Equilibrium properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes.

Abstract

A B S T R A C T This study used messenger RNA encoding each subunit (a, fl, % and 6) 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 a, fl, % and ~ 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 [12sI]a-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 (>l,000-fold range at 1 #M). 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 a-bungarotoxin-binding sites. Five combinations were tested for dtubocurarine inhibition by the dose-ratio method; the apparent dissociation constant ranged from 0.08 to 0.27 #M. 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 fl subunit on voltage sensitivity of the response: gACh(--90 mV)/gAch(+30 mV) is always at least 1, but this ratio increases by an average of 3.5-fold if fl~a replaces fiT. Also, combinations including "YT or 6M usually produce greater receptor assembly than combinations including the homologous subunit from the other species. Finally, EACh is defined as the concentration of ACh inducing 1 #S/fmol at -60 mV; EACh is consistently lower for am. We conclude that receptor assembly, voltage sensitivity, and EACh are governed by different properties.

Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

“…It remains to be seen whether an equivalent number of receptors appears on the oocyte surface and whether channels open with a similar dependence on agonist concentration. The present partial dose-response relations, the more complete measurements of Bertrand et al (1990), and the partial measurements of Yoshii, Yu, Mixter-Mayne, Davidson & Lester (1987) suggest that the o4/32 neuronal receptor has a higher sensitivity to ACh than the muscle receptor.…”

Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region.

“…Sources of the cDNA clones for the mouse and Torpedo et, f~, ~1, and 8 subunits have been described previously (White et al, 1985;Yoshii, Yu, Mixter-Mayne, Davidson, and Lester, 1987). The mouse subunits were cloned from the BC3H-1 cell line.…”

Improved method for heterologous expression of ion channels inXenopusOocyte: A PCR shortcut to Oocyte expression

“…125 I] aBgt, a specific antagonist to the a 7 receptor, according to the method described previously (Yoshii et al, 1987). In short, oocytes were prewashed in a solution of 96 mM NaCI, 2 mM KCI, 1 mM MgCI 2 , 5 mM HEPES, pH 7.6, and then placed in the same solution supplemented with 1 mg/ml bovine serum albumin and 5 nM [ 125 I] aBgt for 1 h. Oocytes were then washed four times and counted individually in a gamma counter (total binding).…”

“…In short, oocytes were prewashed in a solution of 96 mM NaCI, 2 mM KCI, 1 mM MgCI 2 , 5 mM HEPES, pH 7.6, and then placed in the same solution supplemented with 1 mg/ml bovine serum albumin and 5 nM [ 125 I] aBgt for 1 h. Oocytes were then washed four times and counted individually in a gamma counter (total binding). Nonspecific binding was determined from the mock-injected or uninjected oocytes (Yoshii et al, 1987).…”

“…This variation among oocytes is a general phenomenon in many laboratories that study channels and receptors induced by foreign RNA. This is seen, for instance, with Torpedo nAChR (Yoshii et al, 1987). Therefore, we attempted to minimize the cell-to-cell variation in order to draw conclusions.…”