Chimeric Analysis of a Neuronal Nicotinic Acetylcholine Receptor Reveals Amino Acids Conferring Sensitivity to α-Bungarotoxin (original) (raw)
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Subunit Interface Selectivity of the alpha -Neurotoxins for the Nicotinic Acetylcholine Receptor
Journal of Biological Chemistry, 1999
Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short ␣-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other ␣-neurotoxins, binds with high affinity to ␣␥ and ␣␦ subunit interfaces (K D ϳ100 pM) but binds with markedly reduced affinity to the ␣⑀ interface (K D ϳ100 nM). By constructing chimeras composed of portions of the ␥ and ⑀ subunits and coexpressing them with wild type ␣, , and ␦ subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, ␥Pro-175 and ␥Glu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in ⑀, confer low affinity. To identify an interaction between ␥Glu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between ␥Glu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the ␥ subunit surface of the binding site interface.
Biochemistry, 1994
In the a subunit of the Torpedo nicotinic cholinergic receptor (AChR), a sequence region surrounding a pair of adjacent cysteinyl residues at positions 192 and 193 contributes to a binding site for cholinergic ligands, including the snake a-neurotoxins. Synthetic and biosynthetic peptides corresponding to this region bind a-bungarotoxin (a-BTX) in the absence of other structural components of the AChR and, therefore, represent a "prototope" for a-BTX. Using synthetic peptides corresponding to the complete AChR a subunits of Torpedo electroplax and mammalian muscle, we previously defined a sequence segment corresponding to a universal prototope for a-BTX binding between amino acid residues 18 1 and 200 [Conti
Neuronal α-Bungarotoxin Receptors Differ Structurally from Other Nicotinic Acetylcholine Receptors
The Journal of Neuroscience, 1997
We have characterized the ␣-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of ␣7-containing receptors that bind ␣-Bgt with high affinity (K d ϭ 94 pM). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and ␣-Bgt block ϳ90% of the whole-cell current. The binding of nicotinic agonists to cell-surface BgtRs was highly cooperative with four different agonists showing Hill coefficients in the range of 2.3-2.4. A similar agonist binding cooperativity was observed for BgtR homomers formed from chimeric ␣7/5HT3 subunits expressed in tsA 201 cells. Two classes of agonist binding sites, in the ratio of 4:1 for PC12 cell BgtRs and 3:1 for ␣7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and ␣7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and ␣7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.
Proceedings of the National Academy of Sciences of the United States of America, 1991
Neither the structure nor the function of a-bungarotoxin (aBgtx) binding molecules in the nervous system have yet been completely defined, although it is known that some of these molecules are related to cation channels and some are not. Using an improved method of affinity chromatography, we have isolated a toxin binding molecule from chicken optic lobe that contains at least three subunits with apparent Mr values of 52,000, 57,000, and 67,000. The Mr 57,000 subunit binds aBgtx and seems to be present in two copies per receptor. The receptor is recognized by antibodies raised against the aBgtx receptors of human neuroblastoma cells, fetal calf muscle, and chicken optic lobe but not by antibodies raised against Torpedo acetylcholine receptor, the serum of myasthenic patients, or monoclonal antibody 35. '25I-labeled aBgtx binding to the isolated receptor is blocked, with the same potency, by nicotinic agonists and antagonists, such as nicotine, neuronal bungarotoxin and, d-tubocurarine. When reconstituted in a planar lipid bilayer, the purified aBgtx receptor forms cationic channels with a conductance of 50 pS. These channels are activated in a dose-dependent manner by carbamylcholine and blocked by d-tubocurarine.
The EMBO Journal, 2003
The molecular mechanisms of nicotinic receptor activation are still largely unknown. The crystallographic structure of the acetylcholine binding protein (AChBP) reveals a single H-bond between two different acetylcholine binding loops. Within these homologous loops we systematically introduced a4 residues into the a7/5HT 3 chimeric receptor and found that the single point mutations G152K (loop B) and P193I (loop C) displayed a non-additive increase of equilibrium binding af®nity for several agonists compared with the double mutant G152K/P193I. In whole-cell patch±clamp recordings, G152K, P193I and G152K/ P193I mutants displayed an increase up to 5-fold in acetylcholine potency with a large decrease of the apparent Hill coef®cients (signi®cantly smaller than one). Concomitantly, the G152K/P193I mutant showed a dramatic loss of high-af®nity a-bungarotoxin binding (100-fold decrease), thus pinpointing a new contact area for the toxin. Fitting the data with an allosteric±kinetic model, together with molecular dynamic simulations, suggests that the presence of the inter-backbone H-bond between positions 152 and 193, revealed in a4 and in a7 double mutant but not in a7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.
Journal of Biological Chemistry, 2008
Nicotinic acetylcholine receptors (nAChRs) containing ␣3 and 2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related ␣6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog of ␣-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric ␣6/␣323 nAChRs with an IC 50 of 1.2 nM; in contrast, its IC 50 on the closely related ␣32 as well as non-␣6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the ␣3 subunit inserted into the homologous positions of the ␣6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the ␣6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the ␣3 subunit increased the IC 50 of the peptide by >1000-fold. Conversely, when these key residues in ␣3 were replaced with those from ␣6, the IC 50 decreased by almost 150-fold. Similar effects were seen with other ␣6-selective conotoxins, suggesting the general importance of these ␣6 residues in conferring selective binding.