Techniques: Use of concatenated subunits for the study of ligand-gated ion channels.

Members of the pentameric ligand-gated ion channel family, comprising GABA(A) receptors, nicotinic acetylcholine receptors, glycine receptors and 5-HT3 receptors, are involved in information transfer at both synapses and the neuromuscular junction. However, receptors that are composed of five subunits are difficult to analyse by recombinant expression of a mixture of the single subunits because multiple receptor subtypes with different subunit composition or arrangement can be formed. Covalently linking the C-terminus of the preceding subunit with the N-terminus of the following subunit to form a concatenated subunit enables the precise predetermination of subunit arrangement in these receptors. A forced subunit assembly enables the characterization of: (i) receptor architecture; (ii) properties of receptors that contain different subunit isoforms in specific locations; and (iii) selective introduction of a mutation into a specific subunit that occurs multiple times in a receptor. Thus, this method also facilitates the investigation of positional effects of mutations associated with diseases.