The structure-function relationship of the nicotinic acetylcholine receptor (AChR) has been effectively studied by the combination of complementary DNA manipulation and single-channel current analysis1-6. Previous work with chimaeras between the Torpedo californica and bovine AChR δ-subunits has shown that the region comprising the hydrophobic segment M2 and its vicinity contains an important determinant of the rate of ion transport through the AChR channel5. It has also been suggested that this region is responsible for the reduction in channel conductance caused by divalent cations5 and that segment M2 contributes to the binding site of noncompetitive antagonists7,8. To identify those amino acid residues that interact with permeating ions, we have introduced various point mutations into the Torpedo AChR subunit cDNAs to alter the net charge of the charged or glutamine residues around the proposed transmembrane segments9-15. The single-channel conductance properties of these AChR mutants expressed in Xenopus laevis oocytes indicate that three clusters of negatively charged and glutamine residues neighbouring segment M2 of the α-, β-, γ- and δ-subunits, probably forming three anionic rings, are major determinants of the rate of ion transport.
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