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Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes.

Wang S, Zhao C, Liu Z, Wang X, Liu N, Du W, Dai Q - Mar Drugs (2015)

Bottom Line: Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes.Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes.Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Biotechnology, Beijing 100071, China. crosswang@gmail.com.

ABSTRACT
In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH2), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 310-helix from residues Pro7 to His10 and a type I β-turn from residues Pro14 to Cys17. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu2 by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.

No MeSH data available.


Effects of Mr1.7 on rat nAChRs expressed in Xenopus Oocytes. (A) Concentration-dependent response curves of Mr1.7 on various rat nAChR subtypes; (B) Kinetic analysis of the activity of Mr1.7 on nAChR α3β2. The data were fit to a single exponential equation; (C) A representative trace of 100 nM Mr1.7 was applied on nAChR α3β2; (D) Recovery from Mr1.7 block (10 μM) in nAChR α9α10. Peptides were applied by perfusion to oocytes expressing nAChRs as described in Materials and Methods. The error bars denote the S.E.M. of the data from four to nine oocytes for each determination. See Table 1 and Table 4 for a summary of the values obtained.
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marinedrugs-13-03259-f004: Effects of Mr1.7 on rat nAChRs expressed in Xenopus Oocytes. (A) Concentration-dependent response curves of Mr1.7 on various rat nAChR subtypes; (B) Kinetic analysis of the activity of Mr1.7 on nAChR α3β2. The data were fit to a single exponential equation; (C) A representative trace of 100 nM Mr1.7 was applied on nAChR α3β2; (D) Recovery from Mr1.7 block (10 μM) in nAChR α9α10. Peptides were applied by perfusion to oocytes expressing nAChRs as described in Materials and Methods. The error bars denote the S.E.M. of the data from four to nine oocytes for each determination. See Table 1 and Table 4 for a summary of the values obtained.

Mentions: In the present study, we assessed the functional activity of Mr1.7 by activating ACh-evoked membrane currents in Xenopus oocytes. Figure 4A exhibits that it potently inhibited α3β2, α9α10 and α6/α3β2β3 subtypes with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM (Table 1), respectively, but showed no inhibitory activity on other nAChR subtypes. The half time (t1/2) of the recovery of α3β2 subtype from its binding to 100 nM Mr1.7 was 3.710 (3.165–4.480) min (Figure 4B). Figure 4C shows a representative trace of ACh-evoked currents of α3β2 subtype inhibited by 100 nM Mr1.7. However, the recovery rate of Mr1.7 block for α9α10 was significantly slower compared with α3β2 subtype (Figure 4D).


Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes.

Wang S, Zhao C, Liu Z, Wang X, Liu N, Du W, Dai Q - Mar Drugs (2015)

Effects of Mr1.7 on rat nAChRs expressed in Xenopus Oocytes. (A) Concentration-dependent response curves of Mr1.7 on various rat nAChR subtypes; (B) Kinetic analysis of the activity of Mr1.7 on nAChR α3β2. The data were fit to a single exponential equation; (C) A representative trace of 100 nM Mr1.7 was applied on nAChR α3β2; (D) Recovery from Mr1.7 block (10 μM) in nAChR α9α10. Peptides were applied by perfusion to oocytes expressing nAChRs as described in Materials and Methods. The error bars denote the S.E.M. of the data from four to nine oocytes for each determination. See Table 1 and Table 4 for a summary of the values obtained.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4483627&req=5

marinedrugs-13-03259-f004: Effects of Mr1.7 on rat nAChRs expressed in Xenopus Oocytes. (A) Concentration-dependent response curves of Mr1.7 on various rat nAChR subtypes; (B) Kinetic analysis of the activity of Mr1.7 on nAChR α3β2. The data were fit to a single exponential equation; (C) A representative trace of 100 nM Mr1.7 was applied on nAChR α3β2; (D) Recovery from Mr1.7 block (10 μM) in nAChR α9α10. Peptides were applied by perfusion to oocytes expressing nAChRs as described in Materials and Methods. The error bars denote the S.E.M. of the data from four to nine oocytes for each determination. See Table 1 and Table 4 for a summary of the values obtained.
Mentions: In the present study, we assessed the functional activity of Mr1.7 by activating ACh-evoked membrane currents in Xenopus oocytes. Figure 4A exhibits that it potently inhibited α3β2, α9α10 and α6/α3β2β3 subtypes with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM (Table 1), respectively, but showed no inhibitory activity on other nAChR subtypes. The half time (t1/2) of the recovery of α3β2 subtype from its binding to 100 nM Mr1.7 was 3.710 (3.165–4.480) min (Figure 4B). Figure 4C shows a representative trace of ACh-evoked currents of α3β2 subtype inhibited by 100 nM Mr1.7. However, the recovery rate of Mr1.7 block for α9α10 was significantly slower compared with α3β2 subtype (Figure 4D).

Bottom Line: Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes.Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes.Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Biotechnology, Beijing 100071, China. crosswang@gmail.com.

ABSTRACT
In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH2), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 310-helix from residues Pro7 to His10 and a type I β-turn from residues Pro14 to Cys17. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu2 by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.

No MeSH data available.