Multiple functional neurosteroid binding sites on GABAA receptors.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites.

Analysis of GABA Receptor Activation by Combinations of Agonists Acting at the Same or Distinct Binding Sites.

Under both physiologic and clinical conditions GABA receptors are exposed to multiple agonists, including the transmitter GABA, endogenous or exogenous neuroactive steroids, and various GABAergic anesthetic and sedative drugs. The functional output of the receptor reflects the interplay among all active agents. We have investigated the activation of the concatemeric 122L GABA receptor by combinations of agonists.

Enhanced GABAergic actions resulting from the coapplication of the steroid 3α-hydroxy-5α-pregnane-11,20-dione (alfaxalone) with propofol or diazepam.

Many GABAergic drugs are in clinical use as anesthetics, sedatives, or anxiolytics. We have investigated the actions of the combinations of the neuroactive steroid 3α-hydroxy-5α-pregnane-11,20-dione (alfaxalone) with the intravenous anesthetic propofol or the benzodiazepine diazepam. The goal of the study was to determine whether coapplication of alfaxalone reduces the effective doses and concentrations of propofol and diazepam.

High Constitutive Activity Accounts for the Combination of Enhanced Direct Activation and Reduced Potentiation in Mutated GABA Receptors.

GABA receptors activated by the transmitter GABA are potentiated by several allosterically acting drugs, including the intravenous anesthetic propofol. Propofol can also directly activate the receptor, albeit at higher concentrations. Previous functional studies have identified amino acid residues whose substitution reduces potentiation of GABA-activated receptors by propofol while enhancing the ability of propofol to directly activate the receptor.

Mapping two neurosteroid-modulatory sites in the prototypic pentameric ligand-gated ion channel GLIC.

Neurosteroids are endogenous sterols that potentiate or inhibit pentameric ligand-gated ion channels (pLGICs) and can be effective anesthetics, analgesics, or anti-epileptic drugs. The complex effects of neurosteroids on pLGICs suggest the presence of multiple binding sites in these receptors. Here, using a series of novel neurosteroid-photolabeling reagents combined with top-down and middle-down mass spectrometry, we have determined the stoichiometry, sites, and orientation of binding for 3α,5α-pregnane neurosteroids in the ligand-gated ion channel (GLIC), a prototypic pLGIC.

Propofol Is an Allosteric Agonist with Multiple Binding Sites on Concatemeric Ternary GABA Receptors.

GABA receptors can be directly activated and potentiated by the intravenous anesthetic propofol. Previous photolabeling, modeling, and functional data have identified two binding domains through which propofol acts on the GABA receptor. These domains are defined by the (M286) residue at the "+"-"-" interface in the transmembrane region and the (Y143) residue near the "-" surface in the junction between the extracellular and transmembrane domains.

GABA Type A Receptor Activation in the Allosteric Coagonist Model Framework: Relationship between EC and Basal Activity.

The concerted transition model for multimeric proteins is a simple formulation for analyzing the behavior of transmitter-gated ion channels. We used the model to examine the relationship between the EC for activation of the GABA type A (GABA) receptor by the transmitter GABA and basal activity employing concatemeric ternary GABA receptors expressed in oocytes. Basal activity, reflecting the receptor function in the absence of the transmitter, can be changed either by mutation to increase constitutive activity or by the addition of a second agonist (acting at a different site) to increase background activity.

The Actions of Drug Combinations on the GABA Receptor Manifest as Curvilinear Isoboles of Additivity.

Drug interactions are often analyzed in terms of isobolograms. In the isobologram, the line connecting the axial points corresponding to the concentrations of two different drugs that produce an effect of the same magnitude is termed an isobole of additivity. Although the isobole of additivity can be a straight line in some special cases, previous work has proposed that it is curvilinear when the two drugs differ in their maximal effects or Hill slopes.

Determination of the Residues in the Extracellular Domain of the Nicotinic Subunit Required for the Actions of Physostigmine on Neuronal Nicotinic Receptors.

Physostigmine can potentiate and inhibit neuronal nicotinic receptors, in addition to inhibiting the activity of acetylcholinesterase. We found that receptors containing three copies of the 2 subunit are inhibited by low concentrations of physostigmine in contrast to receptors containing three copies of the 4 subunit that are potentiated. We exploited this observation to determine the regions required for the actions of physostigmine.

Activation and modulation of recombinant glycine and GABA receptors by 4-halogenated analogues of propofol.

Background And Purpose: Glycine receptors are important players in pain perception and movement disorders and therefore important therapeutic targets. Glycine receptors can be modulated by the intravenous anaesthetic propofol (2,6-diisopropylphenol). However, the drug is more potent, by at least one order of magnitude, on GABA receptors.

Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol.

Background: Propofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the "+" of the β subunit, in the β-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain.