Yongchang Chang

Stoichiometry of a Recombinant GABAA Receptor

GABA is the main inhibitory neurotransmitter in the mammalian brain. The postsynaptic GABAA receptor/pore complex is presumed to be a pentamer typically composed of a combination of α, β, and γ subunits, although the stoichiometry remains controversial. We probed the stoichiometry of the GABAAreceptor by site-directed mutagenesis of a conserved leucine (to serine) in the putative second membrane-spanning domain of the rat α1(αL263S), β2(αL259S), and γ2(αL274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g., α and αL263S), along with their wild-type counterparts (e.g., β and γ), in Xenopus laevis oocytes resulted in mixed populations of receptors with distinct GABA sensitivities. This is consistent with the interpretation that the leucine mutation increased the GABA sensitivity in proportion to the number of incorporated mutant subunits. The apparent number of incorporated subunits for each class (α, β, and γ) could then be determined from the number of components comprising the compound GABA dose–response relationships. Using this approach, we conclude that the recombinant α1β2γ2 GABAA receptor is a pentamer composed of two α subunits, two β subunits, and one γ subunit.

Channel opening locks agonist onto the GABA(C) receptor

Determination of the activation mechanism of neurotransmitter-operated ion channels has been hindered by a limited understanding of the relationship between agonist binding and the gating of the integral ion pore. Here we describe a [3H]ligand binding assay that enables us to make repeated binding measurements from the same intact oocyte expressing recombinant human ρ1 GABAC receptors and directly correlate the binding kinetics with electrophysiological measurements. We have determined an association rate for GABA of about 105 M–1s–1; this is four orders of magnitude slower than diffusion, indicating GABA has restricted access to its binding site. We also demonstrate that GABA dissociates at two rates. Our data are consistent with the faster rate being the true microscopic dissociation rate of GABA, with the slower rate occurring because the opening of the pore detains agonist release.

Allosteric Activation Mechanism of the α1β2γ2 γ-Aminobutyric Acid Type A Receptor Revealed by Mutation of the Conserved M2 Leucine

A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat alpha 1 beta 2 gamma 2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in alpha1(L263), beta2(L259), and gamma 2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild-type and/or mutant counterparts. The mutation in any one subunit decreased the EC(50) and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC(50) and picrotoxin IC(50) as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (beta 2Y157S; increased the EC(50)) and the conserved M2 leucine (beta 2L259S; decreased the EC(50)) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism.

Mechanism of action of benzodiazepines on GABAA receptors

1 Wild‐type and mutant α1β2γ2 GABAA receptors were expressed in Xenopus laevis oocytes and examined using the two‐electrode voltage clamp. 2 Dose–response relationships for GABA were compared in the absence and presence of 1 μM diazepam (DZP) or methyl‐6,7‐dimethoxy‐4‐ethyl‐beta‐carboline‐3‐carboxylate (DMCM). The dose–current relationships yielded EC50s (concentration for half‐maximal activation) of 41.0±3.0, 21.7±2.7, and 118.3±6.8 μM for GABA, GABA plus DZP, and GABA plus DMCM, respectively. 3 DZP‐ and DMCM‐mediated modulation were examined in GABAA receptors in which the β‐subunit carries the L259S mutation. This mutation has been shown to produce spontaneous opening and impart a leftward shift in the dose–response relationship. In this case, neither DZP nor DMCM produced a significant alteration in the GABA dose–response relationship with GABA EC50s of 0.078±0.005, 0.12±0.03, and 0.14±0.004 μM for GABA, GABA plus 1 μM DZP, and GABA plus 1 μM DMCM. 4 DZP‐ and DMCM‐mediated modulations were examined in GABAA receptors in which the α‐subunit carries the L263S mutation. This mutation also produced spontaneous opening and a leftward shift of the GABA dose–response relation, but to a lesser extent than that of βL259S. In this case, the leftward and rightward shifts for DZP and DMCM were still present with EC50s=0.24±0.03, 0.14±0.02, and 1.2±0.04 μM for GABA, GABA plus 1 μM DZP, and GABA plus 1 μM DMCM, respectively. 5 Oocytes expressing ultrahigh levels of wild‐type GABAA receptors exhibited currents in response to 1 μM DZP alone, whereas DMCM decreased the baseline current. The DZP‐mediated activation currents were determined in wild‐type receptors as well as receptors in which the GABA binding site was mutated (β2Y205S). The EC50s for DZP‐mediated activation were 72.0±2.0 and 115±6.2 nM, respectively, similar to the EC50 for DZP‐mediated enhancement of the wild‐type GABA‐activated current (64.8±3.7 nM). 6 Our results support a mechanism in which DZP increases the apparent affinity of the receptor, not by altering the affinity of the closed state, but rather by shifting the equilibrium towards the high‐affinity open state.

Site-specific fluorescence reveals distinct structural changes with GABA receptor activation and antagonism

Neurotransmitter-operated ion channels, such as the GABA (γ-aminobutyric acid) receptor, are important in fast synaptic transmission between neurons. Using site-specific fluorescent labeling and simultaneous electrophysiological analysis in Xenopus laevis oocytes expressing recombinant ρ1 GABA receptors, we identified agonist-mediated molecular rearrangements at three positions within and near the agonist-binding pocket that were highly correlated with receptor activation. We also show that competitive antagonists induced distinct rearrangements on their own that stabilized the receptor in a closed state. Finally, the allosteric antagonist picrotoxin induced a global conformational change that was sensed in the subunit–subunit interface of the amino (N)-terminal domain, distant from its presumed site of action within the transmembrane domains. This first detection in real time of molecular rearrangements of a ligand-activated receptor provides insights into the structural correlates of activation, antagonism and allosteric modulation.

Mapping the ρ1 GABAC Receptor Agonist Binding Pocket CONSTRUCTING A COMPLETE MODEL

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The GABA receptor type C (GABAC) is a ligand-gated ion channel with pharmacological properties distinct from the GABAA receptor. To date, only three binding domains in the recombinant ρ1 GABAC receptor have been recognized among six potential regions. In this report, using the substituted cysteine accessibility method, we scanned three potential regions previously unexplored in the ρ1 GABAC receptor, corresponding to the binding loops A, E, and F in the structural model for ligand-gated ion channels. The cysteine accessibility scanning and agonist/antagonist protection tests have resulted in the identification of residues in loops A and E, but not F, involved in forming the GABAC receptor agonist binding pocket. Three of these newly identified residues are in a novel region corresponding to the extended stretch of loop E. In addition, the cysteine accessibility pattern suggests that part of loop A and part of loop E have a β-strand structure, whereas loop F is a random coil. Finally, when all of the identified ligand binding residues are mapped onto a three-dimensional homology model of the amino-terminal domain of the ρ1 GABAC receptor, they are facing toward the putative binding pocket. Combined with previous findings, a complete model of the GABAC receptor binding pocket was proposed and discussed in comparison with the GABAA receptor binding pocket.

Permeability and single channel conductance of human homomeric ρ1 GABAC receptors

1 Homomeric human ρ1 GABAC receptors were expressed in Xenopus oocytes and in human embryonic kidney cells (HEK293) in order to examine their conductance and permeability. 2 Reversal potentials of currents elicited by γ‐aminobutyric acid (GABA) were measured in extracellular solutions of various ionic composition to determine relative permeability of homomeric ρ1 receptors. The rank order of anionic permeability was: SCN− > I− > NO3− > Br− > Cl− > formate (For−) > HCO3− > acetate (Ac−) ≈ proprionate (Prop−) ≈ isethionate (Ise−) ≈ F−≈ PO4−. 3 In the oocyte expression system, relative permeabilities to SCN−, I−, NO3−, Br− and HCO3− were higher for ρ1 GABAC receptors than α1β2γ2L GABAA receptors. 4 Expression of ρ1 GABAC receptors in Xenopus oocytes and in HEK293 cells gave similar relative permeabilities for selected anions, suggesting that the expression system does not significantly alter permeation properties. 5 The pore diameter of the homomeric ρ1 GABAC receptor expressed in oocytes was estimated to be 0.61 nm, which is somewhat larger than the 0.56 nm pore diameter estimated for α1β2γ2L GABAA receptors. 6 Homomeric ρ1 GABA receptors expressed in oocytes had a single channel chord conductance of 0.65 ± 0.04 pS (mean ±s.e.m.s) when the internal chloride concentration ([Cl−]i) was 20 mm. With a [Cl−]i of 100 mm, the single channel chord conductance was 1.59 ± 0.24 pS. 7 The mean open time directly measured from 43 GABA‐induced channel openings in six patches was 3.2 ± 0.8 s. The mean open time in the presence of 100 μm picrotoxin was 0.07 ± 0.01 s (77 openings from 3 patches). 8 The differences observed in ionic permeabilities, pore size, single channel conductance and mean open time suggest that the ρ1 homomeric receptor may not be the native retinal GABAC receptor reported previously.

Positive allosteric modulation by ultraviolet irradiation on GABAA, but not GABAC, receptors expressed in Xenopus oocytes

1 Recombinant rat GABAA (α1β2, α1β2γ2, β2γ2) and human GABAC (ρ1) receptors were expressed in Xenopus oocytes to examine the effect of ultraviolet (UV) light on receptor function. 2 GABA‐induced currents in individual oocytes expressing GABA receptors were tested by two‐electrode voltage clamp before, and immediately after, 312 nm UV irradiation. 2 UV irradiation significantly potentiated 10 μm GABA‐induced currents in α1β2γ2 GABA receptors. The modulation was irradiation dose dependent, with a maximum potentiation of more than 3‐fold. 3 The potentiation was partially reversible and decayed exponentially with a time constant of 8.2 ± 1.2 min toward a steady‐state level which was still significantly elevated (2.7 ± 0.3‐fold) compared to the control level. 4 The effect of UV irradiation on GABAA receptors varied with receptor subunit composition. UV irradiation decreased the EC50 of the α1β2, α1β2γ2 and β2γ2 GABAA receptors, but exhibited no significant effect on the ρ1 GABAC receptor. 5 UV irradiation also significantly increased the maximum current 2‐fold in α1β2 GABAA receptors with little effect on the maximum of α1β2γ2 (1.1‐fold) or β2γ2 (1.1‐fold) GABAA receptors. 6 The effect of UV irradiation on GABAA receptors did not overlap the effect of the GABA receptor‐ allosteric modulator, diazepam. 7 The UV effect on GABAA receptors was not prevented by the treatment of the oocytes before and during UV irradiation with one of the following free‐radical scavengers: 40 mmd‐mannitol, 40 mm imidazole or 40 mm sodium azide. In addition, the effect was not mimicked by the free‐radical generator, H2O2. 8 Potential significance and mechanism(s) of the UV effect on GABA receptors are discussed.

Crystallizing our understanding of partial agonists.

A powerful combination of X-ray crystallography and single-channel current measurements provides new insights into the mechanism by which the binding of agonists opens the AMPA-type glutamate receptor in the central nervous system.