Probing function in ligand-gated ion channels without measuring ion transport

Abstract

ABSTRACTAlthough the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion-channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are—by no means—new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled α-bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric α7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two long-standing questions in the field: whether ligand-binding affinities are sensitive to binding-site occupancy, and whether mutations to amino-acid residues in the transmembrane domain can affect the channel’s affinities for ligands that bind to the extracellular domain.