Constraining the expression of nicotinic acetylcholine receptors by using pentameric constructs.

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., beta4_beta4_alpha3_beta4_alpha3). 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 alpha3beta4 nicotinic receptors. We show evidence that the expressed receptors were made up of alpha3 and beta4 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.