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Mar Drugs
2019 Sep 16;179:. doi: 10.3390/md17090535.
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Structure-Function Elucidation of a New α-Conotoxin, MilIA, from Conus milneedwardsi.
Peigneur S
,
Devi P
,
Seldeslachts A
,
Ravichandran S
,
Quinton L
,
Tytgat J
.
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The a-Conotoxins are peptide toxins that are found in the venom of marine cone snails and they are potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies. We describe the isolation and characterization of α-conotoxin MilIA, the first conopeptide from the venom of Conus milneedwardsi. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs that were expressed in Xenopus laevis oocytes. MilIA, which is a member of the α3/5 family, is an antagonist of muscle type nAChRs with a high selectivity for muscle versus neuronal subtype nAChRs. Several analogues were designed and investigated for their activity in order to determine the key epitopes of MilIA. Native MilIA and analogues both showed activity at the fetal muscle type nAChR. Two single mutations (Met9 and Asn10) allowed for MilIA to strongly discriminate between the two types of muscle nAChRs. Moreover, one analogue, MilIA [∆1,M2R, M9G, N10K, H11K], displayed a remarkable enhanced potency when compared to native peptide. The key residues that are responsible for switching between muscle and neuronal nAChRs preference were elucidated. Interestingly, the same analogue showed a preference for α9α10 nAChRs among the neuronal types.
Figure 2. Crude venom of C. milneedwardsi was fractionated by C18 reversed-phase high-performance liquid chromatography (RP-HPLC). The absorbance was monitored at 280 nm (orange line) and at 214 nm (blue line). The red line represents the acetonitrile gradient. The fraction containing the peptide MilIA is indicated with a blue arrow.
Figure 3. Electrophysiological profile of MilIA [M9G, N10K]. The electrophysiological profile showed the ACh-evoked current mediated by α1β1γδ, α4β2, α7, α9α10, α4β4, and α1β1δε nAChRs. The nAChRs were gated by a variable time duration pulse of ACh at 2 mL/min. (red) (200 µM for α1β1γδ, α4β2, α4β4, α1β1δε; 100 µM for α7; 500 µM for α9α10) for different nAChR subtypes. The first and the second peak amplitude represented the absence and presence of 1 µM of MilIA [M9G, N10K], respectively. The conopeptide was applied for 60 s at 2 mL/min. (blue), immediately followed by a variable time duration pulse of ACh (red).
Figure 4. Concentration curves on α1β1γδ (A) and α1β1δε (B) nAChR. The percentage of inhibition of MilIA, MilIA [M9G], MilIA [N10K], MilIA [â1,M2R], MilIA [M9G, N10K], and MilIA [â1,M2R, M9G, N10K, H11K] at α1β1γδ (left) and α1β1δε (right) nAChR was plotted against the logarithm of the different concentrations tested and fitted with the Hill equation. The visualized error bars represent the standard error of the mean (S.E.M). All of the experiments were repeated at least three times (n ⥠3).
Figure 5. Electrophysiological profile of MilIA [â1,M2R, M9G, N10K, H11K]. The electrophysiological profile showed the ACh-evoked current mediated by α1β1γδ, α4β2, α7, α9α10, α4β4 and α1β1δε nAChRs. The nAChRs were gated by a variable time duration pulse of ACh (red) (200 µM for α1β1γδ, α4β2, α4β4, α1β1δε; 100 µM for α7; 500 µM for α9α10) for the different nAChR subtypes at 2 mL/min. The first and the second peak amplitude represented the absence and presence of 1 µM of MilIA [â1,M2R, M9G, N10K, H11K], respectively. The conopeptide was applied for 60 s at 2 mL/min. (blue), immediately followed by the addition of a variable time duration pulse of ACh (red).
Figure 6. (A) Concentration-response curves on α9α10 nAChR. The percentage of inhibition was plotted against the logarithm of the different concentrations tested and fitted with the Hill equation. The visualized error bars represent the standard error of the mean (S.E.M). All experiments were repeated at least three times (n ⥠3). (B) The time duration of the recovery of nAChR response to ACh after inhibition. Blue line: washout kinetic for MilIA [M9G, N10K] on α1β1γδ nAChR; Orange line: washout kinetic for MilIA [â1,M2R, M9G, N10K, H11K] on α9α10 nAChR.
Figure 7. Percentage inhibition of the Ach response after addition of 1 µM MilIA.
Figure 8. Selected traces of the α1β1δε nAChR showing effects induced by MilIA and its mutants. All of the components tested at a concentration of 1 µM could induce a block. The double and triple mutants showed the highest inhibition. The visualized error bars represent the standard error of the mean (S.E.M). All experiments were repeated at least three times (n ⥠3). The key residues are highlighted as lines.
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