Functional stoichiometry at the nicotinic receptor. The photon cross section for phase 1 corresponds to two bis-Q molecules per channel.

Abstract

Changes in the agonist-induced conductance that occur when the agonist-receptor complex is perturbed were examined. Voltage- clamped Electrophorus electroplaques are exposed to the photoisomerizable agonist trans-bis-Q [3,3''-bis-[.alpha.(trimethylammonium)methyl] azobenzene]. A 1-.mu.s laser flash photoisomerizes some trans-bis-Q molecules bound to receptors; because the cis configuration is not an agonist, receptor channels close within a few microseconds. This effect is called phase 1. The fraction of channels that close during phase 1 (parameter a) was compared with the fraction of trans-bis-Q molecules that undergo trans .fwdarw. cis photoisomerization (parameter b). Parameter a is measured as the fractional diminution in voltage-clamp currents during phase 1. Parameter b is measured by changes in the optical spectra of bis-Q solutions caused by flashes. At low flash intensities, a is twice b, which shows that the channel can be closed by photoisomerizing either of 2 bound agonist molecules. Conventional dose-response studies with trans-bis-Q also give a Hill coefficient of 2. As a partial control for changes in the photochemistry caused by binding of bis-Q to receptors, spectral measurements are performed on the photoisomerizable agonist QBr, covalently bound to solubilized acetylcholine receptors [AChR] for Torpedo. The bound and free agonist molecules have the same photoisomerization properties. Evidently, the open state of the AChR channel is much more likely to be associated with the presence of 2 bound agonist molecules than with a single such molecule.