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The roles of aromatic residues in the glycine receptor transmembrane domain.
Tang B
,
Lummis SCR
.
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BACKGROUND: Cys-loop receptors play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular, transmembrane (TM) and intracellular domain. Each TM domain contains 4 α-helices (M1-M4) joined by loops of varying lengths. Many of the amino acid residues that constitute these α-helices are hydrophobic, and there has been particular interest in aromatic residues, especially those in M4, which have the potential to contribute to the assembly and function of the receptor via a range of interactions with nearby residues.
RESULTS: Here we show that many aromatic residues in the M1, M3 and M4 α-helices of the glycine receptor are involved in the function of the receptor. The residues were explored by creating a range of mutant receptors, characterising them using two electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using currently available structural information on the open and closed states of the receptor. For 7 residues function was ablated with an Ala substitution: 3 Tyr residues at the extracellular end of M1, 2 Trp residues located towards the centers of M1 and M3, and a Phe and a Tyr residue in M4. For many of these an alternative aromatic residue restored wild-type-like function indicating the importance of the π ring. EC50s were increased with Ala substitution of 8 other aromatic residues, with those in M1 and M4 also having reduced currents, indicating a role in receptor assembly. The structure shows many potential interactions with nearby residues, especially between those that form the M1/M3/M4 interface, and we identify those that are supported by the functional data.
CONCLUSION: The data reveal the importance and interactions of aromatic residues in the GlyR M1, M3 and M4 α-helices, many of which are essential for receptor function.
Fig. 1. Alignment of the transmembrane domain of the GlyR α1 subunit with those from a range of other pLGIC subunits showing the aromatic residues examined in this study (in red), and the location of residues in the TMD of one of the 5 subunits that constitute the receptor, showing that many are located at the M1/M3/M4 interface
Fig. 2. Example concentration response curves and maximal current traces for WT and two mutant GlyR. Data = mean ± SEM, n = 4–8. Parameters obtained from these curves are shown in Table 1. Scale bars are 2 μA and 10 s
Fig. 3. Maximal currents elicited by glycine are lower in mutants with Ala substitutions to aromatics in M1 and M4. Data = mean ± SEM, n = 4–8, * = significantly different to WT, p < 0.05, ANOVA with Dunnetts post-comparison test
Fig. 4. Structures of Gly, GLIC and GABAA receptors showing possible interactions of Y222 and Y223 or equivalent residues. GlyRo = GlyR in open state; GlyRc = GlyR in closed state
Fig. 5. A Y228 and W286 are well positioned to form a T-type π–π interaction; B These residues, with Y406,W407 and Y410, form an aromatic cluster at the top of the M1/M3/M4 interface. C Y406 could form a hydrogen bond (green dashes) with either A282 or Y228; the data better support the former
Fig. 6. GlyR in the open (A) and closed (B) states show similar interactions between W239, F293 and F399. Structural data show equivalent residue interactions are possible in the GABAA receptor (C) and GLIC (D)
Fig. 7. Distances between W239 and F395 differ in the open (A) and closed (B) states, although those between F395 and F242 do not. Structural data show equivalent residue interactions in this region are possible in the GABAA receptor (C) and GLIC (D)
Fig. 8. Y301 in M3 (A) could form a hydrogen bond with M246 and/or a cation–π interaction with R252 in the open state (B, C) but probably not the former in the closed state (D, E). Similar interactions are possible in the GABAA receptor (F, G)
Fig. 9. F306 could form a cation–π interaction with R392 in the open state (A), but R392 is more likely to do this with W243 in the closed state (B)
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