Photoaffinity Labeling with a Neuroactive Steroid Analogue

Molecular targets of inhaled anesthetics must be represented in the group that specifically bind these drugs, but the paucity of direct binding data has limited the number of candidates for further evaluation. To find candidate targets, we used a combination of photolabeling, two-dimensional gel electrophoresis, and mass spectrometry to identify halothane-binding targets in rat neuronal membranes. Of the 265 spots detected on the two-dimensional gels, 90 were labeled by [14 C]halothane, and 34 were identified. Mitochondrial proteins, especially respiratory complex and voltage-dependent anion channels, dominated the labeled group, and there were several examples of subunit-and state-dependent binding. A significant correlation was found between internal protein cavities and binding in a group of proteins with high resolution structures. Therefore, in addition to identifying novel neuronal targets, these data suggest a general molecular feature, the buried cavity, as a dominant attribute of volatile anesthetic-binding sites found in a limited number of neuronal membrane proteins.

Section: Fig 2 Halothane Labeling Quantitation a Incorporation Ofmentioning

confidence: 81%

Section: Fig 2 Halothane Labeling Quantitation a Incorporation Ofmentioning

confidence: 99%

Inhalational Anesthetic-binding Proteins in Rat Neuronal Membranes

Jin

Liu

Asbury³

et al. 2004

“…Construction of the Myc-tagged rat (r) ␣1 subunit was described previously (24). Restriction sites were introduced into each cDNA for making chimeras between h␤1 and h␤3.…”

Section: Methodsmentioning

confidence: 99%

Multiple Modes for Conferring Surface Expression of Homomeric β1 GABAA Receptors

Bracamontes

Steinbach²

2008

Self Cite

The ␥-aminobutyric acid type A (GABA A ) receptor assembles from individual subunits to form ligand-gated ion channels. Human (h) ␤3 subunits assemble to form homomeric surface receptors in somatic cells, but h␤1 subunits do not. We have identified three distinct sets of amino acid residues in the N-terminal extracellular domain of the h␤1 subunit, which when mutated to the homologous residue in h␤3 allow expression as a functional homomeric receptor. The three sets likely result in three modes of assembly. Mode 1 expression results from a single amino acid change at residue h␤1 Asp-37. Mode 2 expression results from mutations of residues between positions 44 and 73 together with residues between positions 169 and 173. Finally, mode 3 results from the mutations A45V and K196R. Examination of homology-based structural models indicates that many of the residues are unlikely to be involved in physical inter-subunit interactions, suggesting that a major alteration is stabilization of an assembly competent form of the subunit. These mutations do not, however, have a major effect on the surface expression of heteromeric receptors which include the ␣1 subunit.The GABA A 3 receptor is a member of the superfamily of fast acting ligand-gated ion channels, which includes the nicotinic acetylcholine, glycine, and serotonin receptors. They most likely originated from a single receptor subunit active as a homo-oligomer, which then evolved into a variety of subtypes and subunits (1). Individual subunits of these receptors have similar sequences and structural features such as four similarly distributed membrane-spanning regions and a characteristic cysteine loop (2). GABA A receptors are the major fast inhibitory neurotransmitter gated ion channels in the brain (3) and contain diversity of subunit isoforms as follows: ␣(1-6), ␤(1-3), ␥(1-3), ␦, ⑀, , and (4 -6). These and the related receptors formed from subunits are most likely formed as pentamers of subunits (7,8).Studies have been made of the assembly of glycine and GABA A receptors, elucidating regions directing the protein interactions that underlie receptor assembly and revealing specific sequences involved in those interactions. These studies have shown that the N-terminal extracellular domain contains residues critical for the assembly of heteromeric receptors (9 -15) and homomeric receptors (13, 16 -18). Receptors are assembled in the endoplasmic reticulum and retained there by protein chaperones if subunits are incorrectly assembled or folded (19).The GABA A receptor expresses in neurons as a heteromeric pentamer containing two or more different subunits (3). However, studies of homomeric receptors can reveal important requirements for assembly. Previous work has found that the ␤3 subunit can be expressed on the cell surface as a homomeric receptor after transfection into somatic cells (13), whereas the ␤2 and ␤1 subunits are expressed much more poorly (19,20). An analysis of the mouse ␤2 subunit identified four residues in the N-terminal domain, which were required ...

“…In the latter case, two particular methi-onine residues were shown to be labeled. Other workers have synthesized a diazirine derivative of a neuroactive steroid 3␣,5␤-6-azi-3-hydroxypregnan-20-one (6-AziP), which was shown to label the mitochondrial voltage-dependent anion channel VDAC in rat brain membranes (15). Despite the widespread use of diazirines, other photolabeling approaches are possible and in this article we describe the use of a novel etomidate analog, which incorporates an acyl azide moiety.…”

mentioning

confidence: 99%

Identification of Anesthetic Binding Sites on Human Serum Albumin Using a Novel Etomidate Photolabel

Bright¹,

Adham²,

Lemaire³

et al. 2007

We have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as with etomidate, is stereoselective in its actions, with the R(؉) enantiomer being significantly more potent than the S(؊) enantiomer. Its effects on ␣1␤2␥2s GABA A receptors expressed in HEK-293 cells are virtually indistinguishable from the parent compound etomidate, showing stereoselective potentiation of GABA-induced currents, as well as direct mimetic effects at higher concentrations. In addition, a point mutation (␤2 N265M), which is known to attenuate the potentiating actions of etomidate, also blocks the effects of the acyl azide analog. We have investigated the utility of the analog to identify etomidate binding sites by using it to photolabel human serum albumin, a protein that binds ϳ75% of etomidate in human plasma and which is thought to play a major role in its pharmacokinetics. Using HPLC/mass spectrometry we have identified two anesthetic binding sites on HSA. One site is the well-characterized drug binding site I, located in HSA subdomain IIA, and the second site is also an established drug binding site located in subdomain IIIB, which also binds propofol. The acyl azide etomidate may prove to be a useful new photolabel to identify anesthetic binding sites on the GABA A receptor or other putative targets.Although it is now widely accepted that general anesthetics exert their effects by binding directly to their protein targets (1-3), information on the precise molecular locations of these binding sites has been slow in coming. Most information has been derived from in vitro electrophysiological experiments in which putative anesthetic targets, ion channels, or receptors, are genetically modified. What this approach provides is information on the molecular determinants of anesthetic sensitivity, but it is usually impossible to tell whether these determinants represent portions of anesthetic binding sites or regions of the channel or receptor that are responsible for transducing anesthetic binding into changes in channel gating. In principle, the most direct approach would be to determine a high resolution crystal structure of the ion channel or receptor in question in the presence and absence of anesthetics. Unfortunately, because of the difficulties in crystallizing membrane proteins, such experiments are still some way off. An alternative strategy that has recently had some success is to use anesthetics that have been modified so that they contain photoactivatable groups. When these anesthetic analogs are illuminated with a bright light, they are converted into highly reactive intermediates, which then, hopefully, bind irreversibly to their targets.The idea of using photoaffinity labeling to identify anesthetic binding sites was first implemented using the volatile anesthetic halothane (4, 5). Halothane ...