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 neurones. As native receptors can contain more than two different subunits, it is important to be able to express homogeneous populations of such receptors heterologously. In order to restrict the stoichiometry of recombinant nAChRs, tandem constructs were created by linking α3 and β4 subunits and were expressed in Xenopus oocytes along with different monomer subunits. Oocytes were then tested with acetylcholine, using two-electrode voltage clamp, to see which tandem/monomer combinations produced functional receptors.

Tandems of the α3-β4 orientation failed to produce functional receptors either when expressed alone or along with any monomer. Tandems of the β4-α3 orientation did not produce functional receptors when expressed alone or along with α3, α4, α6 or β3 subunits (small responses were observed with monomer α2, α5 or β2 subunits). When the β4-α3 tandem was expressed along with β4 subunits, large inward currents were observed in response to acetylcholine. Dose-response curves were therefore obtained for β4-α3 + β4 receptors (n = 4) and compared with those from α3β4 receptors expressed from monomeric constructs (n = 10). The tandem-containing receptors were found to be macroscopically indistinguishable from α3β4 receptors (EC₅₀ = 122 ± 8 vs. 134 ± 6 µM; n_H = 1.98 ± 0.21 vs. 1.73 ± 0.11, means ± S.D.).

We next wanted to check that receptors expressed from β4-α3 with β4 did actually contain two copies each of the α and β subunits from the tandem construct and one copy of the subunit from the β monomer cRNA. This was done by mutating from leucine to threonine the 9Ô residue in the pore-lining region of the β subunit contained in the tandem construct. In channels of the nicotinic superfamily, incorporation of this mutation increases the receptor agonist sensitivity by an amount that is roughly proportional to the number of mutation copies present. Expressing β4-α3 + β4^LT should give rise to a homogeneous population of receptors with a dose-response curved shifted to the left because of the presence of a single copy of the mutation. Nevertheless, dose-response curves obtained from such mutant combinations typically showed more than one component, indicating the simultaneous presence in the receptor population of channels containing different numbers of mutation copies and therefore different numbers of β subunit produced by the monomer cRNA.

These results indicate that caution has to be exercised in the interpretation of data from the expression of tandem constructs of nicotinic subunits.

This work was supported by the MRC, The Wellcome Trust (project grant 064652) and the School of Pharmacy.