Structurally Diverse GABA Antagonists Interact Differently with Open and Closed Conformational States of the ρ₁ Receptor

Abstract

Ligands acting on receptors are considered to induce a conformational change within the ligand-binding site by interacting with specific amino acids. In this study, tyrosine 102 (Y102) located in the GABA binding site of the ρ₁ subunit of the GABA_C receptor was mutated to alanine (ρ_1Y102A), serine (ρ_1Y102S), and cysteine (ρ_1Y102C) to assess the role of this amino acid in the action of 12 known and 2 novel antagonists. Of the mutated receptors, ρ_1Y102S was constitutively active, providing an opportunity to assess the activity of antagonists on ρ₁ receptors with a proportion of receptors existing in the open conformational state compared to those existing predominantly in the closed conformational state. It was found that the majority of antagonists studied were able to inhibit the constitutive activity displayed by ρ_1Y102S, thus displaying inverse agonist activity. The exception was (±)-4-aminocyclopent-1-enecarboxamide ((±)-4-ACPAM) (8) not exhibiting any inverse agonist activity, but acting explicitly on the closed conformational state of ρ₁ receptors (ρ₁ wild-type, ρ_1Y102C and ρ_1Y102A). It was also found that the GABA antagonists were more potent at the closed compared to the open conformational states of ρ₁ receptors, suggesting that they may act by stabilizing closed conformational state and thus reducing activation by agonists. Furthermore, of the antagonists tested, Y102 was found to have the greatest influence on the antagonist activity of gabazine (SR-95531 (13)) and its analogue (SR-95813 (14)). This study contributes to our understanding of the mechanism of inverse agonism. This is important, as such agents are emerging as potential therapeutics.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS), activating three receptors termed GABA_A, GABA_B, and GABA_C. The GABA_C receptor is found on the retina and at distinct anatomical areas within the CNS, including the superior colliculus,¹ cerebellum,² hippocampus,³ and lateral amygdala,⁴ and has been shown to play an important role in the onset of myopia,⁵ the sleep-waking process,⁶ memory enhancement,⁷ and fear and anxiety disorders.⁴ The design of potent and selective GABA_C receptor antagonists, along with understanding how these agents modulate the receptor, will help characterize these receptors and establish whether GABA_C receptors play a major role in various CNS disorders.^8,9

GABA_C receptors belong to the Cys-loop ligand-gated ion channel (LGIC) superfamily.¹⁰ All members of this superfamily require five subunits to form functional receptors. In mammals, GABA_C receptors are composed of three ρ subunits, ρ₁–ρ₃, which form homomeric receptors or pseudohomomeric receptors, composed of ρ₁ρ₂ or ρ₂ρ₃ subunit combinations.¹¹⁻¹³ The orthosteric binding site of the Cys-loop receptors is located at the interface of two subunits, formed by residues drawn from five discontinuous stretches of amino acids from the N-terminal domain of each subunit. These stretches of residues are referred to as loops A–E. Loops A–C form the principle side of the binding site and loops D and E form the complementary side.¹⁴ GABA_C receptors potentially have five orthosteric or GABA binding sites; GABA binding to these sites induce conformational changes within the receptor that subsequently lead to the opening of the pore, allowing Cl^– ions to pass through.

X-ray crystal structures of related prokaryotic proton-gated ion channels have provided some insights into the structural rearrangement that can occur during receptor gating.¹⁵ ELIC (Erwinia chrysanthem ligand-gated ion channel) represents an inactive receptor conformation and GLIC (Gloebacter violaceus ligand-gated ion channels) represents a desensitized receptor conformation.^16,17 Despite a lack of mammalian crystal structures, there is strong evidence that structural changes occur within the orthosteric binding site upon activation of the receptor by GABA¹⁸ and that the binding site is constricted in the open conformation.¹⁹

Tyrosine at position 102 (Y102) is located on loop D of the ρ₁ subunit within the GABA binding site. This residue has been proposed to be associated with agonist binding and channel gating.²⁰ Recently, this residue was demonstrated not to form a cation−π interaction with GABA²¹ and in an alternative homology model of the ρ₁ GABA binding site implicates that this residue does not directly bind GABA.²² Mutation of Y102 to serine (ρ_1Y102S) in the ρ₁ subunit shifts the equilibrium of the receptor toward the open conformation, producing a constitutively active form of the receptor.²⁰ The competitive antagonist 3-aminopropyl-methyl-phosphinic acid (3-APMPA) inhibits the spontaneous current of ρ_1Y102S receptors in a concentration-dependent manner,²⁰ demonstrating that 3-APMPA acts as an inverse agonist, inducing a conformational change which shifts the equilibrium of the receptor toward the closed conformation.

In this study, the role of Y102 in antagonist activity was assessed. Y102 was mutated to serine (ρ_1Y102S), cysteine (ρ_1Y102C), and alanine (ρ_1Y102A), and the resulting mutant receptors represent channels with a proportion in the open conformational state (ρ_1Y102S) and almost entirely in the closed conformational state (ρ_1Y102C, ρ_1Y102A) of the channel. Twelve known antagonists (1–7 and 9–13) and two novel antagonists (8 and 14) were evaluated on ρ₁ wild-type and mutant receptors to investigate if antagonist activity was altered with receptor conformation.

Results and Discussion

The mechanism by which an agonist binds and subsequently opens the channel of Cys-loop receptors is complex and involves many structural changes throughout the receptor, including changes within the orthosteric binding site. Ligands, for example, 3-APMPA (2), have different affinities for the open or closed conformational states of the receptor, as the conformation of the binding site differs between the two conformational states.²⁰ Constitutively active receptors, such as the ρ_1Y102S receptor, provide an opportunity to assess the activity of antagonists on receptors in equilibrium between the open or closed conformational states of the receptor.

ρ_1Y102S Receptors Are Constitutively Active

Consistent with previous studies,²⁰ the EC₅₀ values for GABA increased by 21-, 233- and 196-fold when ρ₁ Y102 was mutated to cysteine, serine, and alanine, respectively (Figure 1, Table 1). This change in GABA sensitivity suggests that mutation of Y102 on the ρ₁ subunit results in a change in GABA affinity or altered receptor gating. Of the three mutant receptors evaluated, ρ_1Y102S receptors were constitutively active. When clamped at −60 mV, the holding current for cells expressing ρ_1Y102S (−200.3 ± 20.1 nA, n = 11) was greater than that in cells expressing ρ₁ wild-type (−15.2 ± 4.0 nA, n = 11), ρ_1Y102A (−0.2 ± 1.7 nA, n = 11), and ρ_1Y102C (−5.6 ± 4.1 nA, n = 11) mutant receptors. This suggests that while ρ_1Y102S receptors exist in equilibrium between the open and closed conformational states, while ρ₁ wild-type, ρ_1Y102A, and ρ_1Y102C receptors predominantly prefer the closed conformational state.

Figure 1.

GABA concentration response curves for human ρ₁ wild-type (WT) receptors and ρ_1Y102C, ρ_1Y102S and ρ_1Y102A receptors expressed in Xenopus oocytes. Each data point represents the mean ± SEM (n = 3–4). All data are normalized with I_max, which refers to their maximum current. EC₅₀ values are listed in Table 1.

Table 1. EC₅₀ Values for GABA at ρ₁ Wild-Type and Y102 Mutant Receptors^a.

ρ1 Y102 mutation	EC50 (μM)
WT	0.8 ± 0.1
Y102C	17.6 ± 1.2
Y102S	193.7 ± 9.7
Y102A	163.1 ± 2.0

^aAll data are the means ± SEMs (n = 3-4 oocytes).

(±)-4-ACPAM (8) and SR-95813 (14) Are Potent Antagonists at ρ₁ Receptors

In this study, a total of 12 known and 2 novel agents were evaluated at ρ₁ receptors. TPMPA (1), 3-APMPA (2), SGS-742/CGP-36742 (3), (±)-cis-3-ACPBPA (4), (±)-3-trans-ACPBPA (5), S-4-ACPBPA (6), (+)-S-4-ACPCA (7), THIP (9), DAVA (10), 4-GBA (11), ZAPA (12), and SR-93351/Gabazine (13) have been shown previously to act as competitive antagonists at ρ₁ wild-type receptors, indicating that they bind to the GABA binding site. The activities of two novel ligands, (±)-4-ACPAM (8) and SR-95813 (14), were characterized at ρ₁ wild-type receptors recombinantly expressed in Xenopus oocytes. These novel compounds are interesting in that they do not possess an acid moiety (carboxylic or phosphinic acid), a feature common to all ligands that bind to the GABA binding site. Instead of the usual acid moiety, (±)-4-ACPAM (8) has an amide group, while SR-95813 (14) has a nitrile group. To our surprise, these compounds were found to be potent ρ₁ receptor competitive antagonists. Figure 2A demonstrates that (±)-4-ACPAM (8) inhibits the EC₅₀ of GABA (1 μM) in a concentration-dependent manner (Figure 2A, IC₅₀ = 9.6 ± 0.9 μM, n = 4). Schild plot analysis demonstrates that in the presence of increasing concentrations of (±)-4-ACPAM (8) (30, 100, and 300 μM; n = 3–4 oocytes per antagonist concentration), the concentration response curve for GABA is shifted to the right in a parallel manner (Figure 2B, K_B = 30.3 ± 3.1 μM, slope did not differ from 1, see Figure 2Supporting Information), indicating that (±)-4-ACPAM (8) is a competitive antagonist at ρ₁ wild-type receptors.

Figure 2.

Pharmacology of (±)-4-ACPAM (8) and SR-95813 (14) at human ρ₁ wild-type receptors expressed in Xenopus oocytes. (A) Inhibitory concentration response curve for (±)-4-ACPAM (8) against GABA (1 μM) at ρ₁ receptors. Each data point represents the mean ± SEM (n = 3–4). (B) Concentration response curves of GABA alone (black dot, n = 3) and in the presence of (±)-4-ACPAM (8) at 30 (light green dot, n = 3), 100 (green dot, n = 4), and 300 μM (dark green dot, n = 3). Each data point represents the mean ± SEM (n = 4). All data are normalized with I_max, which refers to their maximum current. (C) Inhibitory concentration response curve for SR-95813 (14) against GABA (1 μM) at ρ₁ receptors. Each data point represents the mean ± SEM (n = 4). (D) Concentration response curves of GABA alone (black dot, n = 4) and in the presence of SR-95813 (14) at 30 (light blue dot, n = 3), 100 (blue dot, n = 4), and 300 μM (dark blue dot, n = 3). Each data point represents the mean ± SEM (n = 3–4). All data are normalized with I_max, which refers to their maximum current.

Similarly, SR-95813 (14) inhibits the response produced by GABA (1 μM) in a concentration-dependent manner (Figure 2C, IC₅₀ = 8.0 ± 0.8 μM, n = 4). Schild plot analysis shows that in the presence of increasing concentrations of SR-95813 (14) (30, 100 and 300 μM; n = 3–4 oocytes per antagonist concentration), the GABA concentration response curve is shifted to the right in a parallel manner (Figure 2D, K_B = 12.4 ± 0.4 μM, slope did not differ from 1, see Figure 2 in the Supporting Information), indicating that SR-95813 (14) blocks ρ₁ wild-type receptors in a competitive manner.

Mutagenesis studies of the ρ₁ receptor identified arginine 104 (R104) as important for GABA binding, and this residue is thought to interact via a salt bridge with the carboxylate group of GABA.²³ Homology models of the ρ₁ receptor support this observation.²² As (±)-4-ACPAM (8) and SR-95813 (14) do not possess a carboxylate group, it would be interesting to evaluate R104 in the binding of these ligands.

Assessing the Activity of Antagonists at a Proportion of Receptors in the Open Conformational State

The activities of 14 GABA antagonists (1–14) were evaluated using the constitutively active ρ_1Y102S receptors. Antagonists were tested at 100 and 300 μM in the absence of GABA. The percentage inhibition of the spontaneous current was measured and normalized to the initial resting current for each cell. All antagonists tested inhibited the spontaneous current of ρ_1Y102S receptors to a various extent when evaluated at 300 μM, with the exception of (±)-4-ACPAM (8). (±)-4-ACPAM (8), at either 100 or 300 μM, failed to inhibit the spontaneous current at these receptors (Figure 3B).

Figure 3.

Effect of GABA antagonists at GABA ρ_1Y102S receptor spontaneous currents. (A) A sample current traces showing inverse agonist effects of TPMPA (1) at GABA ρ_1Y102S receptors expressed in Xenopus oocytes. GABA EC₅₀ (180 μM) activates the receptor (black bar), allowing influx of Cl^– ions. Application of TPMPA (1) (200 μM, 1 mM and 5 mM) alone inhibited the resting conductance in a concentration dependent manner (red bar). (B) A sample current trace showing the effect of (±)-4-ACPAM (8) at GABA ρ_1Y102S receptor spontaneous current. (±)-4-ACPAM (8) did not exhibit inverse agonist effects at 100 μM and 300 μM (green bar). (C) Inhibitory concentration–response curves for TPMPA (1) (red), SR-95531 (13) (purple), SR-95813 (14) (blue), (±)-cis-3-ACPBPA (4) (black), and 4-GBA (11) (yellow) on GABA ρ_1Y102S receptors expressed in Xenopus oocytes. All data are normalized with I_max, which refers to the initial resting conductance. Each data point represents the mean ± SEM (n = 3–5). (D) A sample current trace showing weak inhibitions of GABA EC₅₀ (180 μM) (black bar) by (±)-4-ACPAM (8) (300 μM and 1 mM) (green bar) at GABA ρ_1Y102S receptors.

Of the compounds tested, SGS-742 (3), S-4-ACPCA (7), (±)-4-ACPAM (8) and DAVA (10) were the weakest at inhibiting (0–9%) the constitutive current produced by ρ_1Y102S receptors. The remaining antagonists inhibited the current by 10–87% (Table 2). 3-APMPA (2) was the most effective inhibitor of the constitutive current, while TPMPA (1), (±)-cis-3-ACPBPA (4), 4-GBA (11), SR-95331 (13), and SR-95813 (14) displayed moderate inhibition of the constitutive current.

Table 2. Effects of Structurally Diverse Antagonists on Recombinant ρ₁ Wild-Type and ρ_1Y102S Receptors^a.

^aPercentage inhibitions of ρ_1Y102S receptor spontaneous currents by compounds (100 and 300 μM), which were normalized by initial resting conductance. All data are the mean ± SEMs (n = 3–12 oocytes).

^bData from ref (8).

^cData from ref (36).

^dData from ref (37).

^eData from ref (29).

^fData from ref (38).

^gData from ref (39).

^hData from ref (13).

^IData from ref (31).

^jSee Figure 1 in Supporting Information.

^kSee Figure 2 in Supporting Information. NA stands for not active at the concentration.

To explore the relative activity of antagonists on the open conformational state of the ρ₁ receptor, we focused on five structurally different GABA antagonists, TPMPA (1), (±)-cis-3-ACPBPA (4), (±)-4-ACPAM (8), 4-GBA (11), SR-95531 (13), and SR-95813 (14) (Table 2). Application of the competitive antagonist, TPMPA (1), to ρ_1Y102S receptors inhibited the spontaneous current in a concentration-dependent manner (Figure 3A). This indicates that TPMPA shifts the equilibrium of ρ_1Y102S receptors from the open to the closed conformational state, thus acting as an inverse agonist (Table 2). However, TPMPA is weak exhibiting a 600-fold decrease in potency.

Figure 3C shows the concentration response curves for TPMPA (1), (±)-cis-3-ACPBPA (4), (±)-4-ACPAM (8), 4-GBA (11) SR-95531 (13), and SR-95813 (14) inhibiting the spontaneous current of ρ_1Y102S receptors. The affinities of the compounds against the spontaneous current were lower compared to the ρ₁ wild-type (Tables 2 and 3). The order of potency of the compounds at ρ_1Y102S receptors was SR-95813 (14) > SR-95531 (13) > (±)-cis-3-ACPBPA (4) > 4-GBA (11) > TPMPA (1) (Table 3). (±)-4-ACPAM (8) had a very small effect on the constitutive activity of ρ_1Y102S receptors even at the 1 mM concentration (Figure 3C) and failed to significantly inhibit GABA (EC₅₀; 180 μM) at this mutant receptor (Figure 3D).

Table 3. EC₅₀ Values of TPMPA (1), SR-95531 (13), SR-95813,¹⁴ (±)-cis-3-ACPBPA (4), and 4-GBA (11) on GABA ρ_1Y102S Receptors^a.

compd	EC50 (μM)b
TPMPA (1)	1234.3 ± 57.7
SR-95531 (13)	312.9 ± 15.1
SR-95813 (14)	64.3 ± 2.8
(±)-cis-3-ACPBPA (4)	488.3 ± 60.5
4-GBA (11)	568.7 ± 25.3

^aData are the means ± SEMs (n = 4–5 oocytes).

^bConcentration which inhibits 50% of the maximum spontaneous current of ρ_1Y102S receptor.

Interestingly, both SR-95531 (13) and SR-95813 (14) could not completely block the spontaneous current of ρ_1Y102S receptors (Figure 3C), indicating they may act as partial inverse agonists at the mutant receptor. Furthermore, in the presence of GABA, both SR-95531 (13) and SR-95813 (14) inhibited GABA with IC₅₀ values approximately 2-fold weaker than the EC₅₀ value that inhibits the constitutive activity (Table 3 and 4, Figure 4), suggesting the binding affinity of these compounds are similar in the presence or absence of GABA at ρ_1Y102S receptors.

Table 4. Effect of ρ₁Y102 Mutations on the Activity of Selected Antagonists in the Presence of GABA EC₅₀^a.

	% inhibition of GABA EC50 by selected compounds
	WT	Y102S	Y102C		Y102A
compd	300 μMb	300 μMb	300 μMb	IC50 (μM)c	300 μMb
TPMPA (1)	100.0 ± 0.0%	13.0 ± 1.5%	49.7 ± 5.5%	447.2 ± 50.9	25.1 ± 4.4%
(±)-cis-3-ACPBPA (4)	100.0 ± 0.0%	22.5 ± 2.4%	90.8 ± 2.6%	110.7 ± 22.6	74.1 ± 11.5%
(±)-4-ACPAM (8)	100.0 ± 0.0%	inactive at 300 μM	68.5 ± 2.1%	241.8 ± 17.2	21.0 ± 1.0%
4-GBA (11)	98.9 ± 0.6%	9.8 ± 2.8%	50.6 ± 7.1%	460.1 ± 58.1	47.9 ± 7.1%
SR-95531 (13)	96.0 ± 0.9%	775.7 ± 54.9 μMd	7.5 ± 4.1%	50.7 ± 6.4%e	inactive at 300 μM
SR-95813 (14)	98.8 ± 1.2%	135.2 ± 16.0 μMd	inactive at 300 μM	17.3 ± 6.4%e	inactive at 300 μM

^aAll data are the mean ± SEM (n = 3 oocytes).

^bData are percentage inhibition of the current produced by EC₅₀ (submaximal concentration) of GABA by selected compounds (300 μM). EC₅₀ (submaximal concentration) values for GABA at ρ₁ wild-type, ρ_1Y102S, ρ_1Y102C, and ρ_1Y102A mutant receptors are 1, 180, 20, and 200 μM, respectively. All data are the means ± SEMs (n = 3–6 oocytes)

^cCompound concentration of which inhibits EC₅₀ of GABA (20 μM) on ρ_1Y102C receptors.

^dCompound concentration of which inhibits EC₅₀ of GABA (180 μM) at ρ_1Y102S receptors.

^eData are percentage inhibition of the current produced by EC₅₀ of GABA (20 μM) by SR-95531 (13) and SR-95813 (14) (1 mM).

Figure 4.

Inhibitory concentration–response curves for SR-95531 (13) and SR-95813 (14)at GABA ρ_1Y102S receptors expressed in Xenopus oocytes. Each data point represents the mean ± SEM (n = 3–5). All antagonists were tested in the presence of GABA EC₅₀ (180 μM). All data are normalized with I_EC50[GABA].

Assessing the Activity of Antagonists at Receptors in the Closed Conformation State

The activities of the five antagonists were examined at ρ_1Y102C and ρ_1Y102A receptors (Table 4, Figure 5). In contrast to ρ_1Y102S receptors, ρ_1Y102C and ρ_1Y102A receptors were not constitutively active, thus existing predominantly in the closed conformational state. Interestingly, (±)-4-ACPAM (8) at a concentration of 300 μM regained some of its antagonist activity for the ρ_1Y102C and ρ_1Y102A mutant receptors (Table 4). At ρ_1Y102C receptors, (±)-4-ACPAM (8) displayed a 25-fold increase in IC₅₀ compared to ρ₁ wild-type (At ρ₁ wild-type; IC₅₀ = 9.6 ± 0.9 μM: at ρ_1Y102C; IC₅₀ = 241.8 ± 17.2 μM). As (±)-4-ACPAM (8) did not inhibit the constitutive activity of ρ_1Y102S mutant receptors, nor did it inhibit GABA (Figure 3B and D) or the inverse agonist effects of SR-95531 (13) (see Figure 3 in the Supporting Information) at this receptor, we can infer that either tyrosine is crucial for the binding of (±)-4-ACPAM (8) or that (±)-4-ACPAM (8) acts at receptors existing predominantly in the closed over the open conformational state.

Figure 5.

Inhibitory concentration–response curves for TPMPA (1), (±)-cis-3-ACPBPA (4), 4-GBA (11), SR-95531 (13), SR-95813 (14), and (±)-4-ACPAM (8) at GABA ρ_1Y102C receptors expressed in Xenopus oocytes. Each data point represents the mean ± SEM (n = 3–5). All antagonists were tested in the presence of GABA EC₅₀ (20 μM). All data are normalized with I_EC50[GABA].

At ρ_1Y102C receptors, the IC₅₀ values for TPMPA (1), (±)-cis-3-ACPBPA (4) and 4-GBA (11) were also increased by 200-, 22- and 25-fold, respectively, compared to ρ₁ wild-type receptors (Table 4). As the cysteine and alanine mutations did not affect potency of the antagonists to the same extent as the serine mutation, indicating that these compounds have the ability to preferentially bind to the closed conformational state of the receptor. A similar phenomenon is observed with tetracaine at nicotinic acetylcholine (nACh) receptors.²⁴ Tetracaine has a 100-fold higher affinity for the close conformation compared the desensitized conformation of the Torpedo nACh receptor, implicating tetracaine is a closed conformation channel blocker.

In contrast to what was observed at ρ_1Y102S receptors, the inhibitory activity of SR-95531 (13) and its analogue SR-95813 (14) was significantly reduced at both ρ_1Y102C and ρ_1Y102A receptors. SR-95531 (13) (300 μM) inhibited only 7.5% of the current elicited by GABA EC₅₀ (20 μM) at ρ_1Y102C receptors and was inactive at ρ_1Y102A receptors (Figure 6, Table 4). Furthermore, SR-95813 (14) (300 μM) did not inhibit the current elicited by GABA EC₅₀ (20 μM) at both ρ_1Y102C and ρ_1Y102A receptors (Figure 6, Table 4). Thus, the order of potency of the compounds tested at ρ_1Y102C receptors was (±)-cis-3-ACPBPA (4) > (±)-4-ACPAM (8) > 4-GBA (11) ≅ TPMPA (1) ≫ SR-95531 (13) ≅ SR-95318 (14). As SR-95531 (13) and its analogue SR-95813 (14) are more potent on ρ_1Y102S than ρ_1Y102C receptors, may indicate that the compounds are more likely to bind to the open over the closed conformational state of the receptor. While we cannot rule out the possibility of direct interaction between the introduced residues and the antagonists tested, there is no clear structure activity relationship to suggest that either possibility may be the case.

Figure 6.

Sample current trace showing the effect of SR-95531 (13) and SR-95813 (14) at GABA ρ_1Y102C and ρ_1Y102A receptors in Xenopus oocytes. (A) The current produced by GABA (20 μM) (black bar) was inhibited by 7.5% in the presence of SR-95531 (13) (300 μM, purple bar), and SR-95813 (14) (300 μM, dark blue) did not inhibit the current produced by GABA (20 μM, black bar) at ρ_1Y102C mutated receptors. (B) SR-95531 (13) (300 μM, purple bar), and SR-95813 (14) (300 μM, dark blue) did not inhibit the current produced by GABA (200 μM, black bar) at ρ_1Y102A mutated receptor.

Previous studies have shown that mutating Y102 of the ρ₁ subunit to phenylalanine alters the effect of SR-95531 (13)²⁵ and that the mutation of the homologous residue in the GABA_A receptor α₁-subunit (phenylalanine at position 64) to cysteine dramatically changes the affinity of SR-95531.²⁶ The data presented in this study using SR-95531 (13), SR-95813 (14), (±)-cis-3-ACPBPA (4), (±)-4-ACPAM (8), 4-GBA (11), and TPMPA (1) provides further support that Y102 plays a key role in binding/gating. However, the activity of SR-95531 (13) and its analogue SR-95813 (14) is not dramatically changed when ρ_1Y102 is mutated to serine. This indicates that, at least with the gabazine analogues, π–π interactions are not the main interactions affecting the activity of these compounds at ρ₁ receptors, despite an improved affinity of SR-95531 (13) when Y102 is mutated to phenylalanine.²⁵ This supports the homology model which infers that Y102 does not directly interact with GABA²² and is most likely a residue involved in channel gating. In support of this conclusion, the partial agonist imidazole-4-acetic acid (I4AA) activated the ρ_1Y102C mutant receptor with high efficacy and lower potency compared to ρ₁ wild-type,²⁰ consistent with Y102 being a residue involved in gating.

Conclusion

In conclusion, the affinity of ρ₁ receptor antagonists is dependent on the receptor conformation as a result of the introduced mutations. In this study we investigated the potencies of a range of antagonists at ρ₁ wild-type, ρ_1Y102S, ρ_1Y102C, and ρ_1Y102A mutant receptors. It was found that the acid moiety that is a common feature of most ρ₁ antagonists was not found to be critical for antagonist activity, as demonstrated with (±)-4-ACPAM (8) and SR-95813 (14). We also confirmed that Y102 plays important role in the potency of (±)-4-ACPAM (8), SR-95531 (13) and SR-95813 (14). In addition, (±)-4-ACPAM (8) is more potent for closed conformational state of the ρ₁ receptor, while SR-95531 (13) and its analogue SR-95318 (14) are more potent where there are receptors in the open conformational state.

Methods

Chemicals

TPMPA [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid],²⁷ (±)-cis-3-ACPBPA [(±)-cis-(3-aminocyclopentyl)butylphosphinic acid],²⁸ (±)-trans-3-ACPBPA [(±)-trans-(3-aminocyclopentyl)butylphosphinic acid],²⁸ (S)-4-ACPBPA [(S)-4-amino-1-cyclopent-1-enyl(butyl)phosphinic acid],²⁹ (+)-4-ACPCA [(+)-4-aminocyclopent-1-ene-1-carboxylic acid],³⁰ 4-GBA (4-guanidinobutanoic acid),³¹ ZAPA [(Z)-3-[(aminoiminomethyl)thio] prop-2-enoic acid],³² SR-95531 (gabazine),³³ and SR-95813³³ were synthesized according to our previously published methods.²⁷⁻³³

GABA (γ-aminobutyric acid), THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), and DAVA (5-aminovaleric acid) were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO). 3-APMPA (3-aminopropyl(methyl)phosphinic acid) was purchased from Tocris Bioscience (Bristol, U.K.). CGP-36742 or SGS-742 (3-aminopropyl-n-butylphosphinic acid) was a gift from Dr. Wolfgang Froestl (formerly Novartis, Switzerland).

Synthetic Procedure and Characterization Data for (±)-4-ACPAM (8) ((±)-4-Aminocyclopent-1-enecarboxamide)

Methyl 4-tert-butoxycarbonylaminocyclopent-1-enecarboxylate³⁴ (2.90 g, 12 mmol) was added to an aqueous solution of 0.5 M sodium hydroxide (80 mL) and tetrahydrofuran (40 mL) and allowed to stir overnight at room temperature. Excess tetrahydrofuran was removed from this solution under reduced pressure followed by extraction with dichloromethane (60 mL). The remaining aqueous fraction was acidified to pH 3 with 10% aqueous citric acid in the presence of dichloromethane (180 mL). The combined organic phases were dried over magnesium sulfate and evaporated to give 4-tert-butoxycarbonylaminocyclopent-1-enecarboxylic acid (2.59 g, 95% yield). R_f = 0.35 (4:1 ethyl acetate/petroleum ether). ¹H NMR (300 MHz, CDCl₃): δ 6.86 (1H, s, HC=), 4.75 (1H, br s, NH), 4.39 (1H, bs, C(4)H), 2.97 (2H, bt, J = 9 Hz, C(3)H and C(5)H), 2.49–2.39 (2H, m, C(3)H and C(5)H), 1.45 (9H, s, Boc). ¹³C NMR (300 MHz, CDCl₃): δ 168.96 (C=O), 143.94 (=C), 134.27 (HC=), 79.80 (C(CH₃)₃), 50.40 (CHNHBoc), 41.57 (cyclopentene CH₂), 39.03 (cyclopentene CH₂), 28.60 (C(CH₃)₃). CI-MS m/z 154 (52%, MH⁺-C₄H₈O), 126 (100, MH⁺-Boc), 93 (14, MH⁺-Boc-H₂O), 82 (45, MH⁺-Boc-CO₂).

Triethylamine (304 mg, 3 mmol) was added to a solution of 4-tert-butoxycarbonylaminocyclopent-1-enecarboxylic acid (7, 341 mg, 1.5 mmol) in tetrahydrofuran (30 mL) at 0 °C. iso-Butylchoroformate (338 mg, 2.5 mmol) was added dropwise, and the solution left to stir for 15 min. Gaseous ammonia was bubbled through the solution for 20 min and the reaction left to stir at 0 °C for a further 2 h. The reaction was concentrated in vacuo, diluted with ethyl acetate (30 mL), and washed with aqueous sodium hydroxide (1 M, 10 mL), saturated citric acid (10 mL), and brine (10 mL). The organic fraction was dried over sodium sulfate and solvent was removed under reduced pressure. The product was isolated using flash chromatography, eluting with ethyl acetate/dichloromethane (10:1) to give tert-butyl 3-carbamoylcyclopent-3-enylcarbamate (315 mg, 92% yield). R_f = 0.47 (ethyl acetate). ¹H NMR (300 MHz, CDCl₃): δ 6.52 (1H, s, HC=), 5.72–5.18 (2H, br d, NH₂), 4.85–4.64 (1H, m, C(4)H), 4.40 (1H, br s, NHBoc), 3.04–2.84 (2H, m, C(3)H and C(5)H), 2.52–2.33 (2H, m, C(3)H and C(5)H), 1.45 (9H, s, Boc). ¹³C NMR (300 MHz, CDCl₃): δ 166.76 ((C=O)NH₂), 155.57 ((C=O)Ot-Bu), 137.01 (HC=), 136.91 (=C), 79.80 C(CH₃)), 50.65 (CHNHBoc), 41.14 (cyclopentene CH₂), 39.64 (cyclopentene CH₂), 28.60 (C(CH₃)). tert-Butyl 3-carbamoylcyclopent-3-enylcarbamate (315 mg, 1.39 mmol) was dissolved in a saturated solution of hydrochloric acid in ethyl acetate and the resulting solution allowed to stir for 4 h. Solvent was removed in vacuo, and the product isolated using an ion-exchange column of Dowex 50W (H⁺) (10 mL), eluting the amino amide with ammonia (2 M). This gave 4-aminocyclopent-1-enecarboxamide (8, 156 mg, 89% yield). R_f = 0.27 (4:1:1 n-butanol/acetic acid/water). ¹H NMR (300 MHz, D₂O): δ 6.32 (1H, s, HC=), 4.03–3.93 (1H, m, CHNH₂), 3.01–2.84 (2H, m, C(3)H, and C(5)H), 2.56–2.43 (2H, m, C(3)H, and C(5)H). ¹³C NMR (300 MHz, D₂O): δ 171.13 (C=O), 140.23 (HC=), 136.08 (=C), 50.91 (CHNH₂), 42.31 (cyclopentene CH₂), 40.52 (cyclopentene CH₂). ESI-MS m/z positive ion mode: 127 (55%, MH⁺), 110 (5%, MH⁺-NH₃); negative ion mode: 126 (20%, M⁺-H).

Site-Directed Mutagenesis

Serine, cysteine, and alanine mutations were generated at the position 102 of ρ₁ subunit by using sense and antisense oligonucleotide primers (Table 1 in the Supporting Information) and the QuickChange II Site-directed Mutagenesis kit protocol (Stratagene, La Jolla, CA). All mutations were verified by DNA sequencing to confirm fidelity (Australian Genome Research Facility, Australia). The plasmids containing wild-type and mutations inserts were linearized with Xba-I, and T7 mMESSAGE mMACHINE kit (Ambion, Austin, TX) was used for mRNA synthesis.

Expression of Wild-Type and Mutant ρ₁ Receptors in Xenopus Oocytes

Oocytes from Xenopus laevis (South Africa clawed frogs) were harvested as described previously³⁵ in accordance with the National Health and Medical Research Council of Australia’s ethical guidelines and approved by the University of Sydney Animal Ethics Committee. Stage V–VI oocytes were injected with 10–15 ng cRNA and then stored at 18 °C in ND 96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, pH 7.5) supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 μg mL^–1 gentamycin, and 2.5 mg mL^–1 tetracycline.

Electrophysiological Recordings

Two to eight days after injections, the activity was measured by two-electrode voltage clamp recording using a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA), a MacLab 2e recorder (AD Instruments, Sydney, NSW, Australia), and Chart version 5.5.6 program as previously described.⁸ Briefly, oocyte expression receptors were clamped at −60 mV with continuous flow of ND96 buffer. Antagonists were screened for inverse agonist activity by applying increasing concentrations (100 and 300 μM) on ρ₁ receptors. SR-95531 (13) and SR-95813 (14) were dissolved in DMSO, and the compounds concentrations were made with the total concentration of 0.8% DMSO. SR-95531 (13) and SR-95813 (14) were not tested higher than 3 mM concentration due to the solubility issues at high concentrations. Antagonist effects were tested in the presence of GABA EC₅₀ concentration (20 μM for ρ_1Y102C and 200 μM for ρ_1Y102A receptors) on ρ_1Y102C and ρ_1Y102A receptors, and the effects were evaluated for their inhibitory concentration–response actions using ρ_1Y102C receptors. For selected antagonists, concentration–inhibition curves were constructed with a minimum of three cells.

Data Analysis

Current responses were normalized to the maximum GABA-activated current recorded in the same cell and expressed as a percentage of this maximum and fitted by least-squares to Hill equation (eq 1). GABA concentration response curves were generated using GraphPad PRISM 5.02 (GraphPad software San Diego, CA).

1

where I is the current response to a known concentration of agonist, I_max is the maximum current obtained, [A] is the agonist concentration, EC₅₀ is the concentration of agonist at which current response is half maximal, and n_H is the Hill coefficient.

Dissociation equilibrium constants (K_B) were determined via the Schild equation (eq 2), where [B] is the antagonist concentration, [A] is the EC₅₀ of GABA in the presence of antagonist, and [A*] is the EC₅₀ of GABA in the absence of antagonist. The Schild plot of log([A]/[A*] – 1) versus log[B] was fitted, and the slope was sufficiently close to 1 (see Figure 2 in the Supporting Information). Data are expressed as means ± standard error of the mean (SEM).

2

IC₅₀ values were calculated using eq 3. The inhibitory concentration curves were generated using GraphPad PRISM 5.02.

3

I is the peak current at a given concentration of agonist, I_max is the maximal current generated by the concentration of agonist, [A] is the concentration of GABA, IC₅₀ is the antagonist concentration, which inhibits 50% of the maximum GABA response, and n_H is the Hill coefficient.

Glossary

Abbreviations

(+)-4-ACPCA

(+)-4-aminocyclopent-1-ene-1-carboxylic acid

(±)-4-ACPAM

(±)-4-aminocyclopent-1-enecarboxamide

(±)-cis-3-ACPBPA

(±)-cis-(3-aminocyclopentyl)butylphosphinic acid

(±)-trans-3-ACPBPA

(±)-trans-(3-aminocyclopentyl)butylphosphinic acid

(S)-4-ACPBPA

[S)-4-amino-1-cyclopent-1-enyl(butyl)phosphinic acid

3-APMPA

3-aminopropyl(methyl)phosphinic acid

4-GBA

4-guanidinobutanoic acid

CGP-36742 or SGS-742

(3-aminopropyl-n-butylphosphinic acid

DAVA

5-aminovaleric acid

EC₅₀

effective concentration that activates/or inhibits 50% of the maximum response/or spontaneous current

GABA

γ-aminobutyric acid

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IC₅₀

effective concentration that inhibits 50% of GABA EC₅₀

LGIC

ligand-gated ion channel

THIP

4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol

TPMPA

(1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid

ZAPA

(Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid

Supporting Information Available

Information on the oligonucleotide primers, pharmacology of SR-95531 (13), Schild plot analysis, and effect of (±)-4-ACPAM (8). This material is available free of charge via the Internet at http://pubs.acs.org.

Author Contributions

Participated in research design: I.Y., N.G., K.L., R.D.A., J.E.C., M.C.. Conducted experiments: I.Y., N.G., K.L.. Performed data analysis: I.Y., J.E.C., N.A., M.C.. Wrote or contributed to the writing of the manuscript: I.Y., J.E.C., N.A., N.G., K.L., J.R.H., G.A.R.J., M.C..

Both I.Y. and N.G. acknowledge the John A. Lamberton Scholarship, and N.G. acknowledges support from an Endeavor International Postgraduate Research Scholarship.

The authors declare no competing financial interest.