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 > 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 beta- and gamma-subunits, whereas in the alpha-subunit, it changed little. The most dramatic change occurred in the delta-subunit, where it increased to 1.6 within 10 ms but fell to 0.7 during fast desensitization. Four residues in the delta-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 (deltaThr274 and deltaLeu278) are situated toward the extracellular end of helix M2, both contralateral to the ion channel and adjacent to the third residue (deltaPhe232) on M1. The fourth labeled residue (deltaIle288) 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 delta-subunit during gating.
Highlights
General anesthetics often interact more strongly with sites on open than on closed states of ligand-gated ion channels
Torpedo membranes enriched in nicotinic acetylcholine receptors were preincubated with the hydrophobic probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine ([125I]TID) and exposed to agonist for either 0 ms, 1.5 and 10 ms, 1 s, or >1 h and rapidly frozen (
With reference to the nicotinic acetylcholine receptors (nAChRs) closed state structure [6], three of the additional activation-dependent amino acid residues photolabeled by TID are clustered together on M1 and M2, and the fourth is on the M2-M3 loop close to the start of M3, suggesting the formation of a transient hydrophobic pocket in that region during activation
Summary
The fourth labeled residue (␦Ile288) 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 ␦-subunit during gating. General anesthetics exert their primary actions on ion channels of neuronal plasma membranes, at synapses. With reference to the nAChR closed state structure [6], three of the additional activation-dependent amino acid residues photolabeled by TID are clustered together on M1 and M2, and the fourth is on the M2-M3 loop close to the start of M3, suggesting the formation of a transient hydrophobic pocket in that region during activation
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