Estimation of Phi-Values for the Am1 Domain of Neuromuscular Acetylcholine Receptors

Abstract

The Cys-Loop gene super-family of ligand gated ion channels includes nicotinic acetylcholine (nACh), gamma aminobutyric acid (GABA A ), glycine (Gly), and serotonin (5-HT 3A ) receptors. Each receptor is either a homo-or heteropentamer made up of 5 identical or homologous subunits. Each subunit has an extracellular N-terminal domain, which houses the ligand binding site; a transmembrane domain which spans the membrane four times as a-helical segments (M1-M4); and a long loop between M3 and M4 that constitutes the intracellular domain. M2 lines the ion channel, whereas M1, M3 and M4 are abluminal. Muscle nAChRs consist of four different subunits with the clockwise arrangement agabd when viewed from the extracellular side. We used disulphide trapping between individually engineered Cys in the aM2 segments to investigate arrangement and flexibility of the upper part of aM2. Disulfide bond formation was monitored in a 2 bdg nAChR expressed in Xenopus laevis oocytes by two electrode voltage clamp experiments and Western blotting. In properly arranged Cys pairs disulfide bond formation can either occur spontaneously or it can be induced by oxidizing with copper phenanthroline (CuPhen). Cystine bond formation is reversible by reducing with dithiothreitol (DTT). To eliminate interference from the native vicinal disulphide present in the ligand binding site of the a-subunit, we utilized the background mutations aC192S-C193S. Position aE262C that was previously shown to face the channel formed DTT reducible dimers both spontaneously and upon application of the oxidizing agent CuPhen. Based on Unwin's 4-Å resolution model, the formation of disulfide bonds at this channel level would require substantial movement of the channel-lining M2 segments. No dimer formation was observed in aL263C. We are investigating a series of aM2 Cys mutants to determine which positions in M2 can form disulfide bonds.