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

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

Expression of mouse-Torpedo acetylcholine receptor subunit chimeras and hybrids in Xenopus oocytes

In this study, in vitro synthesized mRNA encoding mouse and Torpedo nicotinic acetylcholine receptor subunits was injected into Xenopus oocytes, followed by assays for assembly onto the oocyte surface (using [¹²⁵I]α-bungarotoxin binding) and for acetylcholine-induced conductances (using voltage clamp). We constructed hybrid acetylcholine receptors in Xenopus oocytes by injecting all 8 possible combinations of 4 subunit-specific mRNAs in which a single subunit is derived from the other species. For each hybrid combination, there is detectable assembly and conductance. We also constructed cDNA clones that encode chimeric acetylcholine receptor subunits in which part of the γ subunit from Torpedo was replaced by the homologous region of the δ subunit from mouse. None of the chimeric subunits was able to replace the Torpedo γ, mouse δ, or Torpedo δ subunit with regard to assembly or function. We therefore conclude that widely spaced (and unknown) parts of the protein chain are required for the intersubunit interactions that eventually lead to functional assembly of the receptor

Expression of mouse-Torpedo acetylcholine receptor subunit chimeras and hybrids in Xenopus oocytes

In this study, in vitro synthesized mRNA encoding mouse and Torpedo nicotinic acetylcholine receptor subunits was injected into Xenopus oocytes, followed by assays for assembly onto the oocyte surface (using [¹²⁵I]α-bungarotoxin binding) and for acetylcholine-induced conductances (using voltage clamp). We constructed hybrid acetylcholine receptors in Xenopus oocytes by injecting all 8 possible combinations of 4 subunit-specific mRNAs in which a single subunit is derived from the other species. For each hybrid combination, there is detectable assembly and conductance. We also constructed cDNA clones that encode chimeric acetylcholine receptor subunits in which part of the γ subunit from Torpedo was replaced by the homologous region of the δ subunit from mouse. None of the chimeric subunits was able to replace the Torpedo γ, mouse δ, or Torpedo δ subunit with regard to assembly or function. We therefore conclude that widely spaced (and unknown) parts of the protein chain are required for the intersubunit interactions that eventually lead to functional assembly of the receptor