A continuous-flow, rapid-mixing, photolabeling technique applied to the acetylcholine receptor.
A continuous-flow technique is described in which a photoaffinity label, membrane rich in acetylcholine receptor, and various effectors are rapidly mixed, passed through a delay tube, through a tube in which they are irradiated, and are collected in a tube containing quencher. Delay times as short as 20 ms between mixing and photolysis are achievable. Because the flow is continuous, milliliter volumes of membrane can be labeled in a single run, which is convenient for the analysis of both the functional effects and sites of photolabeling. Using this technique, we have found that receptor in its transitory, active state, in which the channel is open, is more susceptible to photolabeling by the noncompetitive inhibitor analog [3H] quinacrine azide than is receptor in either its resting or desensitized states, in which the channel is closed. This technique should prove generally useful for the photolabeling of transient conformational states of macromolecules.
D000109:Acetylcholine / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002462:Cell Membrane / Q000378:metabolism; D010782:Photolysis / Q000295:instrumentation / Q000379:methods*; D011950:Receptors, Cholinergic / Q000032:analysis* / Q000378:metabolism; D013997:Time Factors; D014101:Torpedo
D000345:Affinity Labels; D001386:Azides; D011950:Receptors, Cholinergic; C032798:quinacrine azide; D000109:Acetylcholine