Gregory W. Sawyer

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 (GABAARs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([3H]azietomidate), which retains anesthetic potency in vivo and enhances GABAAR function in vitro, to identify directly, for the first time, amino acids that contribute to a GABAAR anesthetic binding site. For GABAARs purified by affinity chromatography from detergent extracts of bovine cortex, [3H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC50 = 30 μm). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the αM1 transmembrane helix at α1Met-236 (and/or the homologous methionines in α2,3,5), not previously implicated in etomidate function, and one within the βM3 transmembrane helix at β3Met-286 (and/or the homologous methionines in β1,2), 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 β and α subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket ∼50 Å 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.

SUBTYPES OF THE MUSCARINIC RECEPTOR IN SMOOTH MUSCLE

Muscarinic receptors are expressed in smooth muscle throughout the body. In most instances, the muscarinic receptor population in smooth muscle is composed of mainly the M2 and M3 subtypes in an 80% to 20% mixture. The M3 subtype mediates phosphoinositide hydrolysis and calcium mobilization, whereas the M2 subtype mediates an inhibition of cAMP accumulation. In addition, a variety of ionic conductances are elicited by muscarinic receptors. Muscarinic agonists stimulate a nonselective cation conductance that is pertussis toxin-sensitive and dependent on calcium. The pertussis toxin-sensitivity of this response suggests that it is mediated by M2 receptors. Following agonist induced depolarization of smooth muscle, voltage dependent calcium channels are activated to enable an influx of calcium. In some instances, muscarinic agonists enhance this conductance through a mechanism involving protein kinase C, whereas in other instances, muscarinic agonists suppress this calcium conductance. Smooth muscle often contains calcium activated potassium channels that tend to repolarize the membrane following calcium influx. Activation of muscarinic receptors suppresses this potassium conductance in some smooth muscles. Under standard conditions, muscarinic agonists elicit pertussis toxin-insensitive contractions through activation of the M3 receptor. When most of the M3 receptors are inactivated, it is possible to measure a pertussis toxin-sensitive contractile response to muscarinic agonists that is most likely mediated through M2 receptors. M2 receptors also cause an indirect contraction by inhibiting the relaxant effects of agents that increase cAMP (e.g., forskolin and isoproterenol).

Contractile role of M2 and M3 muscarinic receptors in gastrointestinal smooth muscle.

Muscarinic agonists elicit contraction through M3 receptors in most isolated preparations of gastrointestinal smooth muscle, and not surprisingly, several investigators have identified M3 receptors in smooth muscle using biochemical, immunological and molecular biological methods. However, these studies have also shown that the M2 receptor outnumbers the M3 by a factor of about four in most instances. In smooth muscle, M3 receptors mediate phosphoinositide hydrolysis and Ca2+ mobilization, whereas M2 receptors mediate an inhibition of cAMP accumulation. The inhibitory effect of the M2 receptor on cAMP levels suggests an indirect role for this receptor; namely, an inhibition of the relaxant action of cAMP-stimulating agents. Such a function has been rigorously demonstrated in an experimental paradigm where gastrointestinal smooth muscle is first incubated with 4-DAMP mustard to inactivate M3 receptors during a Treatment Phase, and subsequently, the contractile activity of muscarinic agonists is characterized during a Test Phase in the presence of histamine and a relaxant agent. When present together, histamine and the relaxant agent (e.g., isoproterenol or forskolin) have no net contractile effect because their actions oppose one another. However, under these conditions, muscarinic agonists elicit a highly potent contractile response through the M2 receptor, presumably by inhibiting the relaxant action of isoproterenol or forskolin on histamine-induced contractions. This contractile response is pertussis toxin-sensitive, unlike the standard contractile response to muscarinic agonists, which is pertussis toxin-insensitive. When measured under standard conditions (i.e., in the absence of histamine and without 4-DAMP mustard-treatment), the contractile response to muscarinic agonists is moderately sensitive to pertussis toxin if isoproterenol or forskolin is present. Also, pertussis toxin-treatment enhances the relaxant action of isoproterenol in the field-stimulated guinea pig ileum. These results demonstrate that endogenous acetylcholine can activate M2 receptors to inhibit the relaxant effects of beta-adrenoceptor activation on M3 receptor-mediated contractions. An operational model for the interaction between M2 and M3 receptors shows that competitive antagonism of the interactive response resembles an M3 profile under most conditions, making it difficult to detect the contribution of the M2 receptor.

Identification of the Bovine γ-Aminobutyric Acid Type A Receptor α Subunit Residues Photolabeled by the Imidazobenzodiazepine [3H]Ro15-4513

Ligands binding to the benzodiazepine-binding site in γ-aminobutyric acid type A (GABAA) 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 GABAA receptor α and γ subunits that localize the binding site at the subunit interface. Previous photolabeling studies with [3H]flunitrazepam identified a primary site of incorporation at α1His-102, whereas studies with [3H]Ro15-4513 suggested incorporation into the α1 subunit at unidentified amino acids C-terminal to α1His-102. To determine the site(s) of photoincorporation by Ro15-4513, we affinity-purified (∼200-fold) GABAAreceptor from detergent extracts of bovine cortex, photolabeled it with [3H]Ro15-4513, and identified 3H-labeled amino acids by N-terminal sequence analysis of subunit fragments generated by sequential digestions with a panel of proteases. The patterns of 3H 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 α subunits. Based upon this radiochemical sequence analysis, [3H]Ro15-4513 was found to selectively label the homologous tyrosines α1Tyr-210, α2Tyr-209, and α3Tyr-234, in GABAA 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.

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 γ2 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 γ2 subunit interacts with itself as well as with γ1, γ3 and β1–3 subunits, but not with the α subunits. We further show that γ2 subunits interact with photolabeled pentameric GABAA receptors composed of α1, β2/3 and γ2 subunits, and calculate the dissociation constant to be in the micromolar range. By using deletion constructs of the γ2 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 γ2‐derived peptide. Using similar approaches, we identified the interaction motif in the γ2 subunit mediating interaction with the β2 subunit as a 47‐amino acid motif that includes the γ2 self‐interacting motif. The identified γ2 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.

A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M1 Acetylcholine Receptors Affects Plasma Membrane Expression

We investigated the functional role of a conserved motif, F(x)6LL, in the membrane proximal C-tail of the human muscarinic M1 (hM1) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence 423FRDTFRLLL431. Wild-type and mutant hM1 receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [3H]N-methylscopolamine binding assays. The plasma membrane expression of hM1 receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM1 receptor possessing a L430A/L431A double-point mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant receptor from the ER to the plasma membrane. Atropine treatment also caused an increase in the maximal response and potency of carbachol-stimulated phosphoinositide hydrolysis elicited by the L430A/L431A mutant. The effect of atropine on the L430A/L431A receptor mutant suggests that L430 and L431 play a role in folding hM1 receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V46 and L47, that, when mutated, reduce the plasma membrane expression of hM1 receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM1 receptors to achieve a transport-competent state.

Functional role of muscarinic M2 receptors in α,β‐methylene ATP induced, neurogenic contractions in guinea‐pig ileum

The muscarinic acetylcholine receptors mediating the contractile response elicited to endogenous acetylcholine released by the selective P2X receptor agonist α,β‐methylene ATP (mATP) were investigated in guinea‐pig ileum. mATP (0.1–30 μM) elicited a concentration‐dependent neurogenic contractile response inhibited by tetrodotoxin (TTX) and antagonized by the non‐selective muscarinic receptor antagonist N‐methylscopolamine (NMS). The contractile response to mATP was pertussis toxin‐insensitive, irreversibly antagonized by N‐(2‐chloroethyl)‐4‐piperidinyl diphenylacetate (4‐DAMP mustard), and unaffected by the muscarinic M2/M4 receptor selective antagonist AF‐DX 116 (1 μM). When measured in the presence of histamine and isoproterenol after treatment with 4‐DAMP mustard, mATP elicited a pertussis toxin‐sensitive contractile response potently antagonized by AF‐DX 116. Collectively, our data suggest that endogenous acetylcholine released by mATP can elicit a direct contractile response through the muscarinic M3 receptor and an indirect contractile response through the muscarinic M2 receptor by antagonizing the relaxant effects of isoproterenol on histamine induced contraction.

Comparison of the kinetics and extent of muscarinic M1-M5 receptor internalization, recycling and downregulation in Chinese hamster ovary cells.

We characterized agonist-induced internalization, recycling and downregulation of each muscarinic receptor subtype (M(1)-M(5)) stably expressed in Chinese hamster ovary (CHO) cells. The radioligands [(3)H]QNB and [(3)H]NMS were used to measure the total and plasma membrane populations of muscarinic receptors, respectively. Following carbachol treatment (1 mM), the rank orders for the rate of carbachol-induced internalization of the muscarinic subtypes were M(2)>M(4)=M(5)>M(3)=M(1), respectively. Unlike the M(2) receptor, M(1), M(3), M(4) and M(5) receptors recycled back to the plasma membrane after 1 h carbachol treatment. The receptor downregulation elicited to 24h carbachol treatment was similar for M(2), M(3), M(4) and M(5) receptors, whereas that for the M(1) receptor was greater. Our results indicate that there are subtype-specific differences in the rate and extent of agonist-induced muscarinic receptor internalization, recycling and downregulation in CHO cells.

Pertussis toxin increases isoproterenol induced relaxation in field-stimulated ileum

We investigated the effects of pertussis toxin on contraction in field-stimulated guinea pig ileum in the absence and presence of isoproterenol. Field-stimulation elicited pertussis toxin-insensitive contractions. Cumulative addition of isoproterenol produced a maximal 52% reduction in the contractile response. Following pertussis toxin-treatment, the maximal inhibitory effect of isoproterenol increased to 83%. Pertussis toxin had no effect on the ability isoproterenol to inhibit contractions elicited by histamine agonists. Our results suggest that the increased effectiveness of isoproterenol in pertussis toxin-treated ileum is due to an uncoupling of the muscarinic M2 receptor contractile mechanism.

Mutagenesis of Nucleophilic Residues near the Orthosteric Binding Pocket of M1 and M2 Muscarinic receptors: Effect on the Binding of Nitrogen Mustard Analogs of Acetylcholine and McN-A-343

Investigating how a test drug alters the reaction of a site-directed electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the electrophile is known. In this study, therefore, we mutated nucleophilic residues near and within the orthosteric pockets of M1 and M2 muscarinic receptors to identify where acetylcholine mustard and 4-[(2-bromoethyl)methyl-amino]-2-butynyl-N-(3-chlorophenyl)carbamate (BR384) bind covalently. BR384 is the nitrogen mustard analog of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343). Mutation of the highly conserved aspartic acid in M1 (Asp105) and M2 (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M2 D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M1 D105N mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M2 D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M2 D103N by BR384 was competitively inhibited by N-methylscopolamine and allosterically inhibited by gallamine. Mutation of a variety of other nucleophilic residues, some in combination with D103N, had little effect on M2 receptor alkylation by BR384. Our results suggest that BR384 alkylates at least one residue other than the conserved aspartic acid at the ligand-binding site of M1 and M2 receptors. This additional residue seems to be located within or near the orthosteric-binding pocket and is not part of the allosteric site for gallamine.