Aromatic Residues in the Fourth Transmembrane-Spanning Helix M4

Oct 23, 2017 - in the GABAρ receptor transmembrane domain using site-directed muta- genesis and subsequent expression in HEK293 cells, probing functi...
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Aromatic Residues in the Fourth Transmembrane-Spanning Helix M4 Are Important for GABAρ Receptor Function James Cory-Wright, Mona Alqazzaz, Francesca Wroe, Jenny Jeffreys, Lu Zhou, and Sarah C. R. Lummis* Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB 1QW, United Kingdom ABSTRACT: GABAρ receptors are a subfamily of the GABAA receptor family of pentameric ligand-gated ion channels (pLGICs). Each of the five subunits has four transmembrane α-helices (M1−M4), with M4 most distant from the central pore. Aromatic residues in this M4 helix are important for receptor assembly in pLGICs and also may interact with adjacent lipids and/or residues in neighboring α-helices and the extracellular domain to modify or enable channel gating. This study examines the role of M4 receptor aromatic residues in the GABAρ receptor transmembrane domain using site-directed mutagenesis and subsequent expression in HEK293 cells, probing functional parameters using a fluorescent membrane-potential-sensitive dye. The data indicate that many of the aromatic residues in M4 play a role in receptor function, as substitution with other residues can ablate and/or modify functional parameters. Modeling showed that these residues likely interact with residues in the adjacent M1 and M3 α-helices and/or residues in the Cys-loop in the extracellular domain. We suggest that many of these aromatic interactions contribute to an “aromatic zipper”, which allows interactions between M4 and the rest of the receptor that are essential for function. Thus, the data support other studies showing that M4 does not play a passive role in “protecting” the other transmembrane helices from the lipid bilayer but is actively involved in the function of the protein. KEYWORDS: Cys-loop receptor, M4, mutagenesis, aromatic interaction, hydrophobic interaction

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much attention, and some clarity is emerging,5 but it is becoming apparent that there are other regions of the protein that are of critical importance. One of these is the outermost transmembrane-spanning segment, M4. This highly conserved region (Figure 1) was for many years thought to have its major role in protecting the other transmembrane domains and/or in receptor assembly, and there is good evidence to support the latter (e.g., see ref 6). However, more recent work suggests that this region also contributes significantly to receptor function, maybe in a modulatory but perhaps even an activatory role.6−9 The M4 α-helix faces the lipid, and it has long been known that lipid composition affects pLGIC gating: a lipid-facing mutation in the nACh receptor M4 results in a congenital myasthenic syndrome, and M4 is the site of action of some modulatory compounds, but there is currently no consensus to explain these findings.9,10 Interactions of M4 with adjacent α-helices appear to be important, and recent studies have led to the proposal that interactions of M4 with M1 and M3 are necessary to promote effective interactions between the M4 C-terminus and the Cys-loop; the latter is located at the interface of the ECD and the TMD and is important in coupling agonist binding to channel opening.11,12 Thus, the Cys-loop may adopt a nonfunctional conformation in the absence of effective M4− M1/M3 interactions, preventing channel opening. There have been rather limited studies to test this hypothesis, with the

INTRODUCTION Pentameric ligand-gated ion channels (pLGICs), such as the nicotinic acetylcholine (nACh) or GABAA receptor, are primarily responsible for fast synaptic transmission in the central nervous systems of both vertebrates and invertebrates.1 They are activated by the binding of a neurotransmitter such as acetylcholine (ACh) or γ-aminobutyric acid (GABA), which induces a structural change in the protein that causes the opening of an integral ion channel, allowing ion flux across the postsynaptic membrane. The receptors can be either homomeric or (more usually) heteromeric. Each of the five subunits possesses a large Nterminal extracellular domain (ECD), a transmembrane domain (TMD) consisting primarily of four membrane-spanning segments (M1−M4, the second of which, M2, lines the ionconducting pore), and, usually, an intracellular domain between M3 and M4. Structural details have been slow to arrive, but in the past few years the structures of the first mammalian pLGICs have been solved.2−4 These have largely confirmed details obtained from the invertebrate GluCl receptor and two homologous bacterial receptors, Erwinia ligand-gated ion channel (ELIC) and Gloeobacter ligand-gated ion channel (GLIC), the latter of which has stimulated much interest from structural biologists, with over 40 GLIC structures now in the database. There is still, however, much to be discovered about the relationship between the structure and function of these proteins. The primary activation pathway, leading from the neurotransmitter binding site to the pore, has been the focus of © XXXX American Chemical Society

Received: August 15, 2017 Accepted: October 23, 2017

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DOI: 10.1021/acschemneuro.7b00315 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

Figure 1. (A) M4 sequence alignment for GABAρ1, GABAβ3, GluCl, Gly α1, GLIC, and ELIC receptor subunits, showing their similarity in this region. Those aromatic residues that have been previously substituted with Ala and shown to modify function are colored as follows: blue = loss of function, green = gain of function, red = no function (data from refs 6, 7, and 12 and the current study). * = aromatic residues examined in this study. (B) Model of a GABAρ1 receptor transmembrane domain in one subunit, showing the location of the aromatic residues in M4.

most extensive being performed on GLIC and ELIC. These data suggest that enhanced M4−M1/M3 interactions potentiate pLGIC function while reduced interactions inhibit pLGIC function.11,12 In particular, the data show that aromatic residues are important, with aromatic substitutions that promote M4− M1/M3 interactions enhancing channel function while aromatic substitutions that weaken M4−M1/M3 interactions reduce channel function. However, GLIC is activated by protons at an as-yet undefined but possibly transmembrane site,13,14 and ELIC has atypical gating and conductance properties compared with other pLGICs.14,15 Thus, these proteins may not be ideal model systems to probe this region. Our aim is therefore to explore the role of aromatic residues in the M4 region of the GABAA receptor, a typical eukaryotic GABA-activated pLGIC.16,17

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Figure 2. Functional and immunocytochemical studies on WT and mutant GABAρ receptors. (A) Example Flexstation traces showing [GABA] dependence in WT receptors. HEK293 cells expressing WT GABA receptors were incubated with FMP dye and stimulated at 20 s with GABA. F = fluorescence, arbitrary units. (B) EC50 values derived from Flexstation data for Ala substitutions of aromatic M4 residues (data in Table 1). NF = nonfunctional. (C) Typical immunofluorescence data of nonfunctional Ala-containing mutant GABAρ receptors showing that they are expressed. Scale bars = 50 μm.

RESULTS Molecular Modeling of the GABAρ Receptor M4 αHelix. The crystal structure of the GABAρ receptor has not yet been solved, but there are a number of good templates for homology modeling. A recent study has concluded that the structure of the GluCl receptor is probably the most accurate template,18 and we would agree with this if we were considering the extracellular domain. However, for the transmembrane domain, and especially M4, we consider that the structure of the GABAβ3 receptor provides the best template, as every aromatic residue in the GABAρ receptor M4 has an equivalent aromatic residue in the GABAβ3 receptor. Our model was therefore constructed using the GABAβ3 receptor as a template, and this demonstrates that the majority of the M4 aromatic residues are oriented toward M1 and/or M3, with only Y458 oriented with its side chain completely facing the lipid environment, as in the GABAβ3 receptor (Figure 1B) Functional Characterization of GABAρ Receptors Using a Membrane-Potential-Sensitive Dye. To examine the function of wild-type (WT) and mutant GABAρ receptors, we transfected them into HEK293 cells and probed them using a membrane-potential-sensitive dye. Concentration response curves for WT receptors revealed a GABA EC50 of 0.7 μM (pEC50 = 6.15 ± 0.04) and a Hill coefficient (nH) of 1.5 ± 0.2 (Figure 2A and Table 1), consistent with previously published data (e.g., see ref 19). Ala Scan of Aromatic M4 Residues. As an initial probe of the role of the aromatic M4 residues, each was substituted with Ala and examined after expression in HEK cells. The data (Figure 2 and Table 1) show that this substitution resulted in an increase in EC50 for receptors containing W475A and F478A

substitutions and ablation of function for receptors containing Y467A and Y474A substitutions. The latter effect was not due to loss of expression, as the mutant receptors could be visualized on the cell surface when probed by immunocytochemistry using a GABAρ-specific antiserum (Figure 2C). All of the aromatic residues were then further tested with a range of alternative aromatic and nonaromatic substitutions, and each residue is individually discussed below. Effect of Mutations at Tyr 458. The data show that substitution of Y458 with Ala, Trp, or Phe did not alter the functional parameters of expressed receptors (Table 1 and Figure 3), indicating that neither an aromatic residue nor Tyr is essential at this position. Furthermore, substitution with the charged amino acids Glu and Lys also resulted in parameters similar to those of the WT receptors, indicating that hydrophobicity is not critical here. Y458 is the most N-terminal M4 aromatic residue, and the model indicates that it faces away from the TM region; it is therefore unlikely to be involved in interactions with other regions of the receptor that are important for function. Effect of Mutations at Phe 463 and Phe470. Substitution of F463 with Ala, Trp, or Tyr did not alter the functional parameters of expressed receptors, again indicating B

DOI: 10.1021/acschemneuro.7b00315 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience Table 1a mutant

EC50 (μM)

WT Y458A Y458W Y458F Y458E Y458K

0.7 0.6 0.7 0.8 0.7 0.3

6.15 6.22 6.14 6.10 6.15 6.51

F463A F463W F463Y F463E F463K

0.9 0.8 0.4 NR NR

Y467A Y467W Y467F Y467E Y467K

NR 3.2 1.4 NR NR

F470A F470W F470Y F470E F470K

0.4 0.4 0.3 NR NR

Y474A Y474W Y474F Y474E Y474K

NR NR 7.8 NR NR

W475A W475Y W475F W475E W475K F478A F478W F478Y F478E F478K

nH

pEC50 ± ± ± ± ± ±

0.04 0.09 0.10 0.06 0.03 0.05

1.5 1.4 1.2 1.1 2.7 2.1

± ± ± ± ± ±

n 0.2 0.5 0.3 0.3 0.8 0.4

12 5 6 5 6 6

6.04 ± 0.19 6.11 ± 0.05 6.39 ± 0.05

1.1 ± 0.3 1.6 ± 0.3 1.2 ± 0.4

6 7 5 6 6

5.49 ± 0.06 5.85 ± 0.10

1.5 ± 0.2 1.4 ± 0.3

6.41 ± 0.27 6.40 ± 0.20 6.52 ± 0.07

0.8 ± 0.6 1.2 ± 0.2 1.9 ± 0.7

6 5 6 6 6 6 6 6 6 6

1.4 ± 0.2

8.6 5.2 7.8 NR NR

5.07 ± 0.06 5.29 ± 0.13 5.00 ± 0.02

2.1 ± 0.6 0.7 ± 0.5 2.0 ± 0.2

4 8 6 6 6

1.4 0.8 0.8 0.4 NR

5.86 6.08 6.09 6.39

± ± ± ±

4 8 4 6 6

0.9 1.2 1.4 2.0

0.3 0.2 0.2 0.4

Figure 4. Images of (A, B) F463 and (C) F470 showing their possible interactions with residues in M1 and M3.

and residues in M3, and an aromatic residue, usually a Phe, is found at the equivalent position in many pLGICs. Effects of Mutations at Tyr467. The data suggest that an aromatic residue at Y467 is essential for function, as substitution with Ala, Glu, and Lys ablated function, while substitution with Phe and Trp did not, although the former causes an increase in EC50. The model shows that Y467 is in a location where it has the potential to form interactions with M1 and M3. The model suggests that in M1 there could be a hydrogen bond with T293 and a hydrophobic interaction with V296 (Figure 5A), while in M3 there is the possibility of hydrogen bonds with Y347 and S351 and a π−π interaction with F354 (Figure 5B) .

12 12 4 6 6

5.10 ± 0.08

± 0.09 ± 0.06 ± 0.04 ± 0.04

Figure 3. (A) GABA concentration response curves for WT (black circles) and Y458A (red triangles) containing GABAρ receptors. Parameters obtained from these curves are shown in Table 1. (B) Model showing the location of Y458 facing away from M4 and the rest of the protein.

Data = mean ± SEM. Bold = significantly different from WT (oneway ANOVA, p ≤ 0.05). NR = no response. a

that neither an aromatic residue nor Phe is critical at this position. However, substitution with Glu or Lys ablated function, suggesting that a hydrophobic residue is essential. The model (Figure 4A,B) suggests there could be many potential interactions of F463 with residues on M1 and/or M3, including aromatic or hydrophobic interactions with F303 in M1 and W300 and F356 in M3. A Phe is found at the location equivalent to F463 in most pLGICs, indicating an important functional role. The data for substitution of F470 are similar to those for F463: Ala or an alternative aromatic substitution does not alter functional parameters, but substitution with Glu or Lys ablated function. The model (Figure 4C) suggests the possibility of a number of potential interactions between the π ring of F470

Figure 5. Images of Y467 showing its possible interactions with residues in (A) M1 and (B) M3.

Effect of Mutations at Tyr 474. Y474 was the most sensitive of the aromatic residues we examined. Incorporation of Ala, Glu, Lys, or Trp ablated function, while incorporation of Phe caused an increase in EC50 (Figure 6A). The model shows that Y474 has the potential to form interactions with residues in M1 and M3: with the latter it might form a T-type π−π interaction with Y346 and a C

DOI: 10.1021/acschemneuro.7b00315 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Research Article

DISCUSSION This study shows that most of the aromatic residues in the GABAρ receptor M4 region are important for channel function. This is consistent with several other studies showing that aromatic interactions between M4 and adjacent M1/M3 residues, and also possibly interactions between M4 residues and the Cys-loop, are important for receptor assembly and/or channel gating in pLGICs.8,21,22 The most comprehensive of these studies have been performed using the prokaryotic Cysloop receptor homologues ELIC and GLIC, which are excellent for this type of study because many high-resolution structures of these proteins have been published, allowing molecular details of aromatic residue interactions to be identified. It is not yet clear, however, whether data from this region of ELIC and GLIC can be accurately extrapolated to Cys-loop receptors; structural and functional data suggest that the M2 region of the transmembrane part of GLIC is similar to that of cationselective pLGICs, with some differences being noted with ELIC, but there are few comparative data for M4.23,24 Thus, probing the M4 residues in a Cys-loop receptor is important to reveal whether their roles are similar to those in these prokaryotic receptors. We have used the GABAρ receptor as a representative Cysloop receptor. This protein has the advantage of being able to function as a homopentamer, making the data easier to interpret. A disadvantage is that as yet there is no highresolution GABAρ receptor structure, and we therefore must be cautious in interpreting the data from the model and in particular be aware that there may be differences in the rotamers of the residues and therefore the distances and angles between them; thus, experimental evidence is critical to support the accuracy of the model. Nevertheless the M4 of the GABAβ3 receptor is an excellent template for creating a model of the GABAρ M4 region, as >80% of the residues are identical or have similar chemical properties. Especially important for this study is the fact that all of the aromatic residues in the GABAρ M4 have equivalent aromatic residues in the GABAβ3 structure; they are discussed individually below. Tyr458. Only one of the M4 aromatic residues, Y458, faces away from the protein in the model. Our data are consistent with this, indicating that this residue plays no role in the function of the receptor, as substitution with alternative aromatic, hydrophobic, or hydrophilic residues has no effect on receptor EC50 values. Furthermore, an aromatic residue is not conserved here in most other pLGICs. Phe463 and 470. These Phe residues span the center of M4, and substitution of either has similar results on receptor function, with these data suggesting that aromatics are not essential at these positions as long as the residue is hydrophobic. The model suggests a number of interactions of these residues with residues in adjacent α-helices, and in particular, both could interact with F356 on M3. Thus, it is possible that all of these aromatic residues form part of an aromatic zipper, where replacement of one with an alternative hydrophobic residue has little effect but multiple substitutions prevent zipper action and the receptor does not function. To test this, we created the F463A/F470A double mutant. Neither of these substitutions alone altered function, but the double mutant was nonfunctional, supporting our hypothesis (Table 2). We also explored a range of other double mutants and found that those where we retained one aromatic residue mostly had properties similar to the WT receptors but none of

Figure 6. (A) GABA concentration response curves for WT (black circles) and Y474F (red triangles) containing GABAρ receptors, showing a shift in the curve to the right for the latter. Parameters obtained from these curves are shown in Table 1. (B) Model showing the possible interactions of Y474 with M1 and M3.

backbone hydrogen bond with A343, and there is the possibility of a hydrogen bond with Y289 in M1 (Figure 6B). The data are consistent with the fact that all of these interactions exist and are important for the function of the receptor. Effect of Mutations at Trp475 and Phe478. W475 and F478 are at the extreme C-terminus of the GABAρ subunit (which terminates at S479), a region that has been previously studied.9,20 Our data are consistent with these studies in showing an increased EC50 with W475 substitution by Ala, Phe, or Tyr and no function with Glu or Lys at this position. In our model, W475 has hydrophobic interactions with L207 in the ECD (part of the Cys-loop) and with Y437 in M3. It could also interact with N471 and Y474 in M4 (Figure 7A,B).

Figure 7. Images of W475 and F478 showing their potential interactions with residues in M1, M3, M4, and the ECD.

The receptor retains WT-like function with substitution of F478 with Trp, Tyr, and perhaps surprisingly Glu, but not Lys, and there is an increase in EC50 in F478A-contacting receptors (Table 1). These data can be nicely explained by the model, which shows hydrophobic interactions with residues in M3 and a cation−π interaction and a hydrogen bond with R204, which is again in the Cys-loop of the ECD (Figure 7C,D). D

DOI: 10.1021/acschemneuro.7b00315 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience Table 2a

a

mutant

EC50 (μM)

WT F463A/Y467A F463A/Y467F F463A/Y474F F463A/Y470A Y467A/F478A Y467F/F478A F470A/Y474F

0.7 NR 0.6 0.7 NR NR 0.5 NR

ΔΔG (kcal/mol)

pEC50

nH

6.15 ± 0.04

1.5 ± 0.2

6.22 ± 0.10 6.15 ± 0.08

1.2 ± 0.3 2.8 ± 0.7

−0.7 −1.6

6.31 ± 0.09

2.1 ± 0.6

−0.4

removal results in the loss of hydrophobic interactions with the N-terminal domain, which may be essential for gating of the receptor. They also generated a range of W475 mutations, with W475F resulting in receptors similar to the WT and an Ala substitution causing an increase in EC50 (although no values were reported). A more recent study by this group did report EC50 values, and here W475F resulted in a 5.6-fold increase and W475A a 3.7-fold increase compared with the WT.9 These latter data are consistent with ours, and like them, we found that the introduction of charged residues ablated function. The data therefore support the proposal that there is an important hydrophobic interaction with L207 in the Cys-loop of the ECD as indicated by the model. It is also possible that W475 contributes to associations of M4 with M3, as it has the potential to form a T-type π−π interaction with Y347; it could also possibly interact with N471 and Y474 in M4. Thus, W475 likely has multiple roles in maintaining the structure and/or function of M4. F478 may also have multiple roles. Substitution here with alternative aromatic residues retains the WT properties, while Ala substitution increases the EC50, suggesting that the aromatic/hydrophobic properties of Phe are important. This would be consistent with the hydrophobic interactions with M3 suggested by the model. Introduction of a negatively charged residue here also results in a WT EC50, and although this seems incongruous, it does in fact provide strong support for the existence of a cation−π interaction with the Cys-loop residue R204, which is shown in the model. Indeed, two potential interactions could occur here, a cation−π interaction and a hydrogen bond with the backbone. These may not be critical interactions for function, as previous studies have shown that removal of this Phe does not ablate function (although the EC50 is increased).20 However, it is likely that the structure of this region differs when the terminal residues are removed, and thus, it is possible that interactions with the ECD differ (e.g., W475 may interact with R204). GLIC and ELIC do not have equivalent aromatic residues at these positions, which again indicates differences in the functional roles of residues at the M4 ECD interface in prokaryotic and Cys-loop receptors.

n 12 5 6 6 5 6 6 6

Data = mean ± SEM. ΔΔG = −RT ln(Ω), where

Ω=

EC50(Mut1) × EC50(Mut2) EC50(WT) × EC50(Mut1, 2)

our other double Ala mutants were functional. To further test our hypothesis, we used double-mutant cycle analysis25 where this type of analysis was possible (i.e., with F463A/Y467F, F463A/Y474F, and Y467F/F478A but not with the nonfunctional receptors). The calculated ΔΔG values (Table 2) suggest a weak connections between F463 and Y467 and between Y467 and F478, consistent with a hydrophobic interaction (typically ∼0.7 kcal/mol), and a stronger connection between the more distant F463 and Y474, with a ΔΔG value consistent with an aromatic interaction (1−3 kcal/mol). These data thus provide addition evidence for the existence of a functional network that stretches along a large proportion of M4. Somewhat similar data have been reported for GLIC, where Ala substitution of any of the six aromatic residues in M4, the two in M4, and three out of the four aromatic residues in M1 did not ablate function, but combining two Ala substitutions usually resulted in nonfunctional receptors; those authors proposed a range of interactions of these aromatic residues.12,22 Tyr467 and Tyr474. These residues were more sensitive to substitution than the Phe residues described above, with Ala ablating function and an alternative aromatic residue either ablating function or increasing EC50. The model reveals many potential interactions of these Tyr residues with M1 and M3, suggesting, when combined with the functional data, that these residues are important for the structure and association of αhelices at the extracellular side of the transmembrane domain; if these exist, they could be essential for function. In support of this, substitution of the equivalent aromatic residues in the glycine receptor α-subunit M4 region with Ala also ablated function,6 although the situation with the prokaryotic M4s differs: neither have an aromatic residue at position 474, and while Ala substitution of the residue equivalent to Y467 in GLIC increases EC50, in ELIC it results in a decrease, i.e., a gain of function (although this was less than 2-fold, with the statistical analysis being performed on the EC50 rather than pEC50, and therefore must be viewed with some caution).7,12,22 Thus, these data suggest that there are some differences in the roles of prokaryotic and Cys-loop receptor M4 aromatic residues. Trp475 and Phe478. W475 is the fifth from last and F478 the second from last residue in the carboxy terminus of the p1 subunit, and these last five residues are critical for the stability of the structure: Estrada-Mondragón et al.20 showed that removal of the five terminal residues in the GABAρ subunit results in nonfunctional receptors, and they reported that the structure is “completely broken” without this part of the protein. More specifically, their molecular modeling indicated that the structure of M4 is unstable without W475, whose

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CONCLUSIONS This study shows that many of the aromatic residues in the GABAρ receptor M4 are important for channel function. This supports previous data showing that aromatic interactions between M4 and adjacent M1/M3 α-helices and possibly also interactions between M4 and the Cys-loop are important for receptor assembly and/or channel gating in various pLGICs.6,20−22,26 The GABAρ data presented here show some differences to those obtained from the well-studied prokaryotic receptors ELIC and GLIC. However, it may be that it is not appropriate to compare the GABAρ receptor with both of these, as a recent report suggests that the M4 regions in ELIC and GLIC may represent distinct archetypes.22 Thus, GLIC, along with GABA, glycine, and GluCl, has many aromatic M4 residues that are essential for function and is proposed to represent one archetype, while ELIC, along with the nACh and 5-HT3 receptors, has few aromatic residues that are detrimental to function and is proposed to represent a different archetype. In support of this hypothesis, alignment of the M4 sequences of cationic and anionic Cys-loop receptors shows that the contributing amino acids are significantly more different than those that constitute M1 and M3. It does seem E

DOI: 10.1021/acschemneuro.7b00315 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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surprising that these closely related protein families would have evolved different roles for M4, but it may be that there are aspects of the functional contributions of this region of which we are not yet aware. Perhaps future studies, which will likely have the advantage of using high-resolution structures of all these proteins, will reveal novel roles of this intriguing part of a pLGIC.

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METHODS

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AUTHOR INFORMATION

Research Article

REFERENCES

(1) Thompson, A. J., Lester, H. A., and Lummis, S. C. (2010) The structural basis of function in Cys-loop receptors. Q. Rev. Biophys. 43, 449−499. (2) Hassaine, G., Deluz, C., Grasso, L., Wyss, R., Tol, M. B., Hovius, R., Graff, A., Stahlberg, H., Tomizaki, T., Desmyter, A., Moreau, C., Li, X. D., Poitevin, F., Vogel, H., and Nury, H. (2014) X-ray structure of the mouse serotonin 5-HT3 receptor. Nature 512, 276−281. (3) Huang, X., Chen, H., Michelsen, K., Schneider, S., and Shaffer, P. L. (2015) Crystal structure of human glycine receptor-α3 bound to antagonist strychnine. Nature 526, 277−280. (4) Miller, P. S., and Aricescu, A. R. (2014) Crystal structure of a human GABAA receptor. Nature 512, 270−275. (5) Nemecz, A., Prevost, M. S., Menny, A., and Corringer, P. J. (2016) Emerging Molecular Mechanisms of Signal Transduction in Pentameric Ligand-Gated Ion Channels. Neuron 90, 452−470. (6) Haeger, S., Kuzmin, D., Detro-Dassen, S., Lang, N., Kilb, M., Tsetlin, V., Betz, H., Laube, B., and Schmalzing, G. (2010) An intramembrane aromatic network determines pentameric assembly of Cys-loop receptors. Nat. Struct. Mol. Biol. 17, 90−98. (7) Carswell, C. L., Henault, C. M., Murlidaran, S., Therien, J. P., Juranka, P. F., Surujballi, J. A., Brannigan, G., and Baenziger, J. E. (2015) Role of the Fourth Transmembrane α Helix in the Allosteric Modulation of Pentameric Ligand-Gated Ion Channels. Structure 23, 1655−1664. (8) Carswell, C. L., Sun, J., and Baenziger, J. E. (2015) Intramembrane aromatic interactions influence the lipid sensitivities of pentameric ligand-gated ion channels. J. Biol. Chem. 290, 2496− 2507. (9) Limon, A., Estrada-Mondragon, A., Ruiz, J. M., and Miledi, R. (2016) Dipicrylamine Modulates GABAρ1 Receptors through Interactions with Residues in the TM4 and Cys-Loop Domains. Mol. Pharmacol. 89, 446−456. (10) Baenziger, J. E., Henault, C. M., Therien, J. P., and Sun, J. (2015) Nicotinic acetylcholine receptor-lipid interactions: Mechanistic insight and biological function. Biochim. Biophys. Acta, Biomembr. 1848, 1806−1817. (11) daCosta, C. J., Dey, L., Therien, J. P., and Baenziger, J. E. (2013) A distinct mechanism for activating uncoupled nicotinic acetylcholine receptors. Nat. Chem. Biol. 9, 701−707. (12) Henault, C. M., Juranka, P. F., and Baenziger, J. E. (2015) The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligandgated Ion Channels. J. Biol. Chem. 290, 25118−25128. (13) Rienzo, M., Lummis, S. C., and Dougherty, D. A. (2014) Structural requirements in the transmembrane domain of GLIC revealed by incorporation of noncanonical histidine analogs. Chem. Biol. 21, 1700−1706. (14) Wang, H. L., Cheng, X., and Sine, S. M. (2012) Intramembrane proton binding site linked to activation of bacterial pentameric ion channel. J. Biol. Chem. 287, 6482−6489. (15) Gonzalez-Gutierrez, G., and Grosman, C. (2015) The atypical cation-conduction and gating properties of ELIC underscore the marked functional versatility of the pentameric ligand-gated ionchannel fold. J. Gen. Physiol. 146, 15−36. (16) Chebib, M. (2004) GABAC receptor ion channels. Clin. Exp. Pharmacol. Physiol. 31, 800−804. (17) Martinez-Delgado, G., Estrada-Mondragon, A., Miledi, R., and Martinez-Torres, A. (2010) An Update on GABAρ Receptors. Curr. Neuropharmacol 8, 422−433. (18) Naffaa, M. M., Chebib, M., Hibbs, D. E., and Hanrahan, J. R. (2015) Comparison of templates for homology model of ρ1 GABAC receptors: More insights to the orthosteric binding site’s structure and functionality. J. Mol. Graphics Modell. 62, 43−55. (19) Harrison, N. J., and Lummis, S. C. (2006) Locating the carboxylate group of GABA in the homomeric rho GABAA receptor ligand-binding pocket. J. Biol. Chem. 281, 24455−24461. (20) Estrada-Mondragón, A., Reyes-Ruiz, J. M., Martínez-Torres, A., and Miledi, R. (2010) Structure-function study of the fourth

Cell Culture. Human embryonic kidney (HEK) 293 cells were maintained on 90 mm tissue culture plates at 37 °C and 7% CO2 in a humidified atmosphere. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mix F12 (1:1) with GlutaMAX I medium (Life Technologies, Paisley, U.K.) containing 10% HyClone fetal calf serum (GE Healthcare). For FlexStation studies, cells were transfected using polyethylenimine (PEI) (Polysciences): 30 μL of PEI (1 mg/mL), 5 μL of cDNA (1 mg/mL; subcloned into pcDNA3.1), and 1 mL of DMEM were incubated for 10 min at room temperature, added dropwise to a 70−90% confluent plate, and incubated for 2 days. Cells were then transferred to poly-L-lysine (Cultrex)-coated 96well plates and allowed to adhere overnight before use. FlexStation Studies. The methods were the same as described previously.27 In brief, fluorescent membrane potential dye (Membrane Potential Blue kit, Molecular Devices) was diluted in Flex buffer (10 mM HEPES, 115 mM NaCl, 1 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM glucose, pH 7.4) and added to each well of cells. The cells were incubated at 37 °C for 45 min with the dye and fluorescence measured in a FlexStation 3 (Molecular Devices) at 2 s intervals for 600 s. GABA (Sigma) was added to each well after 20 s. Data were normalized to the maximum ΔF and analyzed using Prism (GraphPad Software, Inc.) Modeling. The GABAρ1 and GABAβ3 subunits were aligned using ClustalW, and the GABAρ1 receptor transmembrane domain homology model was generated using Modeller 9.1328 with default parameters on the basis of the structure of the GABAβ3 receptor (Protein Data Bank entry 4COF). The model was then validated using PROCHECK and RAMPAGE.29,30 Immunofluorescent Localization. The methods were the same as described previously.19 Briefly, transfected cells were washed with Tris-buffered saline (0.1 M Tris/HCl, pH 7.4, 0.9% NaCl), fixed using 4% paraformaldehyde, and incubated in GABAρ antiserum sc-21336 (Santa Cruz Biotechnology, Inc.) at 1:500 dilution overnight at 4 °C. After washing (×3), they were incubated with an Alexa Fluor 594linked secondary antibody (Thermo Fisher Scientific) for 1 h at room temperature, washed (×3), and then observed using a FLoid microscope (Thermo Fisher Scientific).

ORCID

Sarah C. R. Lummis: 0000-0001-9410-9805 Author Contributions

S.C.R.L. participated in research design. J.C.-W., M.A., F.W., S.C.R.L., J.J., and L.Z. conducted experiments and/or data analysis. S.C.R.L. wrote the manuscript. Funding

S.C.R.L. thanks the MRC for support (MR L021676). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; nACh, nicotinic acetylcholine; GABA, γ-aminobutyric acid; ELIC, Erwinia ligand-gated ion channel; GLIC, Gloeobacter ligand-gated ion channel; HEK, human embryonic kidney; AChBP, acetylcholine binding protein F

DOI: 10.1021/acschemneuro.7b00315 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience transmembrane segment of the GABAρ1 receptor. Proc. Natl. Acad. Sci. U. S. A. 107, 17780−17784. (21) Reyes-Ruiz, J. M., Ochoa-de la Paz, L. D., Martinez-Torres, A., and Miledi, R. (2010) Functional impact of serial deletions at the Cterminus of the human GABAρ1 receptor. Biochim. Biophys. Acta, Biomembr. 1798, 1002−1007. (22) Therien, J. P. D., and Baenziger, J. E. (2017) Pentameric ligandgated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function. Sci. Rep. 7, 450. (23) Thompson, A. J., Alqazzaz, M., Ulens, C., and Lummis, S. C. (2012) The pharmacological profile of ELIC, a prokaryotic GABAgated receptor. Neuropharmacology 63, 761−7. (24) Alqazzaz, M., Thompson, A. J., Price, K. L., Breitinger, H. G., and Lummis, S. C. (2011) Cys-loop receptor channel blockers also block GLIC. Biophys. J. 101, 2912−8. (25) Horovitz, A., and Fersht, A. R. (1990) Strategy for analysing the co-operativity of intramolecular interactions in peptides and proteins. J. Mol. Biol. 214, 613−7. (26) Alcaino, C., Musgaard, M., Minguez, T., Mazzaferro, S., Faundez, M., Iturriaga-Vasquez, P., Biggin, P. C., and Bermudez, I. (2017) Role of the Cys Loop and Transmembrane Domain in the Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors. J. Biol. Chem. 292, 551−562. (27) Price, K. L., and Lummis, S. C. (2005) FlexStation examination of 5-HT3 receptor function using Ca2+- and membrane potentialsensitive dyes: advantages and potential problems. J. Neurosci. Methods 149, 172−177. (28) Sali, A., and Blundell, T. L. (1993) Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234 (1993), 779−815. (29) Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283−291. (30) Lovell, S. C., Davis, I. W., Arendall, W. B., III, de Bakker, P. I. W., Word, J. M., Prisant, M. G., Richardson, J. S., and Richardson, D. C. (2003) Structure validation by Cα geometry: ϕ, ψ and Cβ deviation. Proteins: Struct., Funct., Genet. 50, 437−450.

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DOI: 10.1021/acschemneuro.7b00315 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX