General Anesthetic Binding Sites in Human α4β3δ γ-Aminobutyric Acid Type A Receptors (GABAARs)*

Extrasynaptic γ-aminobutyric acid type A receptors (GABAARs),which contribute generalized inhibitory tone to the mammalian brain, are major targets for general anesthetics. To identify anesthetic binding sites in an extrasynaptic GABAAR, we photolabeled human α4β3δ GABAARs purified in detergent with [3H]azietomidate and a barbiturate, [3H]R-mTFD-MPAB, photoreactive anesthetics that bind with high selectivity to distinct but homologous intersubunit binding sites in the transmembrane domain of synaptic α1β3γ2 GABAARs. Based upon 3H incorporation into receptor subunits resolved by SDS-PAGE, there was etomidate-inhibitable labeling by [3H]azietomidate in the α4 and β3 subunits and barbiturate-inhibitable labeling by [3H]R-mTFD-MPAB in the β3 subunit. These sites did not bind the anesthetic steroid alphaxalone, which enhanced photolabeling, or DS-2, a δ subunit-selective positive allosteric modulator, which neither enhanced nor inhibited photolabeling. The amino acids labeled by [3H]azietomidate or [3H]R-mTFD-MPAB were identified by N-terminal sequencing of fragments isolated by HPLC fractionation of enzymatically digested subunits. No evidence was found for a δ subunit contribution to an anesthetic binding site. [3H]azietomidate photolabeling of β3Met-286 in βM3 and α4Met-269 in αM1 that was inhibited by etomidate but not by R-mTFD-MPAB established that etomidate binds to a site at the β3+-α4− interface equivalent to its site in α1β3γ2 GABAARs. [3H]Azietomidate and [3H]R-mTFD-MPAB photolabeling of β3Met-227 in βM1 established that these anesthetics also bind to a homologous site, most likely at the β3+-β3− interface, which suggests a subunit arrangement of β3α4β3δβ3.

␥-Aminobutyric acid type A receptors (GABA A Rs) 2 are the major inhibitory neurotransmitter receptors in the mammalian brain. They are members of the pentameric ligand-gated ion channel superfamily that consists of five homologous subunits, each of which has a large extracellular domain, a transmembrane domain of four transmembrane helices (M1-M4), and an intracellular domain connecting the third and fourth transmembrane helices. GABA A Rs, which are the target of many drugs, among them benzodiazepines and general anesthetics, are heteropentamers, and drug action often depends on the subunit composition. For example, at synaptic receptors, which commonly have a subunit composition of (␣)2(␤)2␥, arranged ␤␣␤␣␥ counterclockwise when viewed from the synaptic or extracellular side of the receptor, benzodiazepines act in the extracellular domain between ␣ ϩ -␥ Ϫ subunits at a site homologous to the GABA binding sites at the two ␤ ϩ -␣ Ϫ subunit interfaces ( Fig. 1) (1-3).
General anesthetics have long been known to bind to sites in the transmembrane domains of pentameric ligand-gated ion channels (reviewed in Refs. 4 -7). Photolabeling of endogenous and heterologous GABA A Rs by [ 3 H]azietomidate located the etomidate binding site in the two ␤ ϩ -␣ Ϫ subunit interfaces (8,9), 50 Å from the GABA site and at a position later shown to overlap with the five ivermectin sites in the crystal structure of the homopentameric glutamate-gated chloride channel (GluCl) (10). More recently, a photoreactive, anesthetic barbiturate, R-mTFD-MPAB, has been shown to bind to sites in the ␥ ϩ -␤ Ϫ and ␣ ϩ -␤ Ϫ subunit interfaces homologous to the etomidate binding sites, introducing the concept of subtype-dependent action of general anesthetics (11). Whereas etomidate and R-mTFD-MPAB bind with high selectivity to their sites, propofol, pentobarbital, and other barbiturates bind with much less selectivity to these two classes of sites.
The in vivo mechanism of action of etomidate has been firmly linked to the GABA A R. Heterologously expressed GABA A Rs that have an N256M mutation on the M2 helix of the ␤3 subunit (␤ ϩ surface of the interface) are relatively insensitive to etomidate (12), and sleep times in knock-in mice bearing the same mutation are much shorter than in wild-type mice (13).
Azietomidate causes normal anesthesia in wild-type mice with the same potency as etomidate, and its action is similarly attenuated in the knock-in mouse (14). R-mTFD-MPAB also causes general anesthesia in mice and is equally potent in wild-type and N256M knock-in mice (15), consistent with the location of its binding sites at the ␤ Ϫ subunit interfaces.
The contrasting subunit-selective actions of these two agents raise questions about the mechanism of general anesthesia itself, because there are 19 known GABA subunits, and which of the possible combinations occur in vivo is not yet fully defined. The state of anesthesia involves many behavioral components (16), so subunit-selective general anesthetics might be associated with specific subsets of the behavioral impairments experienced during anesthesia (17). Of particular interest are the relative contributions of phasic (synaptic) and tonic (extrasynaptic) inhibition actions (18,19). The focus of this study is on the extrasynaptic ␣4␤3␦ GABA A Rs that are sensitive to endogenous neurosteroids and general anesthetics at concentrations lower than necessary to potentiate inhibitory postsynaptic currents (20 -24). Expression studies in fibroblasts and oocytes establish that multiple combinations of ␣4, ␤, and ␦ subunits can combine to form functional receptors, which results in alternative subunit interfaces (25)(26)(27)(28)(29)(30)(31).
When samples of purified human ␣4␤3␦ GABA A R were fractionated by SDS-PAGE and visualized by Coomassie Blue stain, bands were readily visualized at 78 and 58 kDa, along with  fainter bands at 72, 62, and 54 kDa (Fig. 2, lane 1). When extracted materials from in-gel tryptic digests of these bands were characterized by LC/MS/MS ( Table 2), fragments of the GABA A R ␣4 subunit were most enriched in the 72 kDa band, consistent with the expected mobility of the mature subunit (58 kDa ϩ 3 N-linked glycosylations). Fragments from the ␤3 subunit were concentrated in the 62 and 58 kDa bands, as found for ␤3 subunit from expressed ␣1␤3␥2 GABA A Rs (11). Fragments from the ␦ subunit were broadly distributed in the 62, 58, and 54 kDa bands, with ␣4 subunit fragments also recovered from the 54 kDa band. However, in contrast to the recovery of ␣4 subunit fragments from the 72 kDa gel band, for the 54 kDa band, no fragments were recovered from the ␣4 cytoplasmic domain beginning about 30 amino acids after the end of the M3 helix (data not shown). This result suggests that the 54 kDa band contains an N-terminal fragment of the ␣4 subunit containing the M1-M3 helices that was probably produced by proteolytic cleavage during receptor purification. The major component in the 78 kDa band was identified as the chaperone heat shock 70-kDa protein 1A (HSP70-1). When material eluted from the 72 kDa band was characterized by Edman degradation, the primary sequence identified (XXLNXXPGQNQXXXXL . . . ) matched a region near the predicted N terminus of the human ␣4 GABA A R subunit (VCL-NESPGQNQKEEKL . . . ). Multiple amino acids were detected at similar levels at each cycle of Edman degradation of the 62and 58-kDa samples, which precluded de novo identification of the subunits present. Sequence analysis of material from the 54 kDa band identified a primary sequence (XNDIXXYKXD . . . ) matching the N terminus region of the FLAG-tagged human ␦ GABA A R subunit sequence (MNDIGDYKDDDDK . . . , with the underline denoting the FLAG peptide sequence). The N termini of the ␣4 and ␦ subunits identified by Edman degradation are those predicted to be the N termini of the mature subunits by the signal sequence cleavage site prediction program P-signal (34). No N-terminal sequence was detected from the 78-kDa material, consistent with the fact that the N-terminal alanine of 70-kDa heat shock protein is acetylated (35), preventing Edman degradation.
[ 3 H]Azietomidate photolabeling in the 54/58/62 kDa gel bands was inhibited to a greater extent by etomidate than by R-mTFD-MPAB, and, conversely, [ 3 H]R-mTFD-MPAB photolabeling of 58/62 kDa gel bands was inhibited to a greater extent by R-mTFD-MPAB than by etomidate. These findings suggested that 1) there is an etomidate/azietomidate binding site associated with the ␣4 subunit that does not bind R-mTFD-MPAB with high affinity; 2) azietomidate, etomidate, and R-mTFD-MPAB share a common binding site associated with the 58/62 kDa gel band; and 3) there may be an R-mTFD-MPAB binding site associated with the 58/62 kDa gel band that does not bind etomidate.
To further characterize the pharmacological specificity of   (Fig. 4). For both photoreactive anesthetics, the digests of the 58/62 kDa band (␤3 and ␦ subunits) contained peaks of 3 H in hydrophobic fractions (ϳ55 and 70% organic solvent) where fragments beginning at the N termini of the ␤M3 and ␤M1 helices are known to elute (36). For the 72 kDa band (␣4) labeled by [ 3 H]azietomidate, the 3 H eluted in two peaks, a broad hydrophobic peak (55% organic solvent), which for digests of ␣1 subunits contains fragments beginning at the N termini of the M1 and M3 helices, and a peak at 40% organic solvent, where fragments from the ␣1 subunit extracellular domain elute (36). For the 54 kDa band, for each drug, there were peaks of 3 H at 40, 55, and 70% organic solvent, corresponding to the peaks seen in either of the higher molecular weight gel bands.

Etomidate Inhibits [ 3 H]Azietomidate
Photolabeling of ␣4Met-269 (␣M1), ␤3Met-227 (␤M1), and ␤3Met-286 (␤M3)-Aliquots were sequenced of unfractionated EndoLys-C digests from the 72 and 58/62 kDa gel bands from GABA A Rs photolabeled with [ 3 H]azietomidate in the absence and presence of non-radioactive etomidate (Fig. 5). For the 72 kDa band, there was a major peak of etomidate-inhibitable 3 H release in cycle 14 (Fig. 5A). For the digest from the 58/62 kDa band (Fig. 5B), there were peaks of etomidate-inhibitable 3 H release in cycles 7 and 12 (pharmacologically specific photolabeling) and peaks of 3 H release in cycles 3 and 19 that were not inhibited by etomidate (nonspecific labeling). The 72 kDa gel band digest will contain all possible ␣4 subunit proteolytic fragments, including  fragments beginning near the N termini of the M1-M4 helices. For the 58/62 kDa band, digests will include fragments beginning near the N termini of the M1, M3, and M4 helices of the ␤3 and ␦ subunits (Fig. 5C). However, the etomidate-inhibitable peak of 3 H release in cycle 14 for the 72 kDa band digest occurs in the cycle predicted to contain ␣4Met-269, the residue homologous to ␣1Met-236 in the ␣1 subunit M1 helix that was photolabeled by [ 3 H]azietomidate (8,9,11). Similarly, the peaks of etomidate-inhibitable 3 H release in cycles 7 and 12 for the 58/62 kDa band digest occur in the sequencing cycles that will contain ␤3Met-286 in ␤M3 and ␤3Met-227 in ␤M1, respectively, residues also photolabeled by [ 3 H]azietomidate in ␣1␤3 or ␣1␤3␥2 GABA A R (9, 11), as well as the residues from ␦M3 (␦Trp-315 and ␦Phe-320). The peaks of release in cycles 3 and 19 that were not inhibited by etomidate occur in cycles that contain Asp or Glu near the N and C termini of the ␤3 and ␦ subunit M3 helices.

Discussion
In this report, we provide a first characterization of the locations of anesthetic binding sites in a GABA A R subtype expressed extrasynaptically in the CNS. We photolabeled purified human ␣4␤3␦ GABA A Rs with [ 3 H]azietomidate and [ 3 H]R-mTFD-MPAB, photoreactive anesthetics that have been used previously to identify two homologous but pharmacologically distinct classes of anesthetic binding sites in ␣1␤3␥2 GABA A Rs (11). Based upon the identification of photolabeled amino acids and the results of competition photolabeling assays carried out at the level of intact subunits, we demonstrate that etomidate, but not R-mTFD-MPAB, binds with high affinity to a site at the ␤ ϩ -␣ Ϫ subunit interface in ␣4␤3␦ GABA A Rs that is equivalent to its binding site in ␣1␤3␥2 GABA A Rs. In contrast to ␣1␤3␥2 GABA A Rs, which bind R-mTFD-MPAB, but not etomidate, with high affinity to sites at the ␣ ϩ /␥ ϩ -␤ Ϫ interfaces in proximity to ␤3Met-227 in ␤M1, we find that etomidate as well as R-mTFD-MPAB bind with high affinity to a site in ␣4␤3␦ GABA A Rs containing ␤3Met-227. As discussed below, this site is most likely to be at a ␤ ϩ -␤ Ϫ subunit interface. The sites identified by photolabeling with [ 3 H]azietomidate and [ 3 H]R-mTFD-MPAB are distinct from the binding sites for alphaxalone, an anesthetic steroid, or DS-2, a ␦ subunit-selective positive allosteric modulator (32), because neither drug inhibited photolabeling.
␣4␤3␦ GABA A R Composition-Based upon mass spectrometry and Edman degradation, the affinity-purified ␣4␤3␦ GABA A Rs used in this work contain ␣4 and ␤3 subunits as well as the ␦ subunit, whose presence is assured because the FLAG epitope used for purification is attached near the ␦ subunit N terminus. However, we do not know whether the preparation is characterized by a single dominant subunit composition. Whereas receptors having a ␤3␣4␤3␣4␦ subunit arrangement (counterclockwise when viewed from the extracellular side) with two ␤3 ϩ -␣4 Ϫ interfaces containing the agonist sites and a ␦ subunit replacing the ␥ subunit have been reported to be strongly favored in transiently transfected HEK cells (27,28,37), other studies indicate that subunit stoichiometry can be variable and dependent upon the subunit cDNA transfection ratios (26). Also, studies using concatenated subunits provide evidence that the ␦ subunit can assume multiple positions in a receptor pentamer and can contribute to a ␤ ϩ -␦ Ϫ agonist binding site (25,27,30).
In the absence of independent definition of the subunit composition and arrangement in our purified ␣4␤3␦ GABA A Rs, consideration of our photolabeling results suggests a ␤3␣4␤3␦␤3 or ␤3␦␤3␣4␤3 organization for the stably transfected cell line used in our studies. We favor these stoichiometries because 1) they have a ␤3-␤3 interface required for the shared azietomidate/etomidate/R-mTFD-MPAB binding site, and 2) they have three ␤3 subunits to every one ␣4 subunit, consistent with the similar levels of [ 3 H]azietomidate incorporation (cpm/pmol) at the amino acid level in the ␤3 and ␣4 subunits (Fig. 6) in the presence of a higher level of 3 H incorporation in the ␤3 gel band than in the ␣4 band (Fig. 2). However, a ␤2␣4␦␣4␤2 pentameric concatemer, containing a ␤-␤ interface, also forms a functional receptor (30).
Contributions of ␦ Subunit Residues to Etomidate/Barbiturate Binding Sites-In our study, we did not identify any ␦ subunit amino acids photolabeled in an anesthetic-inhibitable manner by [ 3 H]azietomidate or [ 3 H]R-mTFD-MPAB. Based upon sequence analyses of samples containing variable amounts of ␤M3 and ␦M3, any pharmacologically specific photolabeling in ␦M3 is at Ͻ15% the level of ␤M3. It is possible that [ 3 H]azietomidate does bind in a pocket containing ␦M3 residues that is homologous to the ␤ ϩ -␣ Ϫ site without photolabeling any residue in ␦M3, because the pocket would lack the methionine side chains favored by azietomidate's photoreac-␣4␤3␦ GABA A R General Anesthetic Binding Sites DECEMBER 16, 2016 • VOLUME 291 • NUMBER 51 tive intermediate. However, it is unlikely that R-mTFD-MPAB binds without photolabeling any residues in its vicinity because both it and S-mTFD-MPPB, another closely related trifluoromethylphenyldiazirine, have been found to react broadly in GABA A Rs with aliphatic as well as aromatic and nucleophilic side chains (11,36). ␦ subunit fragmentation with EndoLys-C produced a cleavage 19 amino acids before the NH 2 terminus of ␦M1, which was not close enough to allow high sensitivity sequence analysis. In fact, ␦M1 may contribute to a barbiturate binding site, because studies with receptors containing ␣1, ␤3, and chimeric ␥/␦ subunits indicated that pentobarbital sensitivity determinants were contained within a fragment containing the amino terminus and the first 3 amino acids of ␦M1 (44). Further studies will be necessary to clarify whether general anesthetics also bind with high affinity in the pocket at the ␤ ϩ -␦ Ϫ interface in the cell line used in this study or at the ␣ ϩ -␦ Ϫ interface in ␤␣␤␣␦ GABA A Rs.
Functional Significance of the Identified Binding Sites-Photolabeling studies provided a first definition of two classes of pharmacologically distinct binding sites for intravenous general anesthetics at subunit interfaces in the ␣1␤3␥2 GABA A R transmembrane domain (8,11,36) that overlap with the binding sites for ivermectin (45). Mutational analyses of the residues identified by photoaffinity labeling as well as neighboring residues in the shared subunit interface pockets have demonstrated their contributions to GABA A R gating and as determinants of anesthetic efficacy (12,40,43,46,47). In addition, the capacity of anesthetics to protect against modification of substituted cysteines has expanded the definition of residues contributing to anesthetic binding sites (48,49). Mutational analyses will be necessary to determine, for example, whether the ␤ ϩ -␣ Ϫ site and ␤ ϩ -␤ Ϫ sites identified by photoaffinity labeling are equally important for etomidate enhancement of GABA responses in an ␣4␤3␦ GABA A R. However, in view of the difficulty of expressing ␣4␤3␦ GABA A Rs with defined subunit stoichiometry and subunit arrangement, these studies should be carried out using pentameric concatenated receptors.
Purification of ␣4␤3␦ GABA A R-A detailed description of the expression and affinity purification of ␣4␤3␦ GABA A Rs will be presented elsewhere. As described previously for ␣1␤3␥2 GABA A Rs (33), a stably transfected, tetracycline-inducible HEK293-TetR cell line expressing human GABA A R subunits ␣4, ␤3 (splice variant 2), and ␦ containing a FLAG tag near its N terminus (between ␦Gly-29 and ␦Asp-30) was induced and grown for 2-3 days, and then membranes were harvested, flashfrozen in liquid N 2 , and stored at Ϫ80°C until use. GABA A Rs were solubilized with 30 mM n-dodecyl ␤-D-maltopyranoside and affinity-purified as described (11) (8,9,11,36). The dashed line above each alignment denotes the extent of ␣-helices in the GABA A R structure.
eluate fractions from the purifications used for photolabeling contained 50 -70 nM [ 3 H]muscimol sites. Based upon [ 3 H]muscimol binding, the receptor was purified at 10 -25% yield from the starting membranes. Because the receptor was eluted in the presence of 1.5 mM FLAG peptide, it was not possible to estimate purity in terms of pmol of muscimol binding/mg of protein. Based upon analyses by SDS-PAGE and LC/MS/MS (see "Results"), GABA A R subunits were the dominant polypeptides in the preparation.
Radioligand Binding Assays-[ 3 H]Muscimol binding to purified GABA A R was measured by filtration after precipitation with polyethylene glycol (8). The total concentration of sites in eluate fractions was determined at 250 nM [ 3 H]muscimol with 1 mM GABA to determine nonspecific binding. Allosteric modulation of 2 nM [ 3 H]muscimol binding was determined as described (9,11).
Sequence Numbering-For ␣4, residue 1 is the predicted signal sequence Met; for ␤3, residue 1 is the predicted N terminus of the mature protein (splice variant 1, QSNVD . . . ), with ␤3Met-286 at the 15th position in the M2 helix (M2-15Ј); and for ␦, the numbering begins with the signal sequence Met and excludes the inserted FLAG sequence (DYKDDDDK). The primary structure locations of transmembrane helices M1-M4 in the figures correspond to the extent of the individual ␣-helices in the ␤3 monomeric GABA A R crystal structure (Protein Data Bank code 4COF).
Analysis of the ␣4␤3␦ GABA A R Preparation by LC/MS and N-terminal Sequencing-Three aliquots (24 pmol of [ 3 H]muscimol sites each) of ␣4␤3␦ GABA A R were separated by SDS-PAGE. Based upon Coomassie Blue staining, bands migrating at 78, 72, 62, 58, and 54 kDa were excised. The bands from one lane were submitted to the Harvard Medical School Taplin Mass Spectrometry Facility for reduction and alkylation, in-gel trypsin digestion, and peptide extraction for microcapillary LC/MS/MS analysis. The material from the equivalent gel bands from the other two lanes was eluted and subjected to N-terminal sequence analysis.
GABA A R Photolabeling-Aliquots of purified ␣4␤3␦ GABA A R in elution buffer were photolabeled at analytical or preparative scale (150 -200 l or 1-2 ml of GABA A R per condition, respectively) to characterize photoincorporation at the subunit level or to identify individual photolabeled amino acids by protein microsequencing. Aliquots of [ 3 H]azietomidate or [ 3 H]R-mTFD-MPAB were dried under a gentle argon stream and resuspended with GABA A R solutions for 30 min on ice with gentle vortexing. For preparative photolabeling, non-radioactive drugs were added directly to this resuspension, whereas for analytical photolabeling, drug aliquots were added by the use of a 1-l syringe (Hamilton 86200) to 10 l of purified GABA A R, which was then combined with 90 -150 l of GABA A R equilibrated with radioligand. With the exception of studies with alphaxalone, all photolabeling was carried out in the presence of 300 M GABA. Samples were transferred to 96-well plastic plates or 3.5-cm diameter Petri dishes (Corning catalogue numbers 2797 and 3001) for analytical or preparative scale labeling and irradiated on ice with a 365-nm UV lamp (Spectroline EN-280L) for 30 min at a distance of Ͻ1 cm. Samples were then denatured by mixing 2 parts sample with 1 part SDS-PAGE sample buffer, incubated for ϳ30 min, and fractionated by modified Laemmli SDS-PAGE (11).
Stock solutions of non-radioactive R-mTFD-MPAB (60 mM), etomidate (60 mM), and alphaxalone (8 mM) were prepared in methanol. For these drugs, all samples during photolabeling contained methanol at a final concentration of 0.5% (v/v). DS2 was prepared at 6 mM in 90% methanol, 10% DMSO. For assays with DS2, samples during photolabeling contained methanol/ DMSO at final concentrations of 0.45%/0.05% (v/v). To minimize losses of hydrophobic drugs due to adsorption on plastic surfaces, glass syringes, capillary pipettes, and vials were used for all material transfers up to the equilibration with the purified GABA A R in detergent/lipid.
After electrophoresis, gels were stained with Coomassie Brilliant Blue. In analytical scale experiments, 3 H incorporation into subunits was determined either by fluorography or by liquid scintillation counting of excised gel bands as described (11). In preparative scale experiments, material was eluted from the excised stained bands as described (11)  To determine the relative binding affinity for anesthetics at the [ 3 H]azietomidate or [ 3 H]R-mTFD-MPAB binding sites, aliquots of ␣4␤3␦ GABA A R were photolabeled in the presence of various concentrations of a drug, and subunit gel slice counts from these aliquots were fit to the equation, where B(x) represents the gel slice 3 H cpm at total inhibitor concentration x, B 0 is the gel slice 3 H cpm in the absence of competitor, B ns is nonspecific 3 H cpm incorporation in the presence of maximal concentration of a competitor, and IC 50 is the total drug concentration producing 50% inhibition. Data were fit using Sigma Plot version 11.0 (Systat Software, Inc.) with IC 50 and B ns as adjustable parameters; B 0 was fixed at the experimentally observed value. Due to limited quantities of receptor, competition assays were done only once, and the S.E. values given are from the least-squares fits. Proteolysis, Reversed-phase HPLC, and N-terminal Sequence Analysis-Aliquots of labeled subunits isolated from gel bands were digested (2 weeks, 20°C, 0.3-1 units/sample) with EndoLys-C (Roche Applied Science). Digests were fractionated by rpHPLC as described (51), except that the gradient began at 95% aqueous solvent (0.08% TFA) and 5% organic solvent (60% acetonitrile, 40% isopropyl alcohol, 0.05% TFA) and progressed to 100% organic in 75 min by approximating (in 5-min intervals) the quadratic growth curve, f(x) ϭ 5 ϩ 0.017 ϫ x 2 , where x is time in minutes and f(x) is percentage of organic solvent. The flow rate was 200 l/min, and fractions were collected every 2.5 min, with 10% assayed for 3 H. Fractions of interest were pooled and drop-loaded onto glass fiber filters for N-terminal sequence analysis on an Applied Biosystems Procise 492 protein sequencer modified so that two-thirds of each cycle were injected for PTH-derivative detection and quantification, whereas one-third was collected for scintillation counting. Some digested samples were sequenced without rpHPLC separation by loading them onto Applied Biosystems ProSorb TM PVDF filters by diluting the samples 10-fold into 0.1% TFA. The pmol of PTHderivatives detected were calculated by using rpHPLC peak heights at 269 nm compared with a standard injection.
Photolabeling in ␣4M1 or ␣4M3 was determined by sequencing appropriate rpHPLC fractions from digests of the 72 kDa gel band. Labeling in ␤3M1 and ␤3M3 was identified by sequencing fractions from the 58/62 kDa gel bands. In preliminary studies, we established that that fragments containing ␦M1 and ␦M3 were present at the highest level in the fractions containing ␤M3 from the 58/62 kDa gel band, where they were present at ϳ50% the level of the ␤M3 fragment. The ␦M3 fragment was present in the equivalent fractions from the 54 kDa gel band at ϳ200% the level of the ␤M3 fragment. The N termini of ␤M3 (␤3Ala-280) and ␦M3 (␦Ala-309) were each at the first cycle of Edman degradation. However, comparison of 3 H release profiles and relative amounts of ␤M3 and ␦M3 during sequence analyses of fractions from the 54 and 58/62 kDa gel bands established that the anesthetic-inhibitable peaks of 3 H release originated from residues in ␤M3 rather than ␦M3. Sequencing through ␦M1 began only after 19 cycles of Edman degradation, at which point PTH-derivative and 3 H releases were too low to allow characterization of photolabeling in ␦M1.
The detected sequences were quantitated by fitting the background-subtracted pmol of the detected peptide to the equation, where M(x) represents the pmol in cycle x, I 0 is the initial amount of the peptide, and R is the repetitive yield. Cys, Trp, Ser, and His were omitted from the fits due to known problems with their quantitations. The 3 H incorporation E(x), the efficiency of photolabeling (in cpm/pmol) of the amino acid in cycle x, was calculated by the following equation.