Functional Stoichiometry at the Nicotinic Receptor 51 5

These experiments examine changes in the agonist-induced con-ductance that occur when the agonist-receptor complex is perturbed. Voltage-clamped Electrophorus electroplaques are exposed to the photoisomerizable ago-nist trans-Bis-Q A 1-hs 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 hundred microseconds. This effect is called phase 1. We compare (a) the fraction of channels that close during phase 1 with (b) the fraction of trans-Bis-Q molecules that undergo trans--+ cis photoisomerization. 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 two bound agonist molecules. Conventional dose-response studies with trans-Bis-Q also give a Hill coefficient of two. 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 acetyl-choline receptors from Torpedo. The bound and free agonist molecules have the same photoisomerization properties. These results verify the concept that the open state of the acetylcholine receptor channel is much more likely to be associated with the presence of two bound agonist molecules than with a single such molecule