Gating-enhanced accessibility of hydrophobic sites within the transmembrane region of the nicotinic acetylcholine Receptor's δ-subunit : A time-resolved photolabeling study

Abstract General anesthetics often interact more strongly with sites on open than on closed states of ligand-gated ion channels. To seek such sites, Torpedo membranes enriched in nicotinic acetylcholine receptors (nAChRs) were preincubated with the hydrophobic probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine ([125I]TID) and exposed to agonist for either 0 ms (closed state), 1.5 and 10 ms (activated states), 1 s (fast desensitized state), or ≥1 h (equilibrium or slow desensitized state) and then rapidly frozen (<1 ms) and photolabeled. Within 1.5 ms, the fractional change in photoincorporation relative to the closed state decreased to 0.7 in the β- and γ-subunits, whereas in the α-subunit, it changed little. The most dramatic change occurred in the δ-subunit, where it increased to 1.6 within 10 ms but fell to 0.7 during fast desensitization. Four residues in the δ-subunit's transmembrane domain accounted for the enhanced photoincorporation induced by a 10-ms agonist exposure both when TID was added simultaneously with agonist and when it was preincubated with membranes. In the published closed state structure, two residues (δThr274 and δLeu278) are situated toward the extracellular end of helix M2, both contralateral to the ion channel and adjacent to the third residue (δPhe232) on M1. The fourth labeled residue (δIle288) is toward the end of the M2-M3 loop. Contact with these residues occurs on the time scale of a rapid phase of TID inhibition in Torpedo nAChRs, suggesting the formation of a transient hydrophobic pocket between M1, M2, and M3 in the δ-subunit during gating.