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. 2009 Sep 21;588(Pt 1):59–66. doi: 10.1113/jphysiol.2009.179705

The role of Loop F in the activation of the GABA receptor

Alpa Khatri 1, David S Weiss 1
PMCID: PMC2821547  PMID: 20045907

Abstract

Functional studies of the ligand gated ion channel family (nicotinic acetylcholine, serotonin Type 3, glycine and GABA receptors) along with the crystal structure of the acetylcholine binding protein (AChBP) and molecular dynamics simulations of the nAChR structure have resulted in a structural model in which the agonist-binding pocket comprises six loops (A–F) contributed by adjacent subunits. It is presumed that the binding of agonist results in a local structural rearrangement that is then transduced to the gate, causing the pore to open. Efforts are underway to better define the specific roles of the six binding loops. Several studies have suggested Loop F may play a direct role in linking the structural rearrangement within the binding pocket to the gate, although other investigations have indicated Loop F may be crucial for locking the agonist molecule into the binding site. This review will focus on the controversy surrounding the role of Loop F during GABA receptor activation.

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David Weiss (University of Texas HSC at San Antonio, TX, USA) obtained his bachelor's degree from the University of North Carolina at Chapel Hill and his PhD in Neuroscience from Baylor College of Medicine. His first faculty position was at the University of South Florida in the Physiology Department followed by a 10 year stint at the University of Alabama at Birmingham in the Department of Neurobiology. Since 2005 he has been a Professor and Chairman of the Physiology Department at the University of Texas Health Science Center at San Antonio. He has spent most of his career studying the structure and function of GABA receptors and is currently on the editorial boards of Biophysical Journal, Journal of Biological Chemistry and Cell Science.

GABA receptors

The ionotropic GABA receptors (GABAA and GABAC) underlie fast inhibitory synaptic transmission in the central nervous system (Enz et al. 1995, 1996; Wegelius et al. 1998). They are members of the ligand-gated ion channel (LGIC) family, which also includes the nicotinic acetylcholine (nAChR), glycine receptors and the serotonin 5-HT3A receptor (Noda et al. 1982; Grenningloh et al. 1987; Schofield et al. 1987; Maricq et al. 1991). Overall, 19 GABA receptor subunits have been cloned to date: α1–6, β1–3, γ1–3, δ, ɛ, θ, π and ρ1–3 (Schofield et al. 1987; Barnard & Seeburg, 1988; Khrestchatisky et al. 1989; Pritchett et al. 1989; Olsen et al. 1990; Pritchett & Seeburg, 1990; Garret et al. 1997; Hedblom & Kirkness, 1997; Whiting et al. 1997; Hevers & Luddens, 1998) These subunits all have the same basic structure: a large extracellular amino terminal domain, four transmembrane domains, a large intracellular domain and a short extracellular C-terminus. Like all members of the LGIC family, the GABAA and GABAC receptors consist of five subunits with the stoichiometry of the primary GABAA receptor found in the CNS consisting of two α subunits, two β subunits, and one γ subunit (Chang et al. 1996; Tretter et al. 1997; Baumann et al. 2002). The ρ1 GABA receptor subunit, which comprises the GABAC receptor, was cloned from a retinal library and is thought to exist primarily in the retina (Cutting et al. 1991; Amin & Weiss, 1994), but has been speculated to exist in the brain (Rozzo et al. 2002; Liu et al. 2004; Schlicker et al. 2004).

The agonist binding site

Figure 1 shows a structural model of two subunits of the GABAC receptor amino terminal domain based on the AChBP structure (Brejc et al. 2001). GABA's binding pocket is located in the subunit interface of the extracellular amino terminal domain and is formed by six loops (A–F). The loops are highlighted in Fig. 1. Loops A (red), B (yellow) and C (purple) are contributed by the left subunit, whereas Loops D (orange), E (cyan) and F (green) are contributed by the right, neighbouring subunit. In terms of the activation mechanism, the binding of GABA induces a structural rearrangement within the binding pocket. This structural rearrangement is then transduced as a conformational wave to the gate causing it to open thereby allowing chloride ions to move through the pore. Within the binding pocket, residues from Loops A–E have been postulated to interact with the ligand (Amin & Weiss, 1993; Corringer et al. 2000; Brejc et al. 2001; Boileau et al. 2002; Chang et al. 2002; Torres & Weiss, 2002). It has been hypothesized, based on structural and functional data, that Loop F (Fig. 1, green) may play a key role in linking ligand binding to channel opening (Lyford et al. 2003; Hansen et al. 2005; Thompson et al. 2006). Conflicting data not supporting this hypothesis have been recently published (Khatri et al. 2009; Pless & Lynch, 2009), so the precise role of Loop F remains a source of contention within the LGIC field. We will briefly review the Loop F field and then present our data that directly address the role of Loop F in GABAC receptor activation.

Figure 1.

Figure 1

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The GABAC receptor extracellular domain and the sequence alignment of Loop F A, a putative structural model is shown for two subunits of the GABAC receptor extracellular domain based on the AChBP from Brejc et al. (2001). The six loops thought to form the binding pocket are coloured: Loop A, red; Loop B, yellow; Loop C, purple; Loop D, orange; Loop E, cyan; Loop F, green. GABA was docked into the GABAC receptor structural model presenting a preferred orientation of its carboxylic group facing Loop C and amino group facing Loop E. B, sequence alignment of Loop F from three subunits of the GABAA receptor, the GABAC receptor and the AChBP. Note the low sequence homology for Loop F within the GABA receptor family.

Loop F

Structural information

The initial structural resolution of the Lymnaea stagnalis acetylcholine binding protein (L-AChBP) presented a poorly resolved Loop F (Brejc et al. 2001; Hansen et al. 2005). The structure of the Aplysia californica AChBP was crystallized in the apo form (unbound) and bound by two agonists and two antagonists. Comparing these structures suggested Loop F undergoes a ligand induced structural rearrangement (Hansen et al. 2005). Not only did the agonist induce a structural rearrangement in the binding pocket, but antagonists did as well. Several other AChBP structures supported Loop F's flexibility and suggested it adopts a random coil-like structure (Bourne et al. 2005; Celie et al. 2005). The Torpedo acetylcholine receptor structure derived by electron microscopy revealed that Loop F resides in the transduction zone, just above the lipid membrane (Unwin, 2005; Dellisanti et al. 2007). Recently two structures of the prokaryotic LGIC have been published, but very little was mentioned about Loop F except that it is located in the transduction zone similar to what was observed in the Torpedo acetylcholine receptor structure (Hilf & Dutzler, 2008; Bocquet et al. 2009).

Molecular dynamics (MD) simulations

MD suggested Loop F was one of the most flexible regions in the receptor during activation, similar to what was proposed in the crystal structure of the AChBP and nAChR (Law et al. 2005; Szarecka et al. 2007). These MD simulations suggested Loop F moves into a more aqueous environment in the presence of a ligand (Law et al. 2005; Cheng et al. 2006). In addition to Loop F's proposed large range of motion, a recent MD study suggested that it could play a role in maintaining the local structure of the binding interface (Szarecka et al. 2007). Also, structural modelling of the GABAC receptor amino terminal domain based on the L-AChBP proposed that Loop F is approximately 5 Å from GABA suggesting Loop F is too distant to directly interact with GABA (Harrison & Lummis, 2006).

Functional studies

Early functional studies suggested Loop F has two distinct regions (Newell & Czajkowski, 2003; Thompson et al. 2006). The upper portion of Loop F is thought to line the binding pocket and could potentially be important for the affinity and specificity of ligand binding while the lower portion of Loop F was proposed to be a link between ligand binding and channel opening (Newell & Czajkowski, 2003; Shimomura et al. 2003; Thompson et al. 2006; Zhang et al. 2007). In the end, they hypothesized that the structural rearrangement occurring in Loop F played a direct role in controlling the actions of the gate. Studies in the αβγ GABAA receptor also suggested Loop F is important for the binding of benzodiazepines, modulators of the GABAA receptor, as well as important for the allosteric actions (potentiation) of benzodiazepines on GABA receptor activity (Sancar et al. 2007; Hanson & Czajkowski, 2008; Padgett & Lummis, 2008). On the contrary, studies of Loop F in the homomeric ρ1 GABAC receptor demonstrated that most mutations in this region did not affect receptor function. Furthermore, these studies used the substituted cysteine accessibility method (SCAM), which was originally developed to identify pore lining residues in the LGIC family (Akabas et al. 1992; Xu & Akabas, 1996; Wilson & Karlin, 1998). Cysteines are introduced into a region of interest and then bound to a sulfhydryl-reactive compound. Any change in the function of the mutant receptors in the presence of the sulfhydryl-reactive compound could signify a functionally important region of the receptor. SCAM analysis demonstrated that the residues in Loop F do not directly interact with either GABA or the competitive antagonist 3-APA. This latter study, at least for ρ1 GABA receptors, brings into question whether Loop F may play a direct role in ligand binding (Sedelnikova et al. 2005).

To further understand the role of Loop F during activation, a technique called voltage clamp fluorometry (VCF), initially developed to study the structural rearrangement of voltage-gated ion channels (Mannuzzu et al. 1996), was adapted and used in LGICs (Chang & Weiss, 2002). VCF allows real time visualization of the structural rearrangements occurring in the receptor during activation and antagonism along with the simultaneous recording of current by two-electrode voltage clamp. First, a cysteine is introduced into a region of interest in the receptor and then labelled with an environmentally sensitive fluorophore. The fluorescence intensity of this fluorophore is determined by the hydrophobicity of the surrounding environment. When the hydrophobicity surrounding the fluorophore changes then the fluorescence intensity will increase if the fluorophore moves into a more hydrophobic environment and decrease if the fluorophore moves into a more hydrophilic environment. By simultaneously detecting fluorescence and ligand-induced current, structural changes can be correlated with receptor activation. To date, there have been three VCF studies on Loop F, and one study determined that Loop F is along the transduction pathway and plays a key role in activation (Zhang et al. 2009). However, a more recent study concluded the opposite; Loop F does not play a key role in linking ligand binding to the gating of the receptor, nor is it involved in ligand specificity (Pless & Lynch, 2009). The third VCF study on Loop F was preformed by us and will be considered now.

Our work on the GABAC receptor

The overall focus of our study was to better define the role of Loop F during activation. The cysteine-introduced mutant receptors were expressed in Xenopus laevis oocytes and then labelled with the fluorophore Alexa 546 c5 maleimide (A5m). The oocytes were placed in a special chamber to simultaneously record a change in current (ΔI) and fluorescence (ΔF). Figure 2 shows the GABA-induced ΔF for the 14 residues that form the putative structure of Loop F (corresponding currents are not shown). The ΔF for L216C, T218C, R221C, I222C and I229C increases upon receptor activation whereas K217C, E220C, S223C, L224, S225C, Q226C and E227C exhibit a decrease in ΔF. It is remarkable that nearly all the residues (11 out of 14) in Loop F generate a ΔF with GABA application. Residues S223C–F227C of Loop F exhibited a decrease in the ΔF suggesting they move into a more aqueous environment and this agrees with the MD simulations discussed earlier (Law et al. 2005). If Loop F is a link between ligand binding and channel opening, we reasoned that the degree of activation of the receptor should be reflected in the ΔF. To control the degree of activation, we employed full, partial and competitive antagonists. A partial agonist is defined as a ligand that can bind the same site as a full agonist, in this case GABA, but not fully activate the receptor. It is possible that the reduced current induced by the partial agonist is the result of a subconductance state of the GABAC receptor, rather than a decreased open probability. Testing this possibility would be difficult since the dominant conductance state of the GABAC receptor is less then 1 pS (Wotring et al. 1999). However, at least for the highly homologous GABAA receptor, partial agonists do not alter the conductance, but rather decrease the open probability (Mistry & Hablitz, 1990). A competitive antagonist is an extreme example of a partial agonist; it binds the same site as GABA but does not activate the receptor at all. Some type of a correlation between ΔI and ΔF for these ligands would support the hypothesis that Loop F is involved in the transduction of the structural rearrangement from the binding pocket to the gate of the receptor. For these experiments, we focused on five residues that provided the most consistent and robust fluorescence signals in Loop F. Since these residues are distributed in Loop F, their ΔF is assumed to reflect the structural changes throughout the whole domain. Figure 3 compares the ΔI and ΔF recordings for two full agonists (GABA and Trans-aminocrotonic acid (TACA)), two partial agonists (isoguvacine and imidazole-4-acetic acid, or I4AA) and a competitive antagonist (3-aminopropylphosphonic acid, or 3-APA) at saturating concentrations. The top panel is the ligand-induced currents, and below are the corresponding ΔFs. The bar graphs for the ΔI and ΔF for each ligand are normalized to GABA. In general, for all the data, there was no clear correlation between the current and fluorescence. For example, consider the application of I4AA on L216C, K217C, T218C and S223C. In this case, I4AA induced a smaller current compared to GABA whereas the I4AA induced a ΔF similar in magnitude to the GABA-induced ΔF. The ΔF does not seem to correlate with the ligand induced current for either I4AA or isoguvacine. Quite a different picture is observed for L166C of Loop E. In this case, agonists cause an increase in ΔF, while antagonists decrease ΔF. Isoguvacine behaves as a competitive antagonist in this mutant and does not induce a current, but does cause a large decrease in the ΔF, as does the competitive antagonist 3-APA.

Figure 2.

Figure 2

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Fluorescence changes for the residues of Loop F The 14 residues of Loop F were individually mutated to cysteines and then labelled with the fluorophore A5m (see text). The application of a saturating concentration GABA induced a ΔF for 11 of the 14 residues. The corresponding currents are not shown. Reproduced from Khatri et al. (2009) with permission from Elsevier.

Figure 3.

Figure 3

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Correlation of agonists (GABA and TACA), partial agonists (I4AA and isoguvacine), and a competitive antagonist (3-APA) induced ΔI and ΔF for residues L216C, K217C, T218C, S223C and I229C of Loop F as well as L166C of Loop E The top panel shows the ligand-induced currents with the corresponding ΔF below. The bar graphs depict the ΔI (grey) and ΔF (black) normalized to GABA for each residue. In general, there was no clear correlation between the fluorescence and the functional effects of the ligand. Reproduced from Khatri et al. (2009) with permission from Elsevier.

Conclusions

As discussed above, structural information, MD simulations, as well as functional studies have suggested Loop F plays a direct role in linking ligand binding to channel opening. Zhang et al. (2009) also employed VCF to examine the role of Loop F of the GABAC receptor. While there was clear overlap with our studies, they concluded Loop F had two functional regions, with the first half of Loop F coupled to binding and the second half coupled to activation. There are two noteworthy differences in methodology. First, they employed a different fluorophore. And second, their conclusion was based on a SCAM analysis of Loop F as well as the actions of picrotoxin. It is widely accepted that this non-competitive antagonist binds to the pore region (Gurley et al. 1995; Xu et al. 1995; Das & Dillon, 2005; Sedelnikova et al. 2006). These particular results, while certainly intriguing, are difficult to interpret since the mechanism of picrotoxin block remains an enigma (Constanti, 1978; Newland & Cull-Candy, 1992; Pribilla et al. 1992; Erkkila et al. 2008). There have been two recent VCF studies (including ours) that have challenged the hypothesis that Loop F plays a role in channel activation. They both concluded that Loop F is moving in response to ligand binding, but does not seem to play a direct role in linking ligand binding to channel opening (e.g. transduction). These studies have been performed on an array of receptors within the LGIC family and have resulted in a mixed understanding of Loop F's role during activation. It is possible that Loop F could play a different role during activation for each receptor subtype. However, given the regions that form the ligand binding site and the gate are conserved, as well as other regions across the LGIC family, we have assumed that the activation structures and mechanisms are conserved. One possible role we favour for Loop F is that it may be part of the machinery that serves to lock the ligand into the binding pocket. The structural model in Fig. 4 shows that Loop C and Loop F are adjacent in the primary sequence of the subunit with approximately six ‘connecting’ residues. It is conceivable that these two loops are tightly coupled and therefore any structural rearrangements occurring in Loop C would be directly transferred to Loop F (and vice versa). Also note from Fig. 4 that the connected Loop F and Loop C span adjacent binding pockets. Although highly speculative, this connection could functionally couple binding sites leading to changes in affinity in Site A when Site B is agonist-bound, for example. Again, highly speculative, but this feature may underlie the agonist ‘cooperativity’ reported for the activation of these LGICs (Edelstein & Changeux, 1996). Nevertheless, we are now focusing our attention on other domains of the GABA binding pocket as possible links between the rearrangements in the binding pocket and the opening of the gate.

Figure 4.

Figure 4

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View of the structural model depicting the link between Loop C and Loop F Note how the connected Loop C and Loop F each exist in distinct (neighbouring) binding sites. Although speculation, this feature could serve to functionally couple the adjacent binding sites.

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