. Adams
2*
1
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and
Pharmacy, Ocean University of China, Qingdao 266003, China
2
Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong,
New South Wales 2522, Australia
3
Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine
Science and Technology, Qingdao 266003, China
†
These authors contribute equally to this work.
*
Corresponding authors: djadams@uow.edu.au or ryu@ouc.edu.cn

2
ABSTRACT
α9α10 Nicotinic acetylcholine receptors (nAChRs) putatively exist at different stoichiometries. We
systematically investigated the molecular determinants of α-conotoxins Vc1.1, RgIA#, and PeIA
inhibition at hypothetical stoichiometries of the human α9α10 nAChR. Our results suggest that only
Vc1.1 exhibits strong stoichiometric-dependent inhibition at the α9α10 nAChR. The hydrogen bond
between N154 of α9 and D11 of Vc1.1 at the α9(+)-α9() interface is responsible for the
stoichiometric-dependent potency of Vc1.1.

3
Introduction
Nicotinic acetylcholine receptors (nAChRs) are cation-selective pentameric ligand-gated ion
channels belonging to the family of Cys-loop receptors, which also includes γ-aminobutyric acid
type A (GABA
A
), glycine, and serotonin (5-HT
3
) receptors.
1
The vertebrate nAChRs are
homo/heteromeric assembles of α1-α10, β1-β4, γ, δ or ε subunits. Although the nAChRs are
generally featured in the nervous system, they are also expressed in non-neuronal cells participating
in various physiological events and they are important targets for drug design.
2
The first crystal structure of any heteromeric nAChRs, the human(h) α4β2 nAChR subtype,
reveals the overall architecture consisting of a large extracellular domain (ECD), a transmembrane
domain (TMD) and an intracellular domain (ICD) (Figure 1A).
3
The ligand binding site is located at
the ECD interface of two adjacent subunits, comprising of (+) (principal) and (─) (complementary)
components. For hα4β2 nAChR, the α4 subunit loops A, B and C form the (+) site, and the β2
subunit β-sheet contributes to the (─) site (Figure 1B). Five subunits of the nAChR circle a
conducting pore (Figure 1C) where a gate consisting of hydrophobic residues, such as Leu and Ile,
is located in the middle of the TMD.
Heteromeric α9α10 nAChRs play an important physiological role in mediating olivocochlear
and vestibular neurotransmission.
4,5
Transcripts/protein expression of α9 and/or α10 subunits have
been reported in dorsal root ganglion neurons,
6,7
adrenal medulla,
8
and in other non-neuronal cells,
such as skin keratinocytes, pituitary pars tuberalis, lymphocytes, macrophages and bladder
urothelium.
9–15
α9-Containing nAChRs are potential targets for the therapy of several disorders or diseases
such as ear disorders, chronic pain and pemphigus vulgaris.
7,9,11,16,17
In addition, α9-containing
nAChRs are responsible for nicotine-induced transformation of normal human breast epithelial cells,
and inducible overexpression of α9-nAChR substantially increased tumor growth.
18
Conotoxins derived from the venom of Conus sea snails are pharmacologically valuable
peptides with selective potency at nAChR subtypes. α-Conotoxin antagonists of α9α10 nAChR such

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Molecular determinants conferring the stoichiometric-dependent activity of î±-conotoxins at the human î±9î±10 nicotinic acetylcholine receptor subtype" ?

The authors systematically investigated the molecular determinants of α-conotoxins Vc1. 1, RgIA #, and PeIA inhibition at hypothetical stoichiometries of the human α9α10 nAChR. This journal article is available at Research Online: https: //ro. uow. edu. au/ihmri/1269 Their results suggest that only Vc1. 1 exhibits stoichiometric-dependent inhibition at the α9α10 nAChR. 

Q2. What have the authors stated for future works in "Molecular determinants conferring the stoichiometric-dependent activity of î±-conotoxins at the human î±9î±10 nicotinic acetylcholine receptor subtype" ?

However, the authors can not exclude the possibility of the α9 ( + ) -α9 ( ─ ) as an energetically favourable binding site of RgIA # at ( α9 ) 3 ( α10 ) 2 nAChR. Instead, their study suggests that the sensitivity of both α10 ( + ) -α9 ( ─ ) and α9 ( + ) -α9 ( ─ ) interfaces to RgIA # might be comparable. Regardless, stoichiometric-selective inhibitors of α9α10 nAChR would be useful neurochemical tools for further elucidating the functional differences between the stoichiometries in native cells. Conversely, α9α10 nAChR antagonists that do not discriminate between the stoichiometries, may be suitable candidates to use under conditions where different stoichiometries exist. 

Q3. What is the role of 9-containing nAChRs in breast cancer?

In addition, α9-containing nAChRs are responsible for nicotine-induced transformation of normal human breast epithelial cells, and inducible overexpression of α9-nAChR substantially increased tumor growth. 

Q4. What is the stoichiometric-dependent potency of nACh?

The hydrogen bondbetween N154 of α9 and D11 of Vc1.1 at the α9(+)-α9() interface is responsible for thestoichiometric-dependent potency of Vc1.1. 

Q5. What is the DPR motif in -conotoxins?

The DPR (Asp-Pro-Arg) motif is conserved in α-conotoxins ImI, RgIA# (or RgIA) and Vc1.1,and essential to their binding affinities. 

Q6. How did the h910 nAChRs react with Vc?

at α9:α10 mRNA ratio of 1:3, presumably forming the (α9)2(α10)3 nAChR stoichiometry, 1 μM Vc1.1 inhibited ACh-evoked currents by 25.5 ± 1.8% (n = 15). 

Q7. How strong is the coupling between Vc1.1 D11 and 9 N154?

Using thermodynamic mutant cycles, 32 the coupling coefficient (Ω) gave a reciprocal of 28.57 suggesting a relatively strong coupling between Vc1.1 D11 and α9 N154 . 

Q8. What is the effect of Vc1.1 on the h910 nAC?

Injections of higher α9 to α10 mRNA ratios resulted in greater Vc1.1 inhibition at the expressed hα9α10 nAChRs compared to injections with excess α10 mRNA, alluding to the presence of Vc1.1-highsensitivity α9(+)-α9() site/s in the (α9)3(α10)2 or (α9)4(α10)1 configurations. 

Q9. What is the binding preference of RgIA# at the 9(+)?

a high-sensitivity Vc1.1 binding site was proposed at the α9(+)-α9() interfacepresent only in the (α9)3(α10)2 stoichiometry, and a low–sensitivity Vc1.1 binding site contributed by the α10(+)-α9() interface that exists in both stoichiometries. 

Q10. What is the binding site of the -conotoxins?

in their model the aromatic residues of the binding site form compact van der Waals contacts with the α-helix of the α-conotoxins, and the C192-C193 disulfide bond stacks with the C2-C8 disulfide bond of the α-conotoxins. 

Q11. What is the coupling coefficient of Vc1.1 D11 and 9 N154?

Thehydrogen bond formed between the side chains of α9 N154 and Vc1.1 D11 confers the specificity of Vc1.1 at α9(+)-α9(─) interface, and is responsible for the higher sensitivity of the α9α10 nAChR expressed with higher abundance of the α9 mRNA to Vc1.1. 

Q12. What is the role of nAChRs in the nervous system?

Although the nAChRs are generally featured in the nervous system, they are also expressed in non-neuronal cells participating in various physiological events and they are important targets for drug design. 

Q13. What is the binding energy of the -conotoxins at the h9?

Subsequent experiments were carried out using 1 μM Vc1.1, 300 nM RgIA# and 30 nM PeIA, close to the IC50 values of the α-conotoxins at the hα9α10 nAChR. 

Q14. What is the binding preference of the 9 N154?

To validate their prediction that the hydrogen bond interaction between Vc1.1 D11-α9 and N154is the determinant of Vc1.1 binding preference at the hα9(+)-α9() interface, residue N154 of thehα9 subunit was mutated to the corresponding hα10 subunit G154. 

Q15. What is the stoichiometry of rat 910 ?

The stoichiometry of rat(r) α9α10 nAChR heterologously expressed in Xenopus laevis oocytes was initially suggested to consist of two α9 and three α10 subunits ((α9)2(α10)3). 

Q16. What is the sensitivity of the 910 nAChR?

In this study, the authors used the R13-deleted analogue of RgIA (RgIA#) to probe its sensitivity todifferent stoichiometries of α9α10 nAChRs. 

Q17. What is the determinant of Vc1.1 binding?

24,25 Both Vc1.1 binding sites share a common α9 subunit (─) component, therefore, the determinants of Vc1.1 binding should be contributed by non-conserved residues of the α9 and α10 subunit (+) components. 

Q18. What is the coupling coefficient of the second loop of PeIA?

As shown in PeIA-α9(+)α9(─) model (Figure 3F), residues from the second loop of PeIA form few contacts with the side chain of α9 N154.