Individual properties of the two functional agonist sites in GABA(A) receptors

Abstract

The members of the pentameric ligand-gated receptor channel family are involved in information transfer in synapses and the neuromuscular junction. They often contain several copies of the same subunit isoform. Here, we present a method to functionally dissect the role of individual subunits that occur in multiple copies in these receptors. Opening of the inherent chloride channel in the GABA(A) receptor is achieved through the binding of two agonist molecules; however, it has been difficult to obtain information on the contribution of the two individual binding sites. The sites are both located at beta+/alpha- subunit interfaces, suggesting similar properties. One pair of subunits is flanked by gamma and beta (site 1) and the other by alpha and gamma (site 2), the different environment possibly affecting the binding sites. Here, we used concatenated subunits and two point mutations of amino acid residues, each in alpha and beta subunits, both located in the agonist binding pocket, to investigate the properties of these two sites. The sites were individually mutated, and consequences of these mutations on GABA and muscimol-induced channel opening and its competitive inhibition by bicuculline were studied. A model predicts that opening also occurs for receptors occupied with a single agonist molecule but is promoted approximately 60-fold in those occupied by two agonists and that site 2 has an approximately threefold higher affinity for GABA than site 1, whereas muscimol and bicuculline show some preference for site 1.

Figure 5.

A, Bicuculline inhibition curves of γβα/βα (•), γβα₆₅/βα₆₅ (○), γβα₆₅/βα (▪), and γβα/βα₆₅ (□) receptors. Bicuculline was applied in increasing concentrations together with a GABA concentration eliciting 9-11% of the maximal current amplitude. B, Bicuculline inhibition curves of γβα/βα (•), αβ₂₀₅α/γβ (▴), and αβα/γβ₂₀₅ (▾) receptors. Mean values with SEM from four to five oocytes from two batches for each subunit combination are shown. Individual curves were normalized to the current found in the absence of bicuculline and were subsequently averaged.

Figure 1.

Mutations in α or β subunits of the GABA_A receptor. If a point mutation is introduced in an α or β subunit, two mutations are always present in an altered receptor (top row). The availability of concatenated subunits allows construction of linked receptors containing only one mutation (bottom row). This single mutated α1 subunit can be placed between either two β subunits or a β and γ subunit. This way, the point mutation α₁F65L has been investigated. Similarly, the mutation β₂Y205S subunit has been investigated in a differently concatenated channel.

Figure 2.

Western blot analysis. A, Resistance to proteolysis of the fusion protein αβα. Lanes 1-3 were decorated with bd24, reacting with free termini of α subunits. Lane 1, Noninjected oocytes (n.i.). No signal was detected in noninjected oocytes. Lane 2, Oocytes injected with αβα/γβ. The αβα triple construct migrates at 170 kDa. No specific signal was detected at ∼50 or 110 kDa, the size of a monomeric α1 subunit or dimeric subunit carrying α1 at the N-terminus, as expected as a consequence of proteolysis in one of the linker regions. The absence of specific signals in other areas indicates that no N-terminal breakdown product of this triple subunit construct larger than 21 kDa is formed. Lane 3, Oocytes injected with α/β/γ. The two loose α1 subunits from the wild-type α1β2γ2 receptor migrate at ∼50 kDa. B, Lanes 1-3 were decorated with bd17 and reacted with free termini of β subunits. Lane 1, Noninjected oocytes. Lane 2, Oocytes injected with γβα/βα. The βα dual construct migrates at ∼110 kDa. No specific signal was detected at 53 kDa, the size of a monomeric β2 subunit, as expected during proteolysis in the linker region. Lane 3, Oocytes injected with α/β/γ. The two loose β2 subunits from the wild-type α1β2γ2 receptor migrate at ∼53 kDa.

Figure 3.

A, GABA concentration-response curves of γβα/βα (•), γβα₆₅/βα₆₅ (○), γβα₆₅/βα (▪), and γβα/βα₆₅ (□) receptors. B, Muscimol concentration-response curves of γβα/βα (•) and γβα₆₅/βα₆₅ (○) receptors. Mean values with SEM from four to five oocytes from two batches for each subunit combination are shown. Individual curves were first normalized to the observed maximal current amplitude and subsequently averaged.

Figure 4.

A, GABA (•) and muscimol (○) concentration-response curves of αβα/γβ receptors. B, GABA (closed symbols) and muscimol (open symbols) concentration-response curves of αβα/γβ₂₀₅ (square symbols) and αβ₂₀₅α/γβ (round symbols) receptors. Mean values with SEM from four to five oocytes from two batches for each subunit combination are shown. For receptors containing no mutation, individual curves were first normalized to the observed maximal current amplitude and subsequently averaged. For mutated receptors, amplitudes were normalized to the average maximal response elicited by GABA in αβα/γβ receptors.

Figure 6.

Model of the receptor with two agonist binding sites 1 and 2. The receptor (R) can first bind GABA (A) to either site 1 (AR) or site 2 (RA). Analogously, the receptor can first bind bicuculline (I) to either site 1 (IR) or site 2 (RI). The receptor occupied by two agonist molecules (ARA) can isomerize to the open-state ARA^*, and the receptors occupied by a single agonist molecule can isomerize to the open-states AR^* and RA^*. The model at the top left describes a receptor composed of loose subunits. Concentration modifies L and L₁ with fG and fg, respectively. Effects of mutation in site 1 are allosterically transferred to site 2 and vice versa (ta, agonists; tb, antagonists). Constants are taken as dissociation constants and gating constants as closed state divided by open state. Combined Levenberg-Marquardt fit gave the following estimates for the parameters: L, 0.23; L₁, 15.3; L₂, 13.6; K₁, 128 μm; K₂, 42 μm; fA, 4.5; fa, 8.6; fG, 20.4; fg, 31.2; K₁65, 1440 μm; K₂65, 1320 μm; K₁205, 1.3 m; K₂205, 14.9 m; ta65, 0.84; tb65, 1.00; ta205, 4.6; tb205, 2.2; K_1I, 2.2 μm; K_2I, 3.0 μm; m, 97.

Figure 7.

A-D, Simulation of the GABA dose-response curves (A, C) and bicuculline inhibition curves (B, D). A, C, γβα/βα, γβα₆₅/βα₆₅ (sites 1 and 2 mutated), γβα₆₅/βα (site 1 mutated), and γβα/βα₆₅ (site 2 mutated) receptors. B, D, αβα/γβ, αβα/γβ₂₀₅ (site 1 mutated), and αβ₂₀₅α/γβ (site 2 mutated) receptors. The parameters obtained in a fit are given in Figure 6.

Figure 8.

Schematic representation of the GABA_A receptor showing the two functional agonist sites (G) and the benzodiazepine binding site (B). Agonist site 2 has a preference for the agonist GABA, and site 1 has a preference for muscimol and a slight preference for the antagonist bicuculline.