Neuronal nicotinic acetylcholine receptors are pentameric ligand-gated ion channels. The large number of different subunits that can be expressed in vivo makes it difficult to know the exact make-up of the receptors present in neurons. As native receptors can contain multiple different subunits, it is important to be able to obtain efficient heterologous expression of such receptors as a homogeneous population.

In order to restrict the stoichiometry of recombinant nAChRs we originally created tandem constructs by linking 3 and β4 subunits. These tandem constructs, when expressed in Xenopus oocytes along with a β4 monomer subunit, did produce functional 3 β4 channels apparently identical to non-linked 3 β4 receptors expressed in oocytes. However, when we further investigated these channels (by mutating the 9' position in the middle of TM2), we observed multiple components, indicating that tandem constructs do not always fully incorporate into the receptor complex. Thus this approach is useless for studying receptors containing multiple subunits (Groot-Kormelink et al., 2004).

Using standard molecular biology techniques, we have now linked five subunits into a pentameric construct. This pentameric 3_ β4_ 3_ β4_ β4 construct was successfully expressed in the Xenopus oocyte system and had an EC₅₀ value to ACh of 97 ± 4 µM (nH = 1.60 ± 0.10, n=13). This value is not much less of those than observed for non-linked 3 β4 subunits expressed in oocytes, both at 1:1 and 1:9 :β subunit ratios (134 ± 6 µM; nH = 1.73 ± 0.10 and EC₅₀ = 133 ± 8.2 µM; nH = 1.77 ± 0.17, n=4, respectively; see Broadbent et al., this meeting). When we co-expressed the pentameric construct with excess monomer ( 3 or β4) mutated in the 9' position in TM2, we could not detect a shift in the dose response curve. This suggests that no break-down or incomplete incorporation of the pentameric construct takes place. We also successfully introduced 9’TM2 mutations into several subunits of the pentameric construct.

These pentameric constructs seem to be an effective approach to the production of homogeneous populations of receptors containing multiple types of subunit, of receptors mutated in only one of several identical subunit copies and to the assembly of a mutated subunit at a specified position in the receptor pentamer.

Groot-Kormelink et al. (2004) J. Gen. Physiol. 123 , p697-708.
This work has been sponsored by a Wellcome Trust grant (project 064652) and the MRC (to SB).