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Sci Rep
2017 Mar 27;71:450. doi: 10.1038/s41598-017-00573-2.
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Pentameric ligand-gated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function.
Therien JP
,
Baenziger JE
.
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Although transmembrane helix-helix interactions must be strong enough to drive folding, they must still permit the inter-helix movements associated with conformational change. Interactions between the outermost M4 and adjacent M1 and M3 α-helices of pentameric ligand-gated ion channels have been implicated in folding and function. Here, we evaluate the role of different physical interactions at this interface in the function of two prokaryotic homologs, GLIC and ELIC. Strikingly, disruption of most interactions in GLIC lead to either a reduction or a complete loss of expression and/or function, while analogous disruptions in ELIC often lead to gains in function. Structural comparisons suggest that GLIC and ELIC represent distinct transmembrane domain archetypes. One archetype, exemplified by GLIC, the glycine and GABA receptors and the glutamate activated chloride channel, has extensive aromatic contacts that govern M4-M1/M3 interactions and that are essential for expression and function. The other archetype, exemplified by ELIC and both the nicotinic acetylcholine and serotonin receptors, has relatively few aromatic contacts that are detrimental to function. These archetypes likely have evolved different mechanisms to balance the need for strong M4 "binding" to M1/M3 to promote folding/expression, and the need for weaker interactions that allow for greater conformational flexibility.
Figure 1. Side chain chemistry at the M4-M1/M3 interfaces of GLIC and ELIC. Structures of (A) GLIC (PDB: 4HFI) and (B) ELIC (B, PDB: 2VL0) showing on the left the full structure. The boxes on the right show the highlighted regions of GLIC and ELIC. Residues are coloured depending on their properties, aromatic (yellow), hydrogen bonding (green), negatively charged (red) and positively charged (blue). A water molecule in GLIC’s M4-M1/M3 interface is also shown (cyan). Note that aliphatic residues are not shown for clarity. (C) The centroid distances between interacting pairs of aromatic residues at the M4-M1/M3 interfaces of GLIC and ELIC.
Figure 2. Functional characterization of the GLIC and ELIC mutants. Whole cell electrophysiological traces were recorded using two-electrode voltage clamp electrophysiology. Currents were recorded from Xenopus laevis oocytes expressing either GLIC (Left panel) or ELIC (Right panel) in response to protons or cysteamine, respectively. The lower panels presents dose response curves (normalized current (I/Imax) versus ligand concentration) for select Ala mutants, with the number (n) of averaged traces. Error bars represent S.E.
Figure 3. Functional effects of Ala mutations at the M4-M1/M3 interface in GLIC. (A) Changes in the pH50 resulting from Ala mutations of residues on M4 (top right panel), M1 (bottom left) and M3 (bottom right). Residues and bar graphs are colour-coded as in Fig. 1, with aliphatic residues tan. The bar graphs represent the magnitude of change ± the standard deviation. (B) Changes in the pH50 values are heat-mapped onto the GLIC structure (PDB: 4HFI). The magnitude of the shift in pH50 is depicted via colour intensity, with no change in pH50 in white, gain-of-function in red, and loss-of-function in blue. Mutants that failed to express and/or function are shown in black.
Figure 4. Functional effects of Ala mutations at the M4-M1/M3 interface in ELIC. (A) Changes in the −log(EC50) resulting from Ala mutations of residues on M4 (top right panel), M1 (bottom left) and M3 (bottom right). Residues and bar graphs are colour-coded as in Fig. 3. The bar graphs represent the magnitude of change ± the standard deviation. (B) Changes in the −log(EC50) values are heat-mapped onto an ELIC homology model (based on GLIC structure). The magnitude of the shift in pH50 is depicted via colour intensity, as in Fig. 3. P304A mutant is coloured grey, due to altered desensitization kinetics.
Figure 5. The M4-M1/M3 interface of different pLGICs. Structures of GluCl (top left, PDB: 4TNW), GlyR (top right, PDB: 5CFB), nAChR (bottom left, PDB: 2BG9), and α4β2 (bottom right, PDB: 5KXI). Each structure shows a zoomed in region of the M4-M1/M3 interface of a single subunit. Residues are coloured depending on their properties: aromatic (yellow), hydrogen bonding (green), negatively charged (red) and positively charged (blue).
Figure 6. Sequence alignments for M1, M3 and M4 in a number of pLGICs. Residues facing the M4-M1/M3 interface are highlighted and colored depending on their properties: aromatic (yellow), hydrogen bonding (green), negatively charged (red) and positively charged (blue).
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