Lodo-α-conotoxin MI selectively binds the α/δ subunit interface of muscle nicotinic acetylcholine receptors

The embryonic mouse muscle nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel formed by alpha1, beta1, delta, and gamma subunits. The receptor contains two ligand binding sites at alpha/delta and alpha/gamma subunit interfaces. [H-3]Curare preferentially binds the alpha/gamma interface. We describe the synthesis and properties of a high-affinity iodinated ligand that selectively binds the W6 interface. An analogue of a-conotoxin MI was synthesized with an iodine attached to Tyr-12 (iodo-alpha-MI). The analogue potently blocks the fetal mouse muscle subtype of nAChR expressed in Xenopus oocytes. It failed, however, to block alpha3beta4, alpha4beta2, or alpha7 nAChRs. Iodo-alpha-MI potently blocks the alpha1beta1delta but not the alpha1beta1gamma subunit combination expressed in Xenopus oocytes indicating selectivity for the W6 subunit interface. alpha-Conotoxin MI was subsequently radioiodinated, and its properties were further evaluated. Saturation experiments indicate that radioiodinated alpha-conotoxin NIT binds to TE671 cell homogenates with a Hill slope of 0.95 +/- 0.0094. Kinetic studies indicate that the binding of [I-125]alpha-conotoxin MI is reversible (k(off) = 0.084 +/- 0.0045 min(-1)); k(on) is 8.5 x 10(7) min(-1) M-1. The calculated k(d) is 0.98 nM. This potency is similar to20-fold higher than the unmodified alpha-MI peptide. Unlike [I-125]alpha-bungarotoxin, [I-115]alpha-conotoxin MI binding to TE671 cell homogenates is fully displaceable by the small molecule antagonist d-tubocurarine.