Ian L. Martin

Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the α4β3δ GABAA Receptor

The GABAA receptor is a chloride-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor consists of five subunits arranged pseudosymmetrically around a central pore. The predominant form of the receptor in the brain contains α1-, β2-, and γ2-subunits in the arrangement αβαγβ, counter-clockwise around the pore. GABAA receptors containing δ-instead of γ-subunits, although a minor component of the total receptor population, have interesting properties, such as an extrasynaptic location, high sensitivity to GABA, and potential association with conditions such as epilepsy. They are therefore attractive targets for drug development. Here we addressed the subunit arrangement within the α4β3δ form of the receptor. Different epitope tags were engineered onto the three subunits, and complexes between receptors and anti-epitope antibodies were imaged by atomic force microscopy. Determination of the numbers of receptors doubly decorated by each of the three antibodies revealed a subunit stoichiometry of 2α:2β:1δ. The distributions of angles between pairs of antibodies against the α- and β-subunits both had peaks at around 144°, indicating that these pairs of subunits were nonadjacent. Decoration of the receptor with ligands that bind to the extracellular domain (i.e., the lectin concanavalin A and an antibody that recognizes the β-subunit N-terminal sequence) showed that the receptor preferentially binds to the mica extracellular face down. Given this orientation, the geometry of complexes of receptors with both an antibody against the δ-subunit and Fab fragments against the α-subunits indicates a predominant subunit arrangement of αβαδβ, counter-clockwise around the pore when viewed from the extracellular space.

Direct visualization of ligand-protein interactions using atomic force microscopy.

Streptavidin is a 60‐kDa tetramer which binds four molecules of biotin with extremely high affinity (KA∼1014u2003M−1). We have used atomic force microscopy (AFM) to visualize this ligand‐protein interaction directly. Biotin was tagged with a short (152‐basepair; 50‐nm) DNA rod and incubated with streptavidin. The resulting complexes were then imaged by AFM. The molecular volume of streptavidin calculated from the dimensions of the protein particles (105±3u2003nm3) was in close agreement with the value calculated from its molecular mass (114u2003nm3). Biotinylation increased the apparent size of streptavidin (to 133±2u2003nm3), concomitant with an increase in the thermal stability of the tetramer. Images of streptavidin with one to four molecules of DNA‐biotin bound were obtained. When two ligands were bound, the angle between the DNA rods was either acute or obtuse, as expected from the relative orientations of the biotin binding sites. The ratio of acuteu2003:u2003obtuse angles (1u2003:u20033) was lower than the expected value (1u2003:u20032), indicating a degree of steric hindrance in the binding of the DNA‐biotin. The slight under‐representation of higher occupancy states supported this idea. Streptavidin with a single molecule of DNA‐biotin bound was used to tag biotinylated β‐galactosidase, a model multimeric enzyme. The ability to image directly the binding of a ligand to its protein target by AFM provides useful information about the nature of the interaction, and about the effect of complex formation on the structure of the protein. Furthermore, the use of DNA‐biotin/streptavidin tags could potentially shed light on the architecture of multi‐subunit proteins.

Decreased GABA enhancement of benzodiazepine binding after a single dose of diazepam.

Abstract: The GABA and benzodiazepine binding sites on GABAA receptors are allosterically coupled. The in vitro binding of 2 nM [3H]flunitrazepam to cortical and cerebellar membranes prepared from drug‐naive rats was potentiated ∼1.6‐fold by 100 γM GABA. Potentiation in both regions was significantly reduced 4 or 12 but not 24 h after a single dose of 15 mg/kg diazepam. At 24 h after the last of 14 daily doses of diazepam, no differences in GABA poten‐tiation were observed. Diazepam‐induced changes in GABAA receptor δ2‐subunit gene transcription and α1‐, β2‐, and δ2‐subunit steady‐state mRNA levels did not appear to be temporally related to allosteric uncoupling.

Isomerization of the proline in the M2-M3 linker is not required for activation of the human 5-HT3A receptor.

Each subunit of the cation‐selective members of the Cys‐loop family of ligand‐gated ion channels contains a conserved proline residue in the extracellular loop between the second and third transmembrane domains. In the mouse homomeric 5‐hydroxytryptamine type 3A (5‐HT3A) receptor, the effects of substitution of this proline by unnatural amino acids led to the suggestion that trans‐cis isomerization of the protein backbone at this position is integral to agonist‐induced channel opening [Nature (2005) vol. 438, pp. 248–252]. We explored the generality of this conclusion using natural amino acid mutagenesis of the homologous human 5‐HT3A receptor. The conserved proline (P303) was substituted by either a histidine or tryprophan and the mutant receptors were expressed in Xenopus oocytes. These mutations did not significantly affect the magnitude of agonist‐mediated currents, compromise channel gating by 5‐HT or inhibition of 5‐HT‐induced currents by either picrotoxin or d‐tubocurarine. The mutations did, however, result in altered dependence on extracellular Ca2+ concentration and a 10‐fold increase in the rate of receptor desensitization. These results demonstrate an important role for P303 in 5‐HT3A receptor function but indicate that trans‐cis isomerization at this proline is unlikely to be a general mechanism underlying the gating process.

Effects of the Antidepressant/Antipanic Drug Phenelzine on Alanine and Alanine Transaminase in Rat Brain

Abstract1. Phenelzine (PLZ) is an antidepressant with anxiolytic properties. Acute and chronic PLZ administration increase brain GABA levels, an effect due, at least in part, to an inhibition of the activity of the GABA metabolizing enzyme, GABA transaminase (GABA-T).2. Previous preliminary reports have indicated that acute PLZ treatment also elevates brain alanine levels. As with GABA, the metabolism of alanine involves a pyridoxal phosphate-dependent transaminase.3. In the study reported here, the effects of acute PLZ treatment on the levels of various amino acids, some of which are also metabolized by pyridoxal phosphate-dependent transaminases were compared in rat whole brain. Of the 6 amino acids investigated, only GABA and alanine levels were elevated (in a time- and dose-dependent manner).4. The elevation in brain alanine levels could be explained, at least in part, by a time- and dose-dependent inhibitory effect of PLZ on alanine transaminase (ALA-T), although as with GABA the increases are higher than expected from the degree of enzyme inhibition produced. In addition, we also showed that the elevation in alanine levels and the inhibition of alanine transaminase in the brain are retained after 14 days of PLZ treatment, and that PLZ produces a marked increase in extracellular levels of alanine.5. These results are discussed in terms of their relevance to synaptic function and to the pharmacological profile of PLZ.

Atomic force microscopy of ionotropic receptors bearing subunit-specific tags provides a method for determining receptor architecture

We have developed an atomic force microscopy (AFM)-based method for the determination of the subunit architecture of ionotropic receptors, and tested the method using the GABAA receptor as a model system. The most common form of the GABAA receptor probably consists of 2α1-, 2β2- and 1γ2-subunits. We show here that the arrangement of subunits around the central Cl− ion channel can be deduced by AFM of receptors tagged with subunit-specific antibodies. Transfection of cells with DNA encoding α1-, β2- and γ2-subunits resulted in the production of receptors containing all three subunits, as judged by both immunoblot analysis and the binding of [3H]-Ro15-1788, a specific radioligand for the GABAA receptor. A His6-tag on the α1-subunit was used to purify the receptor from membrane fractions of transfected cells. After incubation with anti-His6 immunoglobulin G, some receptors became tagged with either one or two antibody molecules. AFM analysis of complexes containing two bound antibodies showed that the most common angle between the two tags was 135°, close to the value of 144° expected if the two α-subunits are separated by a third subunit. This method is applicable to the complete elucidation of the subunit arrangement around the GABAA receptor rosette, and can also be applied to other ionotropic receptors.

Benzodiazepine recognition site ligands and GABAA receptors

The benzodiazepine recognition site of the mammalian brain GABAA receptor (responsible for the majority of neuronal inhibition in the mammalian CNS) has taken its name from the chemical structures that we now know to interact specifically with this allosteric site. These benzodiazepines were introduced into clinical practice in the 1960s and proved to be enormously successful in the treatment of anxiety and sleep disorders. The pharmacological spectrum of activity for benzodiazepine site ligands is much wider. While it was recognised early that benzodiazepines were efficacious anticonvulsants and muscle relaxants, advances in our understanding of their mechanisms of action over the intervening years have revealed even greater potential for their clinical use. Early attempts to decrease the sedative potential of the benzodiazepine ligands, without losing their anxiolytic activity, proved frustratingly difficult. Meanwhile, the exciting potential of partial inverse agonists at this site to improve performan...