Kachel HS et al. (2016), Block of nicotinic acetylcholine receptors by p...

XB-ART-52823

Sci Rep 2016 Nov 30;6:38116. doi: 10.1038/srep38116.

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Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition.

Kachel HS , Patel RN , Franzyk H , Mellor IR .

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Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50 = 12 nM at -80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ was most sensitive to PhTX-12 along with α3β4 (IC50 values of 100 nM) with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity.

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Species referenced: Xenopus laevis
Genes referenced: was

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	Figure 1. Structures of the naturally occurring PhTX-433 from Philanthus triangulum as well as of the two synthetic analogues, PhTX-343 and PhTX-12, used in this study.The numbers indicate the carbon spacing between nitrogen atoms in the polyamine moiety.
	Figure 2. (A,B) Responses to ACh in the absence (black) or presence (blue) of 1 μM PhTX-343 (A) or PhTX-12 (B) for all of the tested nAChR subtypes. (C,D) Concentration-inhibition curves for PhTX-343 (C) and PhTX-12 (D) inhibition of α3β4 (red ▲), α4β4 (blue ■), α4β2 (green ●), α3β2 (brown ▼), α7 (purple ♦) and α1β1γδ (orange ×) peak (upper) and late (lower) current. The ACh concentrations were 10 μM for α4β4, α4β2 and α1β1γδ, 30 μM for α3β2, and 100 μM for α3β4 and α7. VH = −80 mV. Curves are fitted by Eq. 1 and IC50 values are given in Tables 1 and 2. There is a noticeably greater left-right spread of curves for PhTX-343 and a leftward shift for late current inhibition curves as compared to peak current inhibition for both toxins.
	Figure 3. Alignment of amino acid sequences for the M1-M2 loop and M2 region in all of the nAChR subunits used in the present study.Residues expected to line the nAChR pore (termed rings below) are depicted in bold and numbered 1–8 starting at the extracellular terminus. F in ring 3 of r-β4 (a difference that is conserved in other species including humans) is highlighted in orange, F and G in rings 5 and 6 (selectivity filter) of m-β1 are highlighted in green and the positively charged residues at the external mouth of the pore in m-β1, m-δ and m-γ are highlighted in blue.
	Figure 4. The effect of M2 mutations β2(V253F) and β4(F255V) on PhTX-343 and PhTX-12 inhibition.Concentration-inhibition curves for PhTX-343 (A,B) and PhTX-12 (C,D) inhibition of α3β4 (blue ■), α3β4(F255V) (blue □), α4β2 (red ●) and α4β2(V253F) (red ○) peak (A,C) and late (B,D) current. The ACh concentrations were 10 μM for α4β2 and α4β2(V253F), and 100 μM for α3β4 and α3β4(F255V). VH = −80 mV. Curves are fitted by Eq. 1 and IC50 values are given in Tables 1 and 2.
	Figure 5. Voltage dependence of inhibition of nAChRs by PhTX-343 (A,B) and PhTX-12 (C,D) for peak (A,C) and late (B,D) current. The bars show IC50 (μM) at VH of −60, −80 and −100 mV. (p < 0.05), (p < 0.01), (p < 0.001) and *(p < 0.0001) indicate significant differences in the IC50 values for −60 and −100 mV (NS = not significantly different; p > 0.05).
	Figure 6. Recovery of responses to ACh following antagonism by PhTX-343 (A,B) and PhTX-12 (C,D) for peak current (A,C) and late current (B,D). A pre-toxin control response to ACh was obtained at −6 min, the response at time 0 was in the presence of PhTX, while the responses from 6 min and onwards were applications of ACh only. ACh was applied at 10 μM for α4β2 (green ●), α4β4 (blue ■) and α1β1γδ (orange ), 30 μM for α3β2 (brown ▼) and 100 μM for α3β4 (red ▲) and α7 (purple ♦). PhTX-343 was applied at 10 μM for α4β2, α4β4, α3β4 and α3β2, 30 μM for α7 and 100 μM for α1β1γδ. PhTX-12 was applied at 10 μM for all subunit combinations. (E–G) Currents in response to ACh before PhTX application (black), ACh co-applied with PhTX (light grey), and ACh alone 6 min after co-application with PhTX (dark grey) for PhTX-343 inhibition of α3β4 (E) and α4β2 (F), or PhTX-12 inhibition of α4β2 (G). VH = −80 mV in all cases.
	Figure 7. Peak current ACh concentration-response curves in the absence (●) or presence (■) of PhTX-343 at 1 μM for α4β2, α4β4, α3β2 and α3β4, 10 μM for α1β1γδ and 30 μM for α7.Curves were fitted with Eq. 2 and EC50 values are given in Table 3. The grey symbols () show the % control response for each ACh concentration in the presence of PhTX-343. VH = −80 mV in all cases.
	Figure 8. Peak current (A,B) or late current (C,D) ACh concentration-response curves in the absence (●) or presence (■) of PhTX-12 at 10 μM (peak current) or 0.3 μM (late current) for α3β4 and α1β1γδ.Curves were fitted with Eq. 2 and EC50 values are given in Table 3. The grey symbols () show the % control response for each ACh concentration in the presence of PhTX-12. E-F: α3β4 currents in response to 100 μM (E) or 1 mM ACh in the absence (black) and presence (blue) of 10 μM PhTX-12. VH = −80 mV in all cases.

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