Polly A. Quiram

Congenital Myasthenic Syndrome Caused by Decreased Agonist Binding Affinity Due to a Mutation in the Acetylcholine Receptor ε Subunit

We describe the genetic and kinetic defects for a low-affinity fast channel disease of the acetylcholine receptor (AChR) that causes a myasthenic syndrome. In two unrelated patients with very small miniature end plate (EP) potentials, but with normal EP AChR density and normal EP ultrastructure, patch-clamp studies demonstrated infrequent AChR channel events, diminished channel reopenings during ACh occupancy, and resistance to desensitization by ACh. Each patient had two heteroallelic AChR epsilon subunit gene mutations: a common epsilon P121L mutation, a signal peptide mutation (epsilon G-8R) (patient 1), and a glycosylation consensus site mutation (epsilon S143L) (patient 2). AChR expression in HEK fibroblasts was normal with epsilon P121L but was markedly reduced with the other mutations. Therefore, epsilon P121L defines the clinical phenotype. Studies of the engineered epsilon P121L AChR revealed a markedly decreased rate of channel opening, little change in affinity of the resting state for ACh, but reduced affinity of the open channel and desensitized states.

Mutation causing congenital myasthenia reveals acetylcholine receptor β/δ subunit interaction essential for assembly

We describe a severe postsynaptic congenital myasthenic syndrome with marked endplate acetylcholine receptor (AChR) deficiency caused by 2 heteroallelic mutations in the β subunit gene. One mutation causes skipping of exon 8,truncating the β subunit before its M1 transmembrane domain, and abolishing surface expression of pentameric AChR. The other mutation, a 3-codon deletion (β426delEQE) in the long cytoplasmic loop between the M3 and M4 domains, curtails but does not abolish expression. By coexpressing β426delEQE with combinations of wild-type subunits in 293 HEK cells, we demonstrate that β426delEQE impairs AChR assembly by disrupting a specific interaction between β and δ subunits. Studies with related deletion and missense mutants indicate that secondary structure in this region of the β subunit is crucial for interaction with the δ subunit. The findings imply that the mutated residues are positioned at the interface between β and δ subunits and demonstrate contribution of this local region of the long cytoplasmic loop to AChR assembly. J. Clin. Invest. 104:1403‐1410 (1999).

Structural Elements in α-Conotoxin ImI Essential for Binding to Neuronal α7 Receptors

The neuronal-specific toxin α-conotoxin ImI (CTx ImI) has the sequence Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2, in which each cysteine forms a disulfide bridge to produce a constrained two-loop structure. To investigate the structural basis for bioactivity we mutated individual residues in CTx ImI and determined bioactivity. Bioactivity of the toxins was determined by their competition against 125I-labeled α-bungarotoxin binding to homomeric receptors containing α7 sequence in the major extracellular domain and 5HT-3 sequence elsewhere. The results reveal two regions in CTx ImI essential for binding to the α7/5HT-3 receptor. The first is the triad Asp-Pro-Arg in the first loop, where conservative mutations of each residue diminish affinity by 2–3 orders of magnitude. The second region is the lone Trp in the second loop, where an aromatic side chain is required. The overall results suggest that within the triad of the first loop, Pro positions the flanking Asp and Arg for optimal interaction with one portion of the binding site, while within the second loop, Trp stabilizes the complex through its aromatic ring.

Identification of Residues in the Neuronal α7Acetylcholine Receptor That Confer Selectivity for Conotoxin ImI

To identify residues in the neuronal α7 acetylcholine subunit that confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed α7-α1 chimeras containing segments of the muscle α1 subunit inserted into equivalent positions of the neuronal α7 subunit. To achieve high expression in 293 human embryonic kidney cells and formation of homo-oligomers, we joined the extracellular domains of each chimera to the M1 junction of the 5-hydroxytryptamine-3 (5HT-3) subunit. Measurements of CTx ImI binding to the chimeric receptors reveal three pairs of residues in equivalent positions of the primary sequence that confer high affinity of CTx ImI for α7/5HT-3 over α1/5HT-3 homo-oligomers. Two of these pairs, α7Trp55/α1Arg55 and α7Ser59/α1Gln59, are within one of the four loops that contribute to the traditional non-α subunit face of the muscle receptor binding site. The third pair, α7Thr77/α1Lys77, is not within previously described loops of either the α or non-α faces and may represent a new loop or an allosterically coupled loop. Exchanging these residues between α1 and α7subunits exchanges the affinities of the binding sites for CTx ImI, suggesting that the α7 and α1 subunits, despite sequence identity of only 38%, share similar protein scaffolds.

Molecular dissection of subunit interfaces in the nicotinic acetylcholine receptor.

Ligand binding sites in the muscle nicotinic acetylcholine receptor are generated by pairs of alpha and non-alpha subunits. The non-alpha subunits, gamma, delta and epsilon, contribute significantly to overall affinity of agonists and antagonists, and confer selectivity of these ligands for the two binding sites. By constructing chimeras composed of segments of the various non-alpha subunits and determining ligand selectivity, we have identified four loops, well separated in the linear sequence, that contribute to the non-alpha portion of the binding site. Studies of point mutations in these loops and labeling of engineered cysteines show that the peptide backbones of each non-alpha subunit fold into similar basic scaffolds. Studies of mutations of the peptide antagonists alpha-conotoxin M1 and ImI reveal pairs of residues in the binding site and the toxin that stabilize the complex.

Synthesis of alkylene linked bis-THA and alkylene linked benzyl-THA as highly potent and selective inhibitors and molecular probes of acetylcholinesterase

An efficient and economical synthesis of a series of rationally designed novel 9,9′-(alkane-1,ω-diyldiimino)-1,2,3,4-tetrahydroacridines (ω = 7–10) and a second series of new analogues, 9-(ω-phenylalkylamino)-1,2,3,4-tetrahydroacridines (ω = 4–10), is reported. Compounds in the first series are found to be up to 10 000-fold more selective and 1000-fold more potent in reversibly inhibiting rat acetylcholinesterase (AChE) than the monomer, 9-amino-1,2,3,4-tetrahydroacridine (THA). Some members in the latter series (ω = 7–8) are slightly more potent than THA in inhibiting AChE but still more selective. These compounds can serve as (i) important chemical tools to evaluate the role of AChE inhibition by THA, a clinical drug, in treating Alzheimer’s disease, (ii) effective, safer and low-cost insecticides and parasiticides, (iii) potential blockers of the K+ channel and the N-methyl-D-aspartate receptor channel, and perhaps (iv) improved therapeutics for Alzheimer’s disease.