Stoichiometry of human recombinant neuronal nicotinic receptors containing the β3 subunit expressed in Xenopus oocytes

1. The neuronal nicotinic subunit β3 forms functional receptors when co-expressed with both an α and a β subunit, such as α3 and β4. We examined the subunit stoichiometry of these 'triplet' α3β4β3 receptors by expression in Xenopus oocytes of the α3, β4 and β3 subunits, either in wild-type form or after insertion of a reporter mutation. 2. The mutation chosen was the substitution of a conserved hydrophobic residue in the second transmembrane domain of the subunits (leucine or valine 9′) with a hydrophilic threonine. In other ion channels within the nicotinic superfamily, this mutation type consistently increases the potency of agonists. In muscle-type nicotinic receptors, the magnitude of this effect is approximately constant for each mutant subunit incorporated. 3. In α3β4β3 receptors, the ACh EC₅₀ was decreased by approximately 17-fold when this mutation was in α3 alone and only by fourfold when β3 alone was mutated. Mutating β4 was equivalent to mutating α3, suggesting that the 'triplet' receptor contains one copy of β3 and two copies each of α3 and β4. 4. Mutating β3 and α3 or β3 and β4 reduced the ACh EC₅₀ further, to values two- to threefold lower than those seen when only α3 or β4 carried the mutation. 5. In 'pair' α3β4 receptors (known to contain two α and three β subunits), mutating β4 had a greater effect on the ACh EC₅₀ than mutating α3, in agreement with an α:β ratio of 2:3 and a constant and independent effect of each copy of the mutation. 6. Our results suggest that α3β4β3 neuronal nicotinic receptors contain one copy of β3 and two copies each of α3 and β4 and confirm that in pair α3β4 receptors the α/β subunits are present in a 2:3 ratio.