Electron density maps of the muscle-type nAChR have been obtained at medium resolution
(~6-Å) shortly (~10 ms) after spraying with ACh Torpedo postsynaptic membrane fragments annealed
into 2D tubular crystals, followed by quick-freezing and crystallographic analysis of cryo-EM images
(Unwin and Fujiyoshi 2012). They suggest that the hetero-oligomeric nature of the pentamer induces a
degree of asymmetry in the contribution of the various subunits to opening of the channel and that
changes at the level of the protein/membrane interface remain very limited (Fig. 1.30).
But for the EM data on T. marmorata nAChR, all of the medium- to high-resolution data
discussed thus far bear on homopentameric pLGICs whose natural ligands are not ACh. The X-ray
structure of a heteropentameric ACh receptor has been recently obtained, that of the human central
nervous system α4β2 receptor (the numbers referring to the subunit subtype), whose subunit stoichiometry, in the structure solved, is α 2 β 3 (Morales-Perez et al. 2016). This receptor is the most abundant
nAChR in the brain and is involved in nicotine addiction. Its structure was solved in complex with
either nicotine or an iodinated nicotine analog and was suggested to represent a desensitized state,
with the TM channel closed. In addition to providing the first view of a heteropentameric
pLGIC (Fig. 1.31), and explaining why α/β interfaces bind ACh whereas β/α and β/β ones do not,
the structure offers, by comparison with previously established structures of other pLGICs in various
conformations, further glimpses into the mechanism whereby transconformations of the extracellular
domain induced by ACh binding are transduced into conformational transitions in the TM one
(Fig. 1.32). As noted by the authors, the conclusions have to be taken with precaution, because all
structures but those of T. marmorata nAChR have been obtained in a detergent environment, whereas
the membrane environment is known to influence pLGIC function. Furthermore, whereas the overall
structures of pLGICs are remarkably conserved throughout evolution, there are nevertheless
differences between them, requiring caution when comparing closed, open, and partially or deeply
desensitized conformations observed on different receptors (Nemecz et al. 2016).
Fig. 1.29 Coupling between the extracellular and TM regions of pLGICs. The interlocking of residues at
the interface between the two regions is compared in the active A vs. the resting R state, based on the
structures of GLIC at pH 4 (open, left) (Sauguet et al. 2013) and pH 7 (closed, right) (Sauguet et al. 2014).
A few critical residues (Torpedo numbering) are shown as van der Waals spheres. V46 and V132 from the
extracellular region (blue in the A state, and green in the R one) interact with an absolutely conserved
proline residue in the M2-M3 loop of the TM region, P265 (light orange), to form a pin-in-socket assembly
in the active state, which disassembles in the resting state. This governs the rearrangement of the TM helix
bundle and, in particular, the movement of the outermost part of M2, opening and closing the TM pore
(Adapted from Taly et al. 2014).
1.6 Dynamics of Transmembrane Regions and the Function of Membrane Proteins
41
(~6-Å) shortly (~10 ms) after spraying with ACh Torpedo postsynaptic membrane fragments annealed
into 2D tubular crystals, followed by quick-freezing and crystallographic analysis of cryo-EM images
(Unwin and Fujiyoshi 2012). They suggest that the hetero-oligomeric nature of the pentamer induces a
degree of asymmetry in the contribution of the various subunits to opening of the channel and that
changes at the level of the protein/membrane interface remain very limited (Fig. 1.30).
But for the EM data on T. marmorata nAChR, all of the medium- to high-resolution data
discussed thus far bear on homopentameric pLGICs whose natural ligands are not ACh. The X-ray
structure of a heteropentameric ACh receptor has been recently obtained, that of the human central
nervous system α4β2 receptor (the numbers referring to the subunit subtype), whose subunit stoichiometry, in the structure solved, is α 2 β 3 (Morales-Perez et al. 2016). This receptor is the most abundant
nAChR in the brain and is involved in nicotine addiction. Its structure was solved in complex with
either nicotine or an iodinated nicotine analog and was suggested to represent a desensitized state,
with the TM channel closed. In addition to providing the first view of a heteropentameric
pLGIC (Fig. 1.31), and explaining why α/β interfaces bind ACh whereas β/α and β/β ones do not,
the structure offers, by comparison with previously established structures of other pLGICs in various
conformations, further glimpses into the mechanism whereby transconformations of the extracellular
domain induced by ACh binding are transduced into conformational transitions in the TM one
(Fig. 1.32). As noted by the authors, the conclusions have to be taken with precaution, because all
structures but those of T. marmorata nAChR have been obtained in a detergent environment, whereas
the membrane environment is known to influence pLGIC function. Furthermore, whereas the overall
structures of pLGICs are remarkably conserved throughout evolution, there are nevertheless
differences between them, requiring caution when comparing closed, open, and partially or deeply
desensitized conformations observed on different receptors (Nemecz et al. 2016).
Fig. 1.29 Coupling between the extracellular and TM regions of pLGICs. The interlocking of residues at
the interface between the two regions is compared in the active A vs. the resting R state, based on the
structures of GLIC at pH 4 (open, left) (Sauguet et al. 2013) and pH 7 (closed, right) (Sauguet et al. 2014).
A few critical residues (Torpedo numbering) are shown as van der Waals spheres. V46 and V132 from the
extracellular region (blue in the A state, and green in the R one) interact with an absolutely conserved
proline residue in the M2-M3 loop of the TM region, P265 (light orange), to form a pin-in-socket assembly
in the active state, which disassembles in the resting state. This governs the rearrangement of the TM helix
bundle and, in particular, the movement of the outermost part of M2, opening and closing the TM pore
(Adapted from Taly et al. 2014).
1.6 Dynamics of Transmembrane Regions and the Function of Membrane Proteins
41