Biomedical subjects

Gregory W Sawyer

Publications and source records attributed to Gregory W Sawyer.

4 recordsLinked to original sources

PubMed2006

Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog.

General anesthetics, including etomidate, act by binding to and enhancing the function of GABA type A receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([(3)H]azietomidate), which retains anesthetic potency in vivo and enhances GABA(A)R function in vitro, to identify directly, for the first time, amino acids that contribute to a GABA(A)R anesthetic binding site. For GABA(A)Rs purified by affinity chromatography from detergent extracts of bovine cortex, [(3)H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC(50) = 30 microm). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the alphaM1 transmembrane helix at alpha1Met-236 (and/or the homologous methionines in alpha2,3,5), not previously implicated in etomidate function, and one within the betaM3 transmembrane helix at beta3Met-286 (and/or the homologous methionines in beta1,2), an etomidate sensitivity determinant. The pharmacological specificity of labeling indicates that these methionines contribute to a single binding pocket for etomidate located in the transmembrane domain at the interface between beta and alpha subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket approximately 50 A below the GABA binding site. The localization of the etomidate binding site to an intersubunit, not an intrasubunit, binding pocket is a novel conclusion that suggests more generally that the localization of drug binding sites to subunit interfaces may be a feature not only for GABA and benzodiazepines but also for etomidate and other intravenous and volatile anesthetics.

Animals↗

PubMed2005

Determination of the rate of muscarinic M1 receptor plasma membrane delivery using a regulated secretion/aggregation system.

INTRODUCTION: In this study, we used the regulated secretion/aggregation technology (RPD) to determine the rate of human muscarinic M1 (hM1) receptor plasma membrane delivery. METHODS: hM1 receptors were expressed in CHO cells as C-terminal fusion proteins to a conditional aggregation domain (CAD) consisting of four tandem mutant FKBP12 domains (F(m)). RESULTS: The CAD prevented the plasma membrane expression of hM1 receptors by causing the formation and intracellular retention of CAD-fused receptor aggregates as determined using intact cell [3H]NMS binding assays and epi-fluorescence microscopy, respectively. Aggregates of CAD-fused hM1 receptor could be disrupted in a concentration-dependent manner by the F(m)-selective ligand AP21998, resulting in an increased hM1 receptor plasma membrane expression. A furin cleavage site positioned between the CAD and the hM1 receptor sequence was cleaved by furin once aggregates of fusion protein were disrupted by AP21998, thus ensuring their irreversible dissolution. The plasma membrane delivery of hM1 receptors begins within 30 min of AP21998 exposure and the rate of delivery was constant for up to eight hours. In the continued presence of AP21998, hM1 receptor plasma membrane expression continued to increase for up to 18 h, then began to decrease toward basal levels as incubation continued out to 72 h. Using mathematical models, we determined the rate constants for the plasma membrane delivery of hM1 receptors from these data. Also, hM1 receptors elicited phosphoinositide hydrolysis to carbachol once expressed at the plasma membrane and the pharmacology of the response varied depending upon the concentration of AP21998 used to cause plasma membrane expression. DISCUSSION: Overall, our data indicate that the RPD can be used to characterize the kinetics of receptor plasma membrane delivery and to characterize functional responses elicited to different numbers of plasma membrane expressed receptor.

Animals↗

PubMed2002

Identification of the bovine gamma-aminobutyric acid type A receptor alpha subunit residues photolabeled by the imidazobenzodiazepine [3H]Ro15-4513.

Ligands binding to the benzodiazepine-binding site in gamma-aminobutyric acid type A (GABA(A)) receptors may allosterically modulate function. Depending upon the ligand, the coupling can either be positive (flunitrazepam), negative (Ro15-4513), or neutral (flumazenil). Specific amino acid determinants of benzodiazepine binding affinity and/or allosteric coupling have been identified within GABA(A) receptor alpha and gamma subunits that localize the binding site at the subunit interface. Previous photolabeling studies with [(3)H]flunitrazepam identified a primary site of incorporation at alpha(1)His-102, whereas studies with [(3)H]Ro15-4513 suggested incorporation into the alpha(1) subunit at unidentified amino acids C-terminal to alpha(1)His-102. To determine the site(s) of photoincorporation by Ro15-4513, we affinity-purified ( approximately 200-fold) GABA(A) receptor from detergent extracts of bovine cortex, photolabeled it with [(3)H]Ro15-4513, and identified (3)H-labeled amino acids by N-terminal sequence analysis of subunit fragments generated by sequential digestions with a panel of proteases. The patterns of (3)H release seen after each digestion of the labeled fragments determined the number of amino acids between the cleavage site and labeled residue, and the use of sequential proteolytic fragmentation identified patterns of cleavage sites unique to the different alpha subunits. Based upon this radiochemical sequence analysis, [(3)H]Ro15-4513 was found to selectively label the homologous tyrosines alpha(1)Tyr-210, alpha(2)Tyr-209, and alpha(3)Tyr-234, in GABA(A) receptors containing those subunits. These results are discussed in terms of a homology model of the benzodiazepine-binding site based on the molluscan acetylcholine-binding protein structure.

Affinity Labels↗

PubMed2002

Interaction between GABAA receptor subunit intracellular loops: implications for higher order complex formation.

The majority of fast inhibitory neurotransmission in the CNS is mediated by the GABA type-A (GABAA) receptor, a ligand-gated chloride channel. Of the approximately 20 different subunits composing the hetero-pentameric GABAA receptor, the gamma2 subunit in particular seems to be important in several aspects of GABAA receptor function, including clustering of the receptor at synapses. In this study, we report that the intracellular loop of the gamma2 subunit interacts with itself as well as with gamma1, gamma3 and beta1-3 subunits, but not with the alpha subunits. We further show that gamma2 subunits interact with photolabeled pentameric GABAA receptors composed of alpha1, beta2/3 and gamma2 subunits, and calculate the dissociation constant to be in the micromolar range. By using deletion constructs of the gamma2 subunit in a yeast two-hybrid assay, we identified a 23-amino acid motif that mediates self-association, residues 389-411. We confirmed this interaction motif by inhibiting the interaction in a glutathione-S-transferase pull-down assay by adding a corresponding gamma2-derived peptide. Using similar approaches, we identified the interaction motif in the gamma2 subunit mediating interaction with the beta2 subunit as a 47-amino acid motif that includes the gamma2 self-interacting motif. The identified gamma2 self-association motif is identical to the interaction motif reported between GABAA receptor and GABAA receptor-associated protein (GABARAP). We propose a model for GABAA receptor clustering based on GABARAP and GABAA receptor subunit-subunit interaction.

Amino Acid Motifs↗