Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Sep 1;454(2):311-21.
doi: 10.1042/BJ20130638.

Inter-residue Coupling Contributes to High-Affinity Subtype-Selective Binding of α-Bungarotoxin to Nicotinic Receptors

Affiliations
Free PMC article

Inter-residue Coupling Contributes to High-Affinity Subtype-Selective Binding of α-Bungarotoxin to Nicotinic Receptors

Steven M Sine et al. Biochem J. .
Free PMC article

Abstract

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr¹⁸⁴ in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr¹⁸⁴ depends on local residues, we generated mutations in an α7/5HT(3A) (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured ¹²⁵I-labelled α-btx binding. The results show that mutations of individual residues near Tyr¹⁸⁴ do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr¹⁸⁴ to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr¹⁸⁴ to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr¹⁸⁴ and local residues contributes to high-affinity subtype-selective α-btx binding.

Figures

Figure 1
Figure 1. Sequences of loop C in subtypes of human nicotinic receptor α-subunits
Tyr184 is highlighted in bold.
Figure 2
Figure 2. Crystal structure of the complex between α-btx (purple) and the α7/AChBP chimaera (cyan and orange)
PDB code 4HQP [21]. (a) Top view of the complex with the complementary subunit of one binding site highlighted in orange. (b) Close-up view as in (a) showing the principal (cyan) and complementary (orange) subunits. (c) Stereo view of key residues between the tip of finger II of α-btx (purple) and the principal (cyan) and complementary (orange) subunits of the α7/AChBP chimaera.
Figure 3
Figure 3. Steady-state binding of 125I-labelled α-btx to α7/5HT3 receptors and the indicated mutants expressed in HEK-293 cells
See the Experimental section for more details. Curves through the data are fits of the Hill equation with parameters given in Table 1. alpha7, α7/5HT3.
Figure 4
Figure 4. Binding time courses of 125I-labelled α-btx at the indicated concentrations to α7/5HT3 receptors and the indicated mutants expressed in HEK-293 cells
See the Experimental section for more details. Curves through the data are global fits of the bimolecular binding mechanism (see the main text) to the data with fitted parameters given in Table 2. RT, receptor–toxin complex.
Figure 5
Figure 5. Time courses of dissociation of 125I-labelled α-btx from α7/5HT3 receptors and the indicated mutants expressed in HEK-293 cells
See the Experimental section for more details. Curves through the data are fits of a single exponential plus a variable plateau to the data with fitted parameters in Table 2.
Figure 6
Figure 6. Determinants of target selectivity of α-btx determined from steady-state 125I-labelled α-btx binding to α7/5HT3 receptors with the indicated loop C replacements expressed in HEK-293 cells
See the Experimental section for more details. Curves through the data are fits of the Hill equation with parameters given in Table 3. Bold italic letters indicate substitutions.
Figure 7
Figure 7. Single-channel currents through α7/5HT3 receptors with the indicated loop C replacements expressed in HEK-293 cells
See the Experimental section for more details. For each panel, a trace of single channel currents elicited by 1 mM acetylcholine is shown filtered at a bandwidth of 10 kHz with channel opening corresponding with upward deflections. Underneath each trace are open (left) and closed (right) time histograms plotted on a logarithmic time scale and fitted by the sum of exponentials; individual components are indicated by dashed curves and the sum by solid curves.
Figure 8
Figure 8. Sequence alignment of Finger II in 3-finger α-neurotoxins
Key residues are bold. α-Neurotoxin sequences are from [38,44,45].

Similar articles

See all similar articles

Cited by 9 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback