Desensitization of the Torpedo Nicotinic Acetylcholine Receptor Linked to Movement of the Agonist Site Residues gTrp-55 and dTrp-57.

David C. Chiara and Jonathan B. Cohen
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

The Torpedo nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel consisting on a pentamer of four homologous subunits arranged as agadb.  There are two agonist/competitive antagonist binding sites on each receptor located at the a-g and a-d interfaces (Pedersen and Cohen, PNAS 87: 2785-2789, 1990).  The competitive antagonist d-tubocurarine (dTC) specifically photoincorporates into residues in the a-, g-, and d-subunits.  For the g- and d-subunits, the major sites of [3H]dTC incorporation are gTrp-55 and dTrp-57 (Chiara and Cohen, Biophys. J. 61: A106, 1992).  To investigate structural changes in the agonist sites due to the transition from the resting state to the desensitized state, nAChR-rich membranes were photolabeled with 5 mM [3H]dTC (>99% occupancy of the a-g dTC binding site and ~50% occupancy of the a-d dTC site) in the presence of various concentrations of tetracaine or proadifen, non-competitive antagonists (channel blockers) that stabilize the resting and desensitized states of the nAChR, respectively.  [3H]dTC incorporation into the g- and d-subunits increased 2-fold in the presence on tetracaine (EC50 @ 1 mM).  Proadifen caused a 50% reduction in the [3H]dTC labeling of the g-subunit (EC50 @ 100 nM) with no detectable change in the d-subunit incorporation level.  These results indicate that the photoreactive region of dTC is sensitive to changes in the structure of the binding sites between resting and desensitized states, with enhanced reactivity of the non-alpha residues, gTrp-55 and dTrp-57, a property of of the resting state.