Activation of the Rat α1ß2ε GABAA Receptor by Orthosteric and Allosteric Agonists.

GABAA receptors are a major contributor to fast inhibitory neurotransmission in the brain. The receptors are activated upon binding the transmitter GABA or allosteric agonists including a number of GABAergic anesthetics and neurosteroids. Functional receptors can be formed by various combinations of the nineteen GABAA subunits cloned to date. GABAA receptors containing the ε subunit exhibit a significant degree of constitutive activity and have been suggested to be unresponsive to allosteric agents. In this study, we have characterized the functional properties of the rat α1ß2ε GABAA receptor. We confirm that the α1ß2ε receptor exhibits a higher level of constitutive activity than typical of GABAA receptors and show that it is inefficaciously activated by the transmitter and the allosteric agonists propofol, pentobarbital, and allopregnanolone. Manipulations intended to alter ε subunit expression and receptor stoichiometry were largely without effect on receptor properties including sensitivity to GABA and allosteric agonists. Surprisingly, amino acid substitutions at the conserved 9' and 6' positions in the second transmembrane (TM2) domain in the ε subunit did not elicit the expected functional effects of increased constitutive activity and resistance to the channel blocker picrotoxin, respectively. We tested the accessibility of TM2 residues mutated to cysteine using the cysteine-modifying reagent 4-(hydroxymercuri)benzoic acid and found a unique pattern of water-accessible residues in the ε subunit.

Analysis of Modulation of the ρ1 GABAA Receptor by Combinations of Inhibitory and Potentiating Neurosteroids Reveals Shared and Distinct Binding Sites.

The ρ1 GABAA receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the ρ1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABAA receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5ß-reduced steroids, sulfated or carboxylated steroids, and ß-estradiol, whereas many 5α-reduced steroids potentiate the receptor. In this study, we analyzed modulation of the human ρ1 GABAA receptor by several neurosteroids, individually and in combination, in the framework of the coagonist concerted transition model. Experiments involving coapplication of two or more steroids revealed that the receptor contains at least three classes of distinct, nonoverlapping sites for steroids, one each for the inhibitory steroids pregnanolone (3α5ßP), 3α5ßP sulfate, and ß-estradiol. The site for 3α5ßP can accommodate the potentiating steroid 5αTHDOC. The findings are discussed with respect to receptor modulation by combinations of endogenous neurosteroids. SIGNIFICANCE STATEMENT: The study describes modulation of the ρ1 GABAA receptor by neurosteroids. The coagonist concerted transition model was used to determine overlap of binding sites for several inhibitory and potentiating steroids.

Modulation of the human ρ1 GABAA receptor by inhibitory steroids.

RATIONALE: Modulators of the ρ1 GABAA receptor may be useful in the treatment of visual, sleep, and cognitive disorders. Neuroactive steroids and analogues have been shown to modulate ρ1 receptor function, but the molecular mechanisms are poorly understood. OBJECTIVES: We employed electrophysiology and voltage-clamp fluorometry to compare the actions of several neuroactive steroids and analogues on the human ρ1 GABAA receptor. RESULTS: Results confirmed that P294S and T298F mutations affect modulation by steroids. The P294S mutation abolished inhibition by (3α,5ß)-3-hydroxypregnan-20-one (3α5ßP) while the T298F mutation eliminated inhibition by 17ß-estradiol. Voltage-clamp fluorometry demonstrated that steroids differing in the presence of a charged group on C3 or nature of substituent on C17 uniquely modified fluorescence changes elicited by GABA in the extracellular domain. The I307Q mutation reversed the inhibitory effect of 3α5ßP but was without effect on modulation by (3α,5ß)-3-hydroxypregnan-20-one sulfate or 17ß-estradiol. The effect of 3α5ßP on the fluorescence change generated at Y241C was dependent on whether the steroid acted as an inhibitor or a potentiator. Further, the effect was limited to uncharged 5ß-reduced steroids containing an acetyl group on C17. CONCLUSIONS: The data demonstrate that steroids and analogues differ with respect to conformational changes elicited by these drugs as well as sensitivity to the effects of mutations. Steroids and analogues could be provisionally divided into three major groups based on their actions on the ρ1 GABAA receptor: 5ß-reduced uncharged steroids, sulfated and carboxylated steroids, and 17ß-estradiol. Further division among 5ß-reduced uncharged steroids was based on substituent at position C17.

Impact of human D398N single nucleotide polymorphism on intracellular calcium response mediated by α3ß4α5 nicotinic acetylcholine receptors.

The human CHRNA5 D398N polymorphism (rs16969968) causes an aspartic acid to asparagine change in the nicotinic acetylcholine receptor (nAChR) α5 subunit gene. The N398 variant of CHRNA5 is linked to increased risk for nicotine dependence. In this study, we explored the effect of the CHRNA5 D398N polymorphism on the properties of human α3ß4* nicotinic acetylcholine receptors in human embryonic kidney (HEK) cells. Addition of either D398 or N398 variant of α5 subunit in the α3ß4* receptor did not affect total [(125)I]-epibatidine binding or surface expression of the receptor. However, addition of α5(D398) into α3ß4* receptor decreased the maximal response to agonist without significantly affecting EC(50) in aequorin intracellular calcium assay. α3ß4α5(N398) nAChRs showed further decreased maximal response. The differences in agonist efficacy between the receptor subtypes were found to be dependent upon the concentration of external calcium but independent of external sodium. Moreover, activation of α3ß4α5 nAChRs led to significantly greater intracellular calcium release from IP(3) stores relative to α3ß4 nAChRs although no effect of the α5 polymorphism was observed. Finally, inclusion of the α5 variant caused a small shift to the left in IC(50) for some of the antagonists tested, depending upon α5 variant but did not affect sensitivity of α3ß4* receptors to desensitization in response to incubation with nicotine. In conclusion, addition of either variant of α5 into an α3ß4α5 receptor similarly effects receptor pharmacology and function. However, the N398 variant exhibits a reduced response to agonists when extracellular calcium is high and it may lead to distinct downstream cellular signaling.

Agonist-specific conformational changes in the α1-γ2 subunit interface of the GABA A receptor.

The GABA(A) receptor undergoes conformational changes upon the binding of agonist that lead to the opening of the channel gate and a flow of small anions across the cell membrane. Besides the transmitter GABA, allosteric ligands such as the general anesthetics pentobarbital and etomidate can activate the receptor. Here, we have investigated the agonist specificity of structural changes in the extracellular domain of the receptor. We used the substituted cysteine accessibility method and focused on the γ2(S195C) site (loop F). We show that modification of the site with (2-sulfonatoethyl)methanethiosulfonate (MTSES) results in an enhanced response to GABA, indicating accessibility of the resting receptor to the modifying agent. Coapplication of GABA or muscimol, but not of gabazine, with MTSES prevented the effect, suggesting that GABA and muscimol elicit a conformational change that reduces access to the γ2(S195C) site. Exposure of the receptors to MTSES in the presence of the allosteric activators pentobarbital and etomidate resulted in an enhanced current response indicating accessibility and labeling of the γ2(S195C) site. However, comparison of the rates of modification indicated that labeling in the presence of etomidate was significantly faster than that in the presence of pentobarbital or gabazine or in resting receptors. We infer from the data that the structure of the α1-γ2 subunit interface undergoes agonist-specific conformational changes.

Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence.

Genome-wide association studies have identified common variation in the CHRNA5-CHRNA3-CHRNB4 and CHRNA6-CHRNB3 gene clusters that contribute to nicotine dependence. However, the role of rare variation in risk for nicotine dependence in these nicotinic receptor genes has not been studied. We undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes in African American and European American nicotine-dependent smokers and smokers without symptoms of dependence. Carrier status of individuals harboring rare missense variants at conserved sites in each of these genes was then compared in cases and controls to test for an association with nicotine dependence. Missense variants at conserved residues in CHRNB4 are associated with lower risk for nicotine dependence in African Americans and European Americans (AA P = 0.0025, odds-ratio (OR) = 0.31, 95% confidence-interval (CI) = 0.31-0.72; EA P = 0.023, OR = 0.69, 95% CI = 0.50-0.95). Furthermore, these individuals were found to smoke fewer cigarettes per day than non-carriers (AA P = 6.6 × 10(-5), EA P = 0.021). Given the possibility of stochastic differences in rare allele frequencies between groups replication of this association is necessary to confirm these findings. The functional effects of the two CHRNB4 variants contributing most to this association (T375I and T91I) and a missense variant in CHRNA3 (R37H) in strong linkage disequilibrium with T91I were examined in vitro. The minor allele of each polymorphism increased cellular response to nicotine (T375I P = 0.01, T91I P = 0.02, R37H P = 0.003), but the largest effect on in vitro receptor activity was seen in the presence of both CHRNB4 T91I and CHRNA3 R37H (P = 2 × 10(-6)).

Use of concatemers of ligand-gated ion channel subunits to study mechanisms of steroid potentiation.

Synaptic receptors of the nicotinic receptor gene family are pentamers of subunits. This modular structure creates problems in studies of drug actions, related to the number of copies of a subunit that are present and their position. A separate issue concerns the mechanism of action of many anesthetics, which involves potentiation of responses to neurotransmitters. Potentiation requires an interaction between a transmitter and a potentiator, mediated through the target receptor. We have studied the mechanism by which neurosteroids potentiate transmitter responses, using concatemers of covalently linked subunits to control the number and position of subunits in the assembled receptor and to selectively introduce mutations into positionally defined copies of a subunit. We found that the steroid needs to interact with only one site to produce potentiation, that the native sites for steroid interaction have indistinguishable properties, and that steroid potentiation appears to result from a global effect on receptor function.

Functional characterization of the α5(Asn398) variant associated with risk for nicotine dependence in the α3ß4α5 nicotinic receptor.

Smoking is a major cause for premature death. Work aimed at identifying genetic factors that contribute to nicotine addiction has revealed several single nucleotide polymorphisms (SNPs) that are linked to smoking-related behaviors such as nicotine dependence and level of smoking. One of these SNPs leads to an aspartic acid-to-asparagine substitution in the nicotinic receptor α5 subunit at amino acid position 398 [rs16969968; α5(Asn398)]. The α5 subunit is expressed both in the brain and in the periphery. In the brain, it associates with the α4 and ß2 subunits to form α4ß2α5 receptors. In the periphery, the α5 subunit combines with the α3 and ß4 subunits to form the major ganglionic postsynaptic nicotinic receptor subtype. The α3ß4α5 receptor regulates a variety of autonomic responses such as control of cardiac rate, blood pressure, and perfusion. In this paradigm, the α5(Asn398) variant may act by regulating autonomic responses that may affect nicotine intake by humans. Here, we have investigated the effect of the α5(Asn398) variant on the function of the α3ß4α5 receptor. The wild-type or variant α5 subunits were coexpressed with the α3 and ß4 subunits in human embryonic kidney 293 cells. The properties of the receptors were studied using whole-cell and single-channel electrophysiology. The data indicate that the introduction of the α5(Asn398) mutation has little effect on the pharmacology of receptor activation, receptor desensitization, or single-channel properties. We propose that the effect of the α5(Asn398) variant on nicotine use is not mediated by an action on the physiological or pharmacological properties of the α3ß4α5 subtype.

Site-specific fluorescence reveals distinct structural changes induced in the human rho 1 GABA receptor by inhibitory neurosteroids.

The rho 1 GABA receptor is inhibited by a number of neuroactive steroids. A previous study (J Pharmacol Exp Ther 323:236-247, 2007) focusing on the electrophysiological effects of inhibitory steroids on the rho 1 receptor found that steroid inhibitors could be divided into three major groups based on how mutations to residues in the M2 transmembrane domain modified inhibition. It was proposed that the steroids act through distinct mechanisms. We selected representatives of the three groups (pregnanolone, tetrahydrodeoxycorticosterone, pregnanolone sulfate, allopregnanolone sulfate, and beta-estradiol) and probed how these steroids, as well as the nonsteroidal inhibitor picrotoxinin, modify GABA-elicited fluorescence changes from the Alexa 546 C5 maleimide fluorophore attached to residues in the extracellular region of the receptor. The fluorophore responds with changes in quantum yield to changes in the environment, allowing it to probe for structural changes taking place during channel activation or modulation. The results indicate that the modulators have specific effects on fluorescence changes suggesting that distinct conformational changes accompany inhibition. The findings are consistent with the steroids acting as allosteric inhibitors of the rho 1 GABA receptor and support the hypothesis that divergent mechanisms underlie the action of inhibitory steroids on the rho 1 GABA receptor.

Ethanol modulates the interaction of the endogenous neurosteroid allopregnanolone with the alpha1beta2gamma2L GABAA receptor.

We have examined alpha1beta2gamma2L GABAA receptor modulation by the endogenous steroids allopregnanolone (3alpha5alphaP), pregnenolone sulfate, and beta-estradiol in the absence and presence of ethanol. Coapplication of 0.1 to 1% (17-170 mM) ethanol influenced receptor modulation by 3alpha5alphaP but not that by pregnenolone sulfate or beta-estradiol. One of the three kinetic effects evident in channel potentiation by 3alpha5alphaP, prolongation of the longest-lived open time component (OT3), was affected by ethanol with the midpoint of its dose-response curve moved to lower steroid concentrations by 2 orders of magnitude without significantly affecting the maximal effect. Manipulations designed to affect the ability of 3alpha5alphaP to prolong OT3 also affected OT3 prolongation in the presence of ethanol. A mutation to the gamma2 subunit, which reduces the ability of 3alpha5alphaP to prolong OT3, also reduces the interaction between ethanol and 3alpha5alphaP. And the presence of the competitive steroid antagonist (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17-PA) diminishes the positive interaction between ethanol and 3alpha5alphaP on the GABAA receptor. Together, the findings suggest that steroid interactions with the classic steroid binding site underlie the effect seen in the presence of ethanol, and that ethanol acts by increasing the affinity of 3alpha5alphaP for the site. Tadpole behavioral assays showed that the presence of 3alpha5alphaP at a concentration ineffective at causing changes in tadpole behavior shifted the ethanol dose-response curve for loss of righting reflex to lower concentrations and that this effect was neutralized by coapplication of 17-PA with 3alpha5alphaP.