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

Polly A Quiram,Steven M Sine

doi:10.1074/jbc.273.18.11001

Abstract

To identify residues in the neuronal alpha7 acetylcholine subunit that confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed alpha7-alpha1 chimeras containing segments of the muscle alpha1 subunit inserted into equivalent positions of the neuronal alpha7 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 alpha7/5HT-3 over alpha1/5HT-3 homo-oligomers. Two of these pairs, alpha7Trp55/alpha1Arg55 and alpha7Ser59/alpha1Gln59, are within one of the four loops that contribute to the traditional non-alpha subunit face of the muscle receptor binding site. The third pair, alpha7Thr77/alpha1Lys77, is not within previously described loops of either the alpha or non-alpha faces and may represent a new loop or an allosterically coupled loop. Exchanging these residues between alpha1 and alpha7 subunits exchanges the affinities of the binding sites for CTx ImI, suggesting that the alpha7 and alpha1 subunits, despite sequence identity of only 38%, share similar protein scaffolds.

Highlights

The two neurotransmitter binding sites of muscle nicotinic acetylcholine receptors (AChR)1 are generated by apposition of pairs of nonequivalent subunits, ␣1/␦, ␣1/␥, and ␣1/⑀
The muscle-specific ␣-conotoxins MI, GI, and SI bind with high affinity to muscle receptors, whereas they bind with low affinity to ␣7 neuronal receptors [2]
We probed the binding site of the ␣7 receptor using the neuronal-specific toxin conotoxin ImI (CTx ImI) together with chimeras containing portions of ␣1 sequence substituted into the extracellular domain of ␣7

Summary

EXPERIMENTAL PROCEDURES

Materials—125I-Labeled ␣-bungarotoxin (␣-bgt) was purchased from NEN Life Science Products, d-tubocurarine chloride from ICN Pharmaceuticals, (ϩ)-epibatidine and methyllycaconitine from Research Biochemicals, 293 human embryonic kidney cell line (293 HEK) from the American Type Culture Collection, and unlabeled ␣-bgt from Sigma. Chimera ␣7/5HT-3 (␣7200/5HT-3) was constructed by bridging a 58-bp synthetic oligonucleotide from a TfiI site in ␣7 to an EcoRV site in rat 5HT-3. Chimera ␣1/5HT-3 (␣1205/5HT-3) was constructed by bridging a 69-bp synthetic oligonucleotide from a DraIII site in ␣1 to a StuI site in rat 5HT-3. The cells were briefly centrifuged, resuspended in potassium Ringer’s solution, and divided into aliquots for ligand binding. Binding was terminated by addition of 2 ml of potassium Ringer’s solution containing 600 ␮M of d-tubocurarine chloride. To measure time courses of ␣-bgt dissociation, receptors were incubated with 3.75 nM 125I-labeled ␣-bgt for 120 min to achieve full occupancy, free 125I-labeled ␣-bgt was removed, and 200-␮l aliquots of cells were filtered at specific times. Sucrose Gradient Centrifugation—Two days after transfection, 293 HEK cells expressing various receptors were harvested by gentle agitation in phosphate-buffered saline and resuspended in high potassium. Radioactivity in each fraction was normalized to that of the fraction containing the maximum radioactivity in each gradient

RESULTS

Conotoxin ImI

Point mutants

DISCUSSION