Development of a novel probe for measuring drug binding to the F1*S variant of human alpha 1-acid glycoprotein.

A novel probe was developed to measure drug association with the F1*S variant of the human serum protein alpha 1-acid glycoprotein (AGP). The molecule 2-hydroxy-3,5-diiodo-N-[2(diethylamino)ethyl]benzamide (DEDIC) binds to AGP, quenching its native fluorescence. This quenching was fitted to a two-site model giving apparent dissociation constants of 0.049 +/- 0.005 and 12 +/- 2 microM (mean +/- SEM). Quenching of each of the separate variants of AGP by DEDIC was itself described by a two-site model, giving for the F1*S variant K(D)(1)((F1*S)) = 0.041 +/- 0.010 microM and K(D)(2)((F1*S)) = 29 +/- 7 microM; and for the A variant K(D)(1)((A)) = 0.31 +/- 0.18 microM and K(D)(2)((A)) = 8.8 +/- 0.7 microM. The utility of DEDIC in probing drug interactions with isolated variants was demonstrated in competition experiments with the model drugs amitriptyline and bupivacaine. In addition, the selectivity of DEDIC for variant F1*S rendered it capable of probing the binding of drugs (including the variant A-selective drug amitriptyline) to F1*S in a mixture of variants, such as occurs naturally in whole AGP. DEDIC is unique as an F1*S variant-selective probe of drug binding to whole AGP that is also sufficiently soluble to serve as a probe of drug binding to the lower affinity sites on isolated A and F1*S variants.

Nonhalogenated alkane anesthetics fail to potentiate agonist actions on two ligand-gated ion channels.

BACKGROUND: Although ether, alcohol, and halogenated alkane anesthetics potentiate agonist actions or increase the apparent agonist affinity of ligand-gated ion channels at clinically relevant concentrations, the effects of nonhalogenated alkane anesthetics on ligand-gated ion channels have not been studied. The current study assessed the abilities of two representative nonhalogenated alkane anesthetics (cyclopropane and butane) to potentiate agonist actions or increase the apparent agonist affinity of two representative ligand-gated ion channels: the nicotinic acetylcholine receptor and y-aminobutyric acid type A (GABA(A)) receptor. METHODS: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana, and human GABA(A) receptors (alpha1beta2gamma2L) were expressed in human embryonic kidney 293 cells. The Torpedo nicotinic acetylcholine receptors apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the apparent rates of desensitization induced by a range of acetylcholine concentrations. The GABA(A) receptor's apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the peak currents induced by a range of GABA concentrations. RESULTS: Neither cyclopropane nor butane potentiated agonist actions or increased the apparent agonist affinity (reduced the apparent agonist dissociation constant) of the Torpedo nicotinic acetylcholine receptor or GABA(A) receptor. At clinically relevant concentrations, cyclopropane and butane reduced the apparent rate of Torpedo nicotinic acetylcholine receptor desensitization induced by low concentrations of agonist. CONCLUSIONS: Our results suggest that the in vivo central nervous system depressant effects of nonhalogenated alkane anesthetics do not result from their abilities to potentiate agonist actions on ligand-gated ion channels. Other targets or mechanisms more likely account for the anesthetic activities of nonhalogenated alkane anesthetics.

Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor by reducing the microscopic agonist dissociation constant.

BACKGROUND: Isoflurane increases the apparent agonist affinity of ligand-gated ion channels. This action reflects a reduction in the receptor's agonist dissociation constant and/or the preopen/open channel state equilibrium. To evaluate the effect of isoflurane on each of these kinetic constants in the nicotinic acetylcholine receptor, the authors analyzed isoflurane's actions on (1) the binding of the fluorescent agonist Dns-C6-Cho to the nicotinic acetylcholine receptor's agonist self-inhibition site and (2) the desensitization kinetics induced by the binding of the weak partial agonist suberyldicholine. METHODS: The dissociation constant for Dns-C6-Cho binding to the self-inhibitory site was determined using stopped-flow fluorescence spectroscopy. The values of the kinetic constants for agonist binding, channel gating, and desensitization were determined by modeling the suberyldicholine concentration-dependence of the apparent rate of desensitization. RESULTS: Isoflurane did not significantly alter the dissociation constant for Dns-C6-Cho binding to the self-inhibitory site even at a concentration as high as 1.5 mM, the highest concentration studied. At this concentration, isoflurane substantially reduced the dissociation constant for suberyldicholine binding to its channel opening site by 97% from 17 +/- 5 microM to 0.5 +/- 0.2 microM, whereas the preopen/open channel state equilibrium was reduced only from 19.1 to 5 +/- 1. CONCLUSIONS: Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor primarily by reducing the agonist dissociation constant of the site responsible for channel opening rather than altering channel gating kinetics.

The alkyl chain dependence of the effect of normal alcohols on agonist-induced nicotinic acetylcholine receptor desensitization kinetics.

BACKGROUND: The nAcChoR is the prototypical member of a superfamily of ligand-gated ion channels that are all relevant targets of anesthetics and undergo desensitization upon prolonged exposure to agonist. This study was designed to investigate the effects of representative normal alcohols on the apparent rate of acetylcholine-induced nAcChoR desensitization. METHODS: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana. The apparent rate of acetylcholine-induced desensitization in the presence and absence of normal alcohols was measured using stopped-flow fluorescence. RESULTS: Normal alcohols as long as octanol (the longest studied) increased the apparent rate of desensitization induced by low concentrations of acetylcholine, shifting the agonist concentration-response curve for desensitization to the left Ethanol butanol, and, to a lesser extent, hexanol increased the maximal rate of desensitization induced by high, saturating concentrations of agonist. Beyond hexanol, heptanol and octanol had no effect on this maximal apparent rate of desensitization, even at concentrations that approach those that directly induce desensitization in the absence of agonist. CONCLUSION: Normal alcohols ranging from ethanol to octanol increase the apparent affinity of nAcChoR for agonist with potencies that are proportional to their hydrophobicities. However, normal alcohol effects on the rate constant for desensitization show a cutoff beyond hexanoL This suggests that the effects of normal alcohols on the apparent agonist affinity and rate constant for desensitization of nAcChoR may be modulated by distinct sites that have different steric constraints; the site(s) responsible for increasing the maximal rate of desensitization are predicted to be smaller than those that increase the apparent agonist affinity.

Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor.

BACKGROUND: Volatile general anesthetics increase agonist-mediated ion flux through the gamma-aminobutyric acid(A), glycine, and 5-hydroxytryptamine3 (5-HT3) receptors. This action reflects an anesthetic-induced increase in the apparent agonist affinity of these receptors. In contrast, volatile anesthetics block ion flux through the nicotinic acetylcholine receptor (nAcChoR). The authors tested the hypothesis that in addition to blocking ion flux through the nAcChoR, isoflurane also increases the apparent affinity of the nAcChoR for agonist. METHODS: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana. The apparent agonist affinity of the nAcChoR was determined using a new stopped-flow fluorescence assay. This assay derives the apparent agonist affinity of the nAcChoR from the apparent rates with which agonists convert nAcChoRs from the resting state to the desensitized state. RESULTS: Isoflurane significantly increased the apparent affinity (decreased the apparent dissociation constant) of acetylcholine for the nAcChoR at clinically relevant concentrations. The apparent dissociation constant decreased exponentially with the isoflurane concentration from a control value of 44+/-4 microM to 1.0+/-0.1 microM in the presence of 1.5 mM isoflurane, the highest concentration studied. CONCLUSIONS: Isoflurane increases the apparent agonist affinity of the nAcChoR; however, this effect is poorly resolved in ion flux studies because isoflurane also causes channel blockade. The lack of saturation of isoflurane's effect on the apparent agonist affinity even at relatively high isoflurane concentrations argues against a single site of anesthetic action. However, it is consistent with isoflurane interactions with several receptor sites that exhibit a range of anesthetic affinities, sites within the membrane lipid, or both.

Agonist binding and affinity state transitions in reconstituted nicotinic acetylcholine receptors revealed by single and sequential mixing stopped-flow fluorescence spectroscopies.

The affinity state of nicotinic acetylcholine receptors (nAcChoRs) reconstituted into either dioleoylphosphatidylcholine (DOPC) or a mixture of dioleoylphosphatidylcholine, dioleoylphosphatidic acid, and cholesterol (DOPC/DOPA/cholesterol) has been determined using single and sequential mixing stopped-flow fluorescence spectroscopies. These techniques have millisecond temporal resolution, permitting low- and high-affinity conformational states of the nAcChoR to be resolved following mixing with the fluorescent partial agonist Dns-C6-Cho from their characteristic Dns-C6-Cho dissociation rates. Our studies reveal that prior to agonist-induced affinity state conversion, nAcChoRs reconstituted into either DOPC or DOPC/DOPA/cholesterol are predominantly in a conformational state that has a low affinity for agonist. Prolonged exposure to Dns-C6-Cho converts nearly all DOPC/DOPA/cholesterol-reconstituted nAcChoRs to the high-affinity state. In contrast, Dns-C6-Cho converts only half of all DOPC-reconstituted nAcChoRs to the high-affinity state. The other half persists in a low-affinity state characterized by a Kd for Dns-C6-Cho of 0.61+/-0.07 microM. This Kd is similar to that previously reported for Dns-C6-Cho binding to low-affinity, resting-state nAcChoRs in native membranes. However, affinity state conversion of DOPC-reconstituted nAcChoRs may be facilitated by re-reconstituting them into bilayers composed of DOPC/DOPA/cholesterol. These results indicate that the lipid bilayer composition modulates nAcChoR agonist-induced affinity state transitions.

Nonanesthetic volatile drugs obey the Meyer-Overton correlation in two molecular protein site models.

BACKGROUND: Nonanesthetic volatile compounds fail to inhibit movement in response to noxious stimulation at concentrations predicted to induce anesthesia from their oil-water partitioning. Thus they represent tools to determine whether molecular models behave like the targets that mediate in vivo anesthetic actions. The effects of volatile anesthetics and nonanesthetics were examined in two experimental models in which anesthetics interact directly with proteins: the pore of the nicotinic acetylcholine receptor and human serum albumin. METHODS: Wild-type mouse muscle nicotinic receptors and receptors containing pore mutations (alphaS252I + betaT263I) were studied electrophysiologically in membrane patches from Xenopus oocytes. Patch currents evoked by brief pulses of acetylcholine were measured in the presence of enflurane and two nonanesthetics, 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane. Nonanesthetic interactions with human serum album were assessed by quenching of intrinsic protein fluorescence. RESULTS: Both anesthetic and nonanesthetic volatile compounds inhibited wild-type and alphaS252I + betaT263I mutant nicotinic channels but displayed different selectivity for open versus resting receptor states. Median inhibitory concentrations (IC50s) in wild-type nicotinic receptors were 870+/-20 microM for enflurane, 37+/-3 microM for 1,2-dichlorohexafluorocylcobutane, and 11.3+/-5.6 microM for 2,3-dichlorooctafluorobutane. For all three drugs, ratios of wild-type IC50s to mutant IC50mut ranged from 7-10, and ratios of wild-type IC50s to predicted anesthetic median effective concentrations (EC50s) ranged from 1.8-2.3. 1,2-Dichlorohexafluorocyclobutane quenched human serum albumin with an apparent dissociation constant (Kd) of 160+/-11 microM. The ratios of dissociation constants to predicted EC50s for the nonanesthetics were within a factor of two of the dissociation constant:EC50 ratios calculated for halothane and chloroform from previous published results. CONCLUSIONS: In two models in which anesthetics bind to protein sites, both anesthetic and nonanesthetic volatile drugs cause similar steady state effects with potencies that are predicted by hydrophobicity. These protein sites do not sterically discriminate between anesthetic and nonanesthetic drugs. However, differential state-selective actions on ion channel targets may underlie the distinct in vivo effects of anesthetics and nonanesthetics.

Transient low-affinity agonist binding to Torpedo postsynaptic membranes resolved by using sequential mixing stopped-flow fluorescence spectroscopy.

We have detected the binding of the fluorescent agonist Dns-C6-Cho to both low- and high-affinity states of the nicotinic acetylcholine receptor (nAcChoR) using sequential mixing stopped-flow fluorescence spectroscopy. Our approach to resolving low- and high-affinity binding was to first preincubate receptor membranes with the fluorescent partial agonist Dns-C6-Cho for 15 ms to 1000 s and then to follow the fluorescence decay upon chemical dilution into excess acetylcholine. The fast and slow decays, reflecting Dns-C6-Cho dissociation from low- and high-affinity receptors, had rates of 140 +/- 27 s-1 and 0.1 +/- 0.02 s-1, respectively. With increasing preincubation times, the number of low-affinity receptors decreased while the number of high-affinity receptors increased in a Dns-C6-Cho concentration-dependent manner consistent with current models for agonist-induced affinity state conversion. At receptor-activating concentrations of Dns-C6-Cho, the apparent rates with which high-affinity receptors formed approximated those of ion flux desensitization, implying that the fast desensitized state has an agonist dissociation rate that is indistinguishable from the equilibrium slow desensitized state. The KD for the low-affinity binding site was determined to be 1.1 microM from the increase in the amplitude of the fast decay with Dns-C6-Cho concentration with preincubation times that were sufficiently brief to minimize affinity state conversion. Assuming a bimolecular association rate of 10(8) M-1 s-1, a second estimate of 1.4 microM was made for low-affinity binding. We also detected a fluorescence enhancement consistent with a conformational isomerization of Dns-C6-Cho-inhibited nAcChoRs.

Conformational transitions of the nicotinic acetylcholine receptor as a model for anesthetic actions on ligand-gated ion channels: single and sequential mixing stopped-flow fluorescence studies.

(1) The effects of general anesthetic and nonanesthetic compounds on nicotinic acetylcholine receptor (nAcChoR) desensitization kinetics were characterized with stopped-flow fluorescence spectroscopy. (2) Anesthetics were found to increase the apparent rate of agonist-induced desensitization and shift the receptor equilibrium towards the desensitized state. (3) In contrast, nonanesthetics had little effect on either the apparent rate of desensitization or receptor equilibrium. (4) Octanol, but not isoflurane, decreases the rate of agonist dissociation from resting state nAcChoRs. (5) These results suggest that anesthetics alter nAcChoR desensitization kinetics by increasing either agonist binding affinity to the resting state or the channel opening probability.

Halothane interactions with nicotinic acetylcholine receptor membranes. Steady-state and kinetic studies of intrinsic fluorescence quenching.

BACKGROUND: Although it has been suggested that anesthetics alter protein conformational states by binding to nonpolar sites within the interior regions of proteins, the rate and extent to which anesthetics penetrate membrane proteins has not been characterized. The authors report the use of steady-state and stopped-flow spectroscopy to characterize the interactions of halothane with receptor membranes. METHODS: Steady-state and stopped-flow fluorescence spectroscopy was used to characterize halothane quenching of nicotinic acetylcholine receptor (nAcChoR)-rich membrane intrinsic fluorescence and the rate of isoflurane-induced nAcChoR desensitization. RESULTS: At equilibrium, halothane quenched only 54 +/- 1.4% of all tryptophan fluorescence. Diethyl ether failed to reduce fluorescence quenching by halothane, suggesting that it does not bind to the same protein sites as halothane. Stopped-flow fluorescence traces defined two kinetic components of quenching: a fast component that occurred in less than 1 ms followed by a slower biphasic fluorescence decay. Protein unfolding with sodium dodecyl sulfate reduced halothane's Stern-Volmer quenching constant, eliminated the biphasic decay, and rendered fluorescence accessible to quenching by halothane within 1 ms. Functional studies indicate that anesthetic-induced desensitization of nAcChoR occurs in less than 2 ms. CONCLUSIONS: Unquenchable fluorescence arises from tryptophan residues that are buried within the protein and protected from halothane. Sodium dodecyl sulfate unfolds membrane proteins and allows previously buried fluorescence protein residues to be rapidly and homogeneously quenched by halothane. Halothane quenches protein components of nAcChoR membranes over the same concentration range and time scale that it exerts its functional effects, a finding that is generally consistent with a protein site of action.

Anesthetic management and outcome following noncardiac surgery in nonparturients with Eisenmenger's physiology.

STUDY OBJECTIVE: To evaluate the perioperative risk to nonparturients with Eisenmenger's physiology for noncardiac surgical procedures. DESIGN: Retrospective chart review. SETTING: University-affiliated hospital. PATIENTS: 12 nonparturients with Eisenmenger's physiology who underwent 25 noncardiac surgical procedures requiring care by an anesthesiologist. MEASUREMENTS AND MAIN RESULTS: Preoperative, intraoperative, and postoperative records were retrospectively analyzed. Data examined included patient age, gender, symptoms, laboratory values, monitors used, surgical procedure, and outcome. Twenty-five procedures were performed on 12 patients; 13 procedures were performed with general anesthesia, 6 with peripheral nerve blocks, 5 with sedation by an anesthesiologist with or without local anesthetic infiltration, and one with epidural anesthesia. One patient died perioperatively. Review of the literature revealed two deaths in 32 procedures for nonparturients with Eisenmenger's physiology undergoing noncardiac surgery. CONCLUSIONS: A variety of anesthetic techniques and drugs may be used successfully in nonparturients with Eisenmenger's physiology undergoing noncardiac surgery. Although the study group is small, the perioperative mortality risk is lower than that for parturients undergoing either labor and delivery or cesarean section and is probably in the range of approximately 10%.

Anesthetic and nonanesthetic halogenated volatile compounds have dissimilar activities on nicotinic acetylcholine receptor desensitization kinetics.

BACKGROUND: The Meyer-Overton rule predicts that an anesthetic's potency will correlate with its oil solubility. A group of halogenated volatile compounds that disobey this rule has been characterized. These compounds do not induce anesthesia in rats at partial pressures exceeding those predicted by the Meyer-Overton rule to be anesthetic. The observation that potentiation of GABA(A) receptor responses by anesthetic and nonanesthetic halogenated volatile compounds correlates with their abilities to induce general anesthesia suggests that this receptor is involved in the mechanism of general anesthesia. However, the GABA(A) receptor is only one member of a superfamily of structurally similar ligand-gated ion channels. This study compares the actions of both anesthetic and nonanesthetic halogenated volatile compounds on another member of this super family of receptors, the nicotinic acetylcholine receptor (nAcChoR). METHODS: The actions of both anesthetic and nonanesthetic compounds on desensitization kinetics were characterized from the time-dependent binding of the fluorescent acetylcholine analogue, Dns-C6-Cho, to the nAcChoR. RESULTS: At concentrations predicted by the Meyer-Overton rule to be equianesthetic, the anesthetics isoflurane and enflurane were significantly more effective than the nonanesthetics 1,2-dichlorohexafluorocyclobutane and 2, 3-dichlorooctafluorobutane in enhancing the fraction of receptors preexisting in the slow desensitized state and increasing the apparent rates of agonist-induced fast and slow desensitization. CONCLUSIONS: The potencies with which anesthetic and nonanesthetic compounds enhance desensitization kinetics in the nAcChoR parallel their in vivo anesthetic potencies. These results support the use of desensitization of the nAcChoR as a mechanistic model for studies of general anesthesia and suggest that an insensitivity to nonanesthetic compounds may be a feature common to members of the superfamily of ligand-gated ion channels.

Electron spin resonance studies of acyl chain motion in reconstituted nicotinic acetylcholine receptor membranes.

The electron spin resonance spectra of spin-label positional isomers of stearic acid (n-SASL) incorporated into nicotinic acetylcholine receptors (nAcChoR) reconstituted into dioleoylphosphatidylcholine (DOPC) were deconvoluted into bilayer- and protein-associated components by subtraction under conditions of slow exchange. The selectivity of n-SASL (n = 6, 9, 12, and 14) for the lipid-protein interface of the nAcChoR was threefold greater than that of DOPC and independent of the spin label position. The temperature at which exchange became apparent as judged from lineshape broadening of the mobile lipid component spectrum was dependent upon the position of the spin-label moiety; near the bilayer center, exchange broadening occurred at lower temperatures than it did closer to the lipid headgroup. This suggests that the lipid headgroup region of boundary lipids is relatively fixed, whereas its acyl chain whips on and off the protein with increasing frequency near the bilayer center. Motions on the microsecond time scale were examined by microwave power saturation. Each n-SASL saturated more readily when incorporated into vesicles containing the nAcChoR than when in pure DOPC liposomes. Therefore, lipid mobility is perturbed by the nAcChoR on the microsecond time scale with an apparent magnitude that is relatively modest, probably due to exchange on this time scale.

General anesthetics modify the kinetics of nicotinic acetylcholine receptor desensitization at clinically relevant concentrations.

BACKGROUND: General anesthetics are thought to induce anesthesia through their actions on ligand-gated ion channels. One such channel, the nicotinic acetylcholine receptor (nAcChoR), can be found in different subtypes in the central nervous system and at the periphery in the neuromuscular junction. The latter subtype of the nAcChoR is a useful model for examining interactions between general anesthetics and ligand-gated ion channels, because it can be isolated and purified in sufficient quantities to allow for biophysical and biochemical studies. This study examines the actions of general anesthetics on agonist-induced conversion of the nAcChoR to inactive desensitized conformational states. METHODS: Nicotinic acetylcholine receptor membranes were purified from the electric organ of Torpedo nobiliana. Agonist-induced desensitization was characterized from the time-dependent increase in fluorescence intensity that results from the binding of the fluorescent acetylcholine analog, Dns-C6-Cho, to the nAcChoR. RESULTS: Mixing Dns-C6-Cho with nAcChoR-rich membranes results in an increase in fluorescence that is characterized by four rate processes. Concentrations of isoflurane and butanol, which range from subclinical to toxic increase the rates of the third and fourth components of fluorescence, corresponding to fast and slow desensitization, respectively. At concentrations that are twice their EC50s for anesthesia, isoflurane, butanol, chloroform, methanol, and cyclopentanemethanol increase the apparent rates of fast and slow desensitization by an average of 92 +/- 22% and 108 +/- 22%, respectively. CONCLUSIONS: The concentration range over which general anesthetics modify the kinetics of nAcChoR desensitization is similar to those reported for anesthetic actions on the GABAA receptor. Thus, the nAcChoR, like other members of this superfamily, is a sensitive target of general anesthetics.

A hydrophobic inhibitor of the nicotinic acetylcholine receptor acts on the resting state.

3-(Trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) has recently been found to be a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAcChoR) by both photolabeling and flux assays (White et al., 1991). However, these experiments were done when TID was in equilibrium with the nAcChoR, and thus only its interactions with the resting and the desensitized states of the nAcChoR were studied. In this work we characterized the interaction between TID and nAcChoR in the open and resting states using a flux assay. When TID and acetylcholine were simultaneously mixed with the nAcChoR in native Torpedo vesicles, TID did not inhibit agonist-induced 86Rb+ flux. However, following prolonged preincubation (4 min) of TID with nAcChoR, complete inhibition was observed with a half-inhibition constant of 0.4 microM TID and a Hill coefficient of 0.9. This suggested that TID might act either on the resting or the desensitized state in preference to the open state of the nAcChoR. Preincubation of nAcChoR with TID, followed by a 7 ms agonist-induced flux assay, showed that the flux response declined exponentially with preincubation time. Assuming a pseudo-first-order process, analysis revealed the rate constant for the onset of inhibition of nAcChoR in the resting state to be in the range of (1.2-3.4) x 10(6) M-1 s-1. To test if fast desensitization was enhanced by TID under these conditions, we used a fluorescent analog of acetylcholine. Stopped-flow fluorimetry showed that the fraction of nAcChoR in the predesensitized state did not increase during preincubation with TID.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid-protein interactions and protein dynamics in vesicles containing the nicotinic acetylcholine receptor: a study with ethanol.

Electron paramagnetic resonance (EPR) spectroscopy was used to study the action of ethanol on the protein side chain motions of the nicotinic acetylcholine receptor (nAcChoR) in alkaline extracted membranes from Torpedo nobiliana. EPR spectra of the nAcChoR derivatized with maleimide spin label contain both strongly and weakly immobilized components. The rotational correlation time of the strongly immobilized component decreases by a factor of 2-3-fold with the addition of 1.6 M ethanol, while that of the weakly immobilized component is not significantly altered. EPR spectroscopy was also used to probe the lipid environment immediately surrounding the nAcChoR with stearic acid and phosphatidylcholine spin labeled at the fourteenth acyl carbons (14-SASL and 14-PCSL, respectively), and the steroid spin label androstanol (ASL). EPR spectra of these probes reveal a component corresponding to lipids that are motionally restricted by the receptor (annular lipids) in addition to a more fluid component arising from bulk lipid. Using spectral subtraction, the order of selectivity of these spin labels for the nAcChoR was determined to be ASL > or = 14-SASL > 14-PCSL. The estimated rotational correlation times of the high affinity 14-SASL and ASL probes ranged from approx. 20 to 35 ns. The correlation times of the lower affinity 14-PCSL were generally shorter than those for 14-SASL and ASL and ranged from about 10 to 25 ns. The addition of up to 0.9 M ethanol altered neither the affinity nor the mobility of the motionally restricted EPR component. This suggests that ethanol's actions on the nAcChoR are not mediated via changes at the lipid/protein interface near the center of the bilayer.