Equilibrium Properties of Mouse-Torpedo Acetylcholine Receptor Hybrids Expressed in Xenopus Oocytes

Abstract

This study used messenger RNA encoding each subunit (α, β, γ and δ) of the nicotinic acetylcholine (ACh) receptor from mouse BC3H-1 cells and from Torpedo electric organ. The mRNA was synthesized in vitro by transcription with SP6 polymerase from cDNA clones. All 16 possible combinations that include one mRNA for each of α, β, γ and δ were injected into oocytes. After allowing 2-8 d for translation and assembly, we assayed each oocyte for (a) receptor assembly, measured by the binding of [^125]α-bungarotoxin to the oocyte surface, and (b) ACh-induced conductance, measured under voltage clamp at various membrane potentials. All combinations yielded detectable assembly (30-fold range among different combinations) and ACh-induced conductances (>1,000-fold range at 1 µM). On double-logarithmic coordinates, the dose-response relations all had a slope near 2 for low concentrations of ACh. Data were corrected for variations in efficiency of translation among identically injected oocytes by expressing ACh-induced conductance per femtomole of α-bungarotoxin-binding sites. Five combinations were tested for d-tubocurarine inhibition by the dose-ratio method; the apparent dissociation constant ranged from 0.08 to 0.27 µM. Matched responses and geometric means are used for describing the effects of changing a particular subunit (mouse vs. Torpedo) while maintaining the identity of the other subunits. A dramatic subunit-specific effect is that of the β subunit on voltage sensitivity of the response: g_ACh(-90 mV)/g_Ach(+30 mV) is always at least 1, but this ratio increases by an average of 3.5-fold if β_M replaces β_T. Also, combinations including γ_T or δ_M usually produce greater receptor assembly than combinations including the homologous subunit from the other species. Finally, E_ACh is defined as the concentration of ACh inducing 1 µS/fmol at -60 mV; E_ACh is consistently lower for α_m. We conclude that receptor assembly, voltage sensitivity, and E_ACh are governed by different properties.