Time-Resolved Photolabeling of the Nicotinic Acetylcholine Receptor Identifies the Site of Action of a Resting State Inhibitor

David C. Chiara†, George H. Addona‡*, Marek A. Kloczewiak*, Jonathan B. Cohen†, and Keith W. Miller‡*.
†Department of Neurobiology; ‡Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115; and *Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, Massachusetts 02114

TID (3-(trifluoromethyl)-3-(m-iodophenyl) diazirine) is a resting state inhibitor of cation flux through the nicotinic acetylcholine receptor (nAChR) with an apparent on-rate constant of 1.2 to 3.4 x 106 M-1 s-1.  This hydrophobic, photoactivatable drug binds and photoincorporates within the ion channel domain and at the lipid-protein interface.  To determine which of these is responsible for resting state inhibition, a time-resolved photolabeling apparatus was devised.  At the subunit level, about half the [125I]TID photolabeling occurred at the shortest preincubation time (~2 ms).  The remainder increased exponentially with preincubation time, having a rate of 12 to 14 s-1 at 4 M TID (equivalent to an apparent second order rate constant of 3 x 106 M-1 s-1).  Preparative labelings were undertaken for preincubation times of 2 and 150 ms and at equilibrium.  The data at 150 ms and equilibrium were similar.  At 2 ms, labeling in the M4 hydrophobic segment was already close to the equilibrium value.  In contrast, at 2 ms, labeling at the 9' and 13' residues of delta M2 was a tenth that at equilibrium.  We conclude that TID's access to the ion channel is  more restricted than to the lipid-protein interface, and that TID pre-bound within the ion channel is responsible for flux inhibition upon activation of the receptor.