FIG. 2.

The ligand binding site and the proposed mechanism for gating the ion pore. A: in this depiction, an agonist-binding α subunit (dark blue) and a structural β subunit (in light blue) are shown with a solid surface looking from the extracellular side with the subunit pair slightly tipped away from the pore. When agonist is bound (as shown for nicotine, red), the α C-loop is moved towards the structural subunit to cap the agonist-binding site and effectively encase the ligand in the deep cleft formed between the subunits. The α Cys-Cys pair (187–188) is in yellow. Other residues interacting with the ligand from the α subunit are colored green and from the β subunit are colored in orange. The circled region is enlarged and the surface removed to reveal in B the amino acids within the agonist-binding site that interact with nicotine. The same color scheme is used, and the residues interacting to form the agonist-binding site are named and numbered. The arrows indicate β-strand structure. The weak lines interacting with nicotine (whose electrostatic surface is in light red) are hydrogen bonds. Certain key residues include tryptophan 143 (W143) from the α subunit which contributes to forming the base of the agonist-binding site and α-tyrosine 185 (Y185), which is important to stabilize the ligand within the pocket upon entry. In the α5 nAChR subunit, this residue is an aspartic acid that introduces a potentially negatively charged group into the pocket to inhibit ligand binding. As indicated by the extent of the molecular surface of nicotine (shown in transparent red), these hydrophobic residues from both subunit faces further stabilize the ligand in the pocket through van der Waals interactions, and other residues not shown (including D85, located near W143) also contribute to ligand binding through stabilizing the position of pocket residues. [Adapted from the 2.7-Å resolution X-ray structure of the AChBP (Protein Data Bank ID 1I9B.pdb) and the images generated in UCSF Chimera by Pettersen et al. (375).] B: upon binding of agonist and capping of the ligand-binding site (1), rotational motion in the β-strands is transmitted through the subunit (2) to residues that are near the TM domain-membrane interface. At this point, the rotational motion imparts two important interactions. The first is to move the loop between β-strands β1 and β2 towards the linking sequence of TM2 and TM3. This positions an invariant valine (V44) into the hydrophobic pocket that is created by the proximity of proline-272 (P272) and serine-269 (S269). These amino acids, or conservative changes, are present in most nAChRs. At the same time, the β10 strand moves counterclockwise to position arginine-209 (R209) towards glutamic acid-45 (E45; also β1 strand) to form an ionic (salt) bond. These interactions result in the rotation of TM4 ~15° to move the hydrophobic gating residues [valines (V255) and (V259) and leucine (L251)] away from the pore and the polar S248 and S252 toward the widened channel. The relief of the gate allows the channel to completely hydrate and conduct ions (5). Residues at the extracellular and intracellular faces (e.g., E241) ring the channel. These residues vary among subunits and receptors as polar and/or charged and contribute to determining the relative ion current through the pore. Also, highly charged rings of amino acids such as E241 enhance certain ion permeability such as by Ca²⁺. [Model shown is based on the original study of Unwin (475) taken from electron microscopy studies of channel gating from the Torpedo nAChR (Protein Data Bank code 2BG9) and from high-resolution studies of the AChBPs (see text for details).]