Time-resolved photolabeling of membrane proteins: application to the nicotinic acetylcholine receptor

An apparatus has been developed that allows photoaffinity ligands to be crossed-linked to milligram quantities of membrane proteins with maximum attainable yield following contact times of approximately 1 ms. The apparatus consisted of three parts: a conventional rapid mixing unit, a novel freeze-quench unit, and a photolabeling unit. The freeze-quench unit consisted of a rapidly rotating metal disk which was precooled in liquid nitrogen. Correct alignment of the exit jet from the sample mixer allowed up to 2 ml of sample to be frozen in a thin film on the disk. Experiments with colorimetric reactions showed the combined dead time of mixing and freeze-quenching to be submillisecond. Photoincorporation was maximized by prolonged irradiation of the freeze-quenched sample. Using this apparatus we determine the binding kinetics of the resting state channel inhibitor 3-[125I](trifluoromethyl)-3-(m-iodophenyl) diazirine (TID) to nicotinic acetylcholine receptor-rich membranes from Torpedo. The binding kinetics for the 125I-labeled alpha and delta subunits were biphasic; about half the binding was complete by 2.4 ms, and the remainder could be resolved and occurred with a pseudo-first-order rate constant determined at 4 microM [125I]TID of 12.0 +/- 2.3 and 13.6 +/- 4.0 s-1, respectively. This compares well to the same constant determined for the inhibition of agonist-induced cation flux in Torpedo membranes.