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Constraining the Expression of Nicotinic Acetylcholine Receptors by Using Pentameric Constructs

Department of Pharmacology, University College London, London, United Kingdom

Much of our understanding of ligand-gated ion channels comes from heterologous expression studies. However, this technique cannot produce receptors with a predetermined subunit composition for channels formed by several different subunits and cannot insert a single mutation copy if the subunit of interest is present in several copies in the channel. Here, we describe a novel approach that overcomes these problems by expressing pentameric constructs, in which the code of the five subunits is linked (i.e., 4_4_3_4_3). This is the first time that a concatemer of the complete pentameric receptor has been expressed for channels in the cysteine-loop superfamily. The presence of the linker did not change the agonist or antagonist sensitivity of 34 nicotinic receptors. We show evidence that the expressed receptors were made up of 3 and 4 subunits in one pentameric fusion protein as designed in the construct. This approach can be applied to any nicotinic superfamily receptor to produce channels with a defined subunit arrangement and to introduce specific mutations at any desired location of the pentameric fusion protein.

Address correspondence to: Dr. Lucia Sivilotti, Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT, United Kingdom. E-mail: l.sivilotti{at}ucl.ac.uk

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