Biomedical subjects

K W Miller

Publications and source records attributed to K W Miller.

At least 91 records · Page 5Linked to original sources

PubMed1990

A 91-day feeding study in rats with heated olestra/vegetable oil blends.

A 91-day feeding study in rats was conducted to assess the potential toxicity of heated olestra/vegetable oil blends. Olestra is a mixture of the hexa-, hepta- and octa- long-chain fatty acid esters of sucrose. The blends tested were 35/65 olestra/vegetable oil (w/w), pan-fried at 380 degrees F for 30 min to simulate home-use conditions and 75/25 olestra/vegetable oil, deep-fried at 365 degrees F for 84 hr to simulate extended food-service use. Vegetable oil, prepared using both heating conditions, unheated vegetable oil and unheated olestra/vegetable oil blends served as controls. The olestra/vegetable oil blends and vegetable-oil control were generally fed at 10% (w/w) of the diet. Two further groups received the heated olestra/vegetable oil blends at 5% of the diet. Survival, clinical signs, body weight, feed consumption, feed conversion efficiency, organ weights, organ-to-body-weight ratios, haematological parameters and histomorphology were evaluated. No adverse effects from the ingestion of heated olestra/vegetable oil blends were detected. These findings indicate that heated olestra was non-toxic and, in this respect, no different from unheated olestra or heated or unheated vegetable oil.

Animals↗

PubMed1990

The effect of olestra on systemic levels of oral contraceptives.

The effect of olestra, a nonabsorbable, noncaloric fat replacement, on the absorption and efficacy of a highly lipophilic oral contraceptive was investigated in a double-blind, placebo-controlled crossover study with 28 women. Subjects consumed 18 gm/day olestra for 28 days while taking an oral contraceptive containing 300 micrograms of norgestrel and 30 micrograms ethinyl estradiol (Lo/Ovral-28). Blood taken on days 12 to 14 of the treatment cycles was analyzed for ethinyl estradiol and norgestrel. There was no statistically significant difference in time to attain maximum concentration, maximum concentration, or area under the concentration-time curve between the olestra and placebo treatments for either drug component. Measurements of serum progesterone indicated that olestra ingestion did not reduce efficacy as indicated by ovulation. The data show that ingestion of 18 gm/day olestra did not affect the absorption or efficacy of the highly lipophilic oral contraceptive.

Adult↗

PubMed1990

Relative potencies for barbiturate binding to the Torpedo acetylcholine receptor.

1. The structural requirements of an allosteric barbiturate binding site on acetylcholine receptor-rich membranes isolated from Torpedo electroplaques have been characterized by the ability of fourteen barbiturates to displace [14C]-amobarbitone binding. 2. The barbiturates could be grouped into two classes with ten barbiturates producing a strong inhibition of [14C]-amobarbitone binding (class one) and with four exerting minimal effects (class two). 3. Eight of the ten class one barbiturates displaced essentially all of the [14C]-amobarbitone from its binding site, while, at their respective aqueous solubility limits, two of these barbiturates (thiopentone and dimethylbutylbarbitone (DMBB) inhibited [14C]-amobarbitone binding by nearly 80%. The apparent inhibition constants (KI) for the class one barbiturates ranged from 13 microM for amobarbitone to 2.8 mM for barbitone with the other eight agents lying in the range 100-600 microM, and having the rank order pentobarbitone approximately secobarbitone greater than thiopentone greater than DMBB greater than butabarbitone approximately phenobarbitone greater than aprobarbitone greater than allylbarbitone. 4. By contrast, the class two barbiturates had minimal effects even at close to saturating concentrations. [14C]-amobarbitone binding was reduced slightly (less than 30%) by hexobarbitone, mephobarbitone and methohexitone and was enhanced slightly (less than 20%) by metharbitone. 5. All of the class two, but none of the class one barbiturates, were N-methylated.

Allosteric Site↗

PubMed1989

Ethanol increases agonist affinity for nicotinic receptors from Torpedo.

The presence of ethanol increases the apparent affinity with which acetylcholine and carbamylcholine elicit 86Rb+ flux from Torpedo nicotinic acetylcholine receptor-rich vesicles at 4 degrees C. Affinity increased exponentially with ethanol concentration, reaching nearly 200-fold by 3.0 M ethanol without sign of saturation. At submaximal agonist concentrations 50-100 mM ethanol enhanced flux by 15-35%, but the maximum agonist-induced flux was unaffected in quenched-flow assays. The effect was independent of the agonist and of the time over which flux was measured (5 ms to 10 s), indicating that ethanol acts before agonist-induced desensitization occurs. Ethanol also caused an increase in the apparent affinity with which acetylcholine caused fast desensitization. This affinity increase was equal to that for flux-response curves, but the maximum fast desensitization rate was increased 50% at 0.5 M ethanol. This was the most pronounced of ethanol's actions and has not been reported before. Prolonged preincubation with 1.0 M ethanol alone reduced agonist-induced flux activity by only 25%. The rate of agonist-induced slow desensitization was also increased, but neither of these effects was as marked as those on fast desensitization and cation flux.

Acetylcholine↗

PubMed1989

Procaine rapidly inactivates acetylcholine receptors from Torpedo and competes with agonist for inhibition sites.

The relationship between the high-affinity procaine channel inhibition site (apparent dissociation constant Kp congruent to 200 microM) and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid 86Rb+ quenched-flux assays at 4 degrees C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with alpha-bungarotoxin (alpha-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations (10 microM-10 mM) alone, AChR undergoes one phase of inactivation (fast desensitization, rate = kd) in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting (greater than 10 mM) ACh concentrations [Forman & Miller (1988) Biophys. J. 54, 149-158]--rapid inactivation (rate = kr) complete in 30-75 ms is followed by fast desensitization at the same kd observed without procaine. The dependence of kr on [procaine] is consistent with a bimolecular association between procaine and its AChR site with kon = 2.5 X 10(5) M-1 s-1, koff = 36 s-1, and Kp = 145 +/- 36 microM). Inhibition of AChR function by mixtures of procaine (up to 12Kp) plus self-inhibiting concentrations of ACh or suberyldicholine ([SubCh] up to 13 X the 50% self-inhibiting agonist concentration, KB) was studied by reducing the level of alpha-BTX block in vesicles. The apparent KB increased in the presence of procaine, and the apparent KP increased linearly with [SubCh], indicating mutually exclusive actions at a common AChR site. Our data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and kr's dependence on [ACh] in the channel-activating range closely parallels that of 86Rb+ flux response to ACh.

Acetylcholine↗

PubMed1989

Molecular sites of anesthetic action in postsynaptic nicotinic membranes.

Theories of general anesthesia have traditionally been based on correlations between potency and the properties of simple models such as apolar solvents, lipid bilayers and soluble proteins. However, mechanisms can now be determined directly by studying excitable proteins in their membrane environment. Stuart Forman and Keith Miller describe the physiological, biophysical and molecular biological evidence pointing to the location of a discrete allosteric site on the nicotinic acetylcholine receptor at which local anesthetics act. General anesthetics, while superficially resembling local anesthetics in their actions on the receptor, do not appear to act upon such a site.

Anesthetics↗

PubMed1989

Nonanesthetic alcohols dissolve in synaptic membranes without perturbing their lipids.

While many theories of general anesthesia postulate a lipid site of action, there has been no adequate explanation for the lack of anesthetic potency of the highly hydrophobic primary alkanols with more than 12 carbons (the cut-off). Some work suggests that these nonanesthetic alcohols do not dissolve in membranes. Other work contradicts this and suggests that an anesthetic site on a protein provides a better explanation. Here we show that both the anesthetic dodecanol and the nonanesthetic tetradecanol are taken up equally well into the tissues of animals and into isolated postsynaptic membranes. When a group of Rana pipiens tadpoles were treated with dodecanol, half were anesthetized by 4.7 microM (free aqueos concentration), and the corresponding concentration in the tissues was found to be 0.4 mmol per kg wet weight. Prolonged exposure (92 hr) to tetradecanol produced even higher tissue concentrations (0.7 mmol per kg wet weight), yet no anesthetic effects were observed. Furthermore, general anesthetics are thought to act on postsynaptic membranes but both alkanols partitioned into postsynaptic membranes from Torpedo electroplaques. The spin label, 12-doxyl stearate, was incorporated into these membranes. The lipid order parameter it reported was decreased by the anesthetic alcohols (octanol, decanol, and dodecanol), whereas the nonanesthetic alcohols either did not change it significantly (tetradecanol) or actually increased it (hexadecanol and octadecanol). Thus, although lipid solubility is unable to account for the pharmacology of the cut-off in potency of the long-chain alcohols, lipid perturbations provide an accurate description.

Alcohols↗

PubMed1989

Anaesthetic potencies of primary alkanols: implications for the molecular dimensions of the anaesthetic site.

1. We have redetermined the anaesthetic potencies (EC50S) for a series of primary alkanols, to resolve uncertainties about the molecular dimensions of the anaesthetic site resulting from the use of data from different laboratories. 2. For each alkanol, concentration-response relationships for loss of righting reflex (LRR) were plotted for over one hundred tadpoles, and the median effective concentrations determined. Aqueous concentrations present during potency assays were determined independently, and for alkanols with chain length greater than nonanol, correction was made for depletion from the aqueous phase. 3. The EC50S were found to decrease logarithmically with increasing number of carbon atoms in the hydrocarbon chain of the alkanol (CN), such that, on average, each additional methylene group was associated with an approximately four fold increase in potency. 4. The relationship between log EC50 and CN was best described by the quadratic equation, log EC50 = 0.022 (+/- 0.0038) CN2 + 0.76 (+/- 0.051) CN + 3.7 (+/- 0.14) (r2 = 0.9951). 5. A previously described correlation between the apparent changes in the free energy of binding of an additional methylene group both to luciferase and to the sites for LRR in tadpoles was not confirmed. 6. A cut-off in potency beyond dodecanol was established in experiments where tadpoles were maintained in supersaturated solutions of tridecanol for 20 h without demonstrable LRR. 7. These findings indicate that the soluble enzyme firefly luciferase does not adequately model the anaesthetic site. Specifically, there are discrepancies in the position of cut-off, and the apparent changes in the free energy of binding, per methylene group, of an alkanol to luciferase do not parallel that for tadpoles.

Alcohols↗

PubMed1989

Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli.

The expression of the positively charged human protein secretory leukocyte protease inhibitor (SLPI) in Escherichia coli causes severe cellular toxicity. After induction of SLPI synthesis in a high-level-expression strain, SGE61, the growth of the strain is arrested and total protein and RNA synthesis rates decline by 60 to 70%. The mechanism of SLPI-mediated inhibition of macromolecular synthesis was examined in cell-free transcription-translation systems. SLPI proved to be a potent inhibitor of translation in vitro. When SLPI was added to translation reactions at SLPI/mRNA ratios attained during maximal SLPI accumulation in SGE61, translation of a test mRNA was inhibited by 75%. The mechanism of translation inhibition was deduced from in vitro experiments showing that SLPI bound to mRNA and interfered with the interaction of RNA-metabolizing enzymes, such as RNase. In addition, SLPI bound to DNA in vitro, but transcription was not inhibited as strongly in cell-free reactions as it was in SGE61. Similar nucleic acid-binding and translation inhibition properties were displayed in vitro by another basic protein, chicken egg white lysozyme, but were not displayed by the relatively acidic protein bovine serum albumin. On the basis of these results, we concluded that SLPI binds to nucleic acids via charge interactions and inhibits translation by competing with ribosomes for binding to mRNA. Since SLPI-mRNA and SLPI-DNA binding occurred at SLPI/mRNA and SLPI/DNA ratios existing in SGE61, nucleic acid binding may contribute to the toxicity of SLPI to E. coli. These results indicate that, in general, high-level expression of basic recombinant proteins in E. coli may be problematic.

Bacterial Proteins↗

PubMed1989

Actions of pentobarbital enantiomers on nicotinic cholinergic receptors.

The enantiomers of pentobarbital had four different actions on the nicotinic receptor-rich membranes from Torpedo electroplaques. (i) Both inhibited cholinergically stimulated cation flux through the receptor's channel, with IC50 values of approximately 25 microM and extremely weak stereoselectivity. (ii) (R)-(+)-[14C]Pentobarbital bound to a saturable site with an apparent dissociation constant of 100 microM, a Hill coefficient of 1.2, and a stoichiometry of 1:1 with the acetylcholine binding sites. (S)-(-)-Pentobarbital also displaced (+)-[14C]pentobarbital but its IC50 was 4-fold higher than that of the (+)-enantiomer under the same conditions. (iii) Both enantiomers caused a stereoselective allosteric inhibition of [3H]acetylcholine binding, which occurred over the same concentration range and with the same stereoselectivity as barbiturate binding. (iv) Above 1 mM, pentobarbital caused an unexpected and sudden increase in [3H]acetylcholine binding, which lacked significant stereoselectivity. These results are consistent with a model where low concentrations of pentobarbital act on the receptor by binding to allosteric sites that have higher affinity but lower stereoselectivity for the open channel conformation than for the resting conformation, whereas the highest concentrations of pentobarbital act by nonspecific mechanisms mediated by general membrane perturbations.

Acetylcholine↗

PubMed1989

The modes of binding of ligands to cardiac muscarinic receptors.

Ionizable groups on the cardiac M2 muscarinic receptor which regulate the binding of ligands have been examined by studying the pH dependence of the ligand affinity constants. The presence of three titratable residues (approximate pK values, 5.4, 6.8 and 7.5) whose protonation modulates antagonist binding has been demonstrated. Cardioselective antagonists are selectively affected by the protonation state of the pK 6.8 residue, whereas the binding of antagonists having differing selectivities is more strongly affected by protonation of the pK 5.4 residue on cardiac receptors. Methoctramine is capable of binding to both the pK 5.4 and 6.8 residues simultaneously. Protonation of the residue of highest pK produces a conformational change at the receptor which can affect both agonist and antagonist binding. It is now possible to demonstrate differences both in the way ligands bind to a given receptor subtype and in the way a given ligand binds to different subtypes.

Animals↗

PubMed1988

Effect of thromboxane synthetase inhibition on platelet function and morphology during ovine pregnancy-induced hypertension.

Arterial blood pressure, serum fibrin/fibrinogen degratory products, plasma thromboxane B2, in vitro platelet aggregation, and platelet ultrastructure were studied in ten gravid ewes during fast-triggered ovine pregnancy-induced hypertension and subsequent administration of the thromboxane synthetase inhibitors CGS13080 and CGS12970. During the hypertensive period, blood pressure (p less than 0.005) and plasma thromboxane B2 levels (p less than 0.005) were significantly altered. Collagen-induced in vitro platelet aggregation lag times increased (p less than 0.01), and percent aggregation (p less than 0.05), primary (p less than 0.01), and secondary (p less than 0.005) aggregatory slopes decreased. Collagen also failed to induce aggregation in some ewes. Primary slopes of ADP-induced in vitro platelet aggregation decreased (p less than 0.01) during hypertension. Degranulation and open canalicular tubule system swelling were observed in platelets which produced abnormal or no aggregation response. However, these ultrastructural abnormalities did not necessarily correspond to hypertensive periods. Thromboxane synthetase inhibitor administration lowered blood pressure (p less than 0.005) and plasma thromboxane B2 levels (p less than 0.005). Abnormalities in collagen and ADP-induced platelet aggregation curves were also corrected, and ultrastructural abnormalities were not detected. Marked elevations in plasma thromboxane levels during ovine pregnancy-induced hypertension may have had an "exhaustive" effect on thrombocytes which was reversed by thromboxane synthetase inhibition.

Animals↗

PubMed1988

Erythrocyte morphologic features and serum chemistry studies in ovine pregnancy-induced hypertension treated with thromboxane synthetase inhibitors.

Erythrocyte morphologic characteristics and serum chemistry results were studied in 10 gravid ewes during experimental ovine pregnancy-induced hypertension and subsequent administration of the thromboxane synthetase inhibitors CGS13080 and CGS12970. During the hypertensive period mean arterial blood pressure, plasma thromboxane B2 levels, and serum chemistry results, and electrolyte levels were significantly altered. Parameters returned to baseline values or were improved after drug administration. Erythrocyte morphologic features did not change significantly with the onset of the syndrome. Echinocytosis was present during baseline measurement and persisted throughout hypertension. However, after thromboxane synthetase inhibition, percentages of discocytes increased (p less than or equal to 0.005) with the same frequency that echinocyte numbers decreased (p less than or equal to 0.05). Schistocytes were present throughout the study, and changes in their numbers were not detected. Serum phosphorus, blood urea nitrogen, and bilirubin levels and anion gap rose significantly during hypertension and returned to normal levels after drug treatment. We speculate that CGS13080 or CGS12970, by decreasing thromboxane levels and blood pressure, promoted the normalization of erythrocyte membranes.

Animals↗

PubMed1988

High acetylcholine concentrations cause rapid inactivation before fast desensitization in nicotinic acetylcholine receptors from Torpedo.

By using both a 3 to 4 ms quenched-86Rb+ flux assay and native acetylcholine receptor (AChR) rich electroplaque vesicles on which 50-60% of acetylcholine activation sites were blocked with alpha-BTX, we determined apparent rates of agonist-induced inactivation in AChR from Torpedo under conditions where measured flux response was directly proportional to initial 86Rb+ influx rate. Inactivation kinetics with acetylcholine in both the activating range (10 microM-10 mM) and the self-inhibiting range (15-100 mM) were measured at 4 degrees C. In the presence of 10 microM-1 mM acetylcholine, inactivation is characterized by a single exponential rate constant, kd (fast desensitization). Plots of kd vs. acetylcholine concentration display maximum kds [kd(max)] of 6.6-8.0 s-1, half-maximal kd at 102 +/- 16 microM, and a Hill coefficient of 1.6 +/- 0.3, closely paralleling the initial ion flux response of AChR. Thus, fast desensitization probably occurs from a doubly-liganded preopen state or the open channel state. In the self-inhibiting acetylcholine concentration range, inactivation is biphasic. A "rapid inactivation" phase is complete within 30 ms, followed by fast desensitization at a rate close to kd(max). Both the rate and extent of rapid inactivation increase with acetylcholine concentration, indicating that acetylcholine binds to its self-inhibition site with apparent kon approximately equal to 10(3) M-1s-1 and koff approximately equal to 40 s-1. This slow kon suggests either hindered access to the inhibitory allosteric site or that a fast binding step is followed by a slower conformational change leading to channel inhibition. Overall, our data suggest that acetylcholine binds preferentially to its inhibitory site when the receptor is in the open-channel conformation and that fast desensitization can occur from all multiple-liganded states.

Acetylcholine↗

PubMed1987

Identification of the genes in the Escherichia coli ileS-lsp operon. Analysis of multiple polycistronic mRNAs made in vivo.

The genes encoding isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) of Escherichia coli were previously shown to be co-transcribed (Miller, K. W., and Wu, H. C. (1987) J. Biol. Chem. 262, 389-393). However, the boundaries of this transcriptional unit have not been established. In this regard, DNA sequence determination has shown that ileS and lsp are closely flanked by four open reading frames, i.e. x-ileS-lsp-orf149-orf316-orf304. To define the boundaries of the operon, we applied Northern blotting hybridization and mRNA 5'-end mapping to analyze mRNA from a wild-type strain (SM31) and a mutant strain (SM31-2B4) that exhibits an increased expression of prolipoprotein signal peptidase. Four ileS-lsp co-transcripts were detected in RNA from the strain SM31. In addition to these four mRNAs, two new, highly abundant co-transcripts were also detected in RNA from the mutant. Based upon the determination of the 5'-ends of the mRNAs and analysis of their coding sequences, we conclude that the six mRNAs actually are comprised of three pairs of related mRNAs. The two mRNAs in a given pair have the same 5'-termini (all located upstream of or within gene x), but vary with respect to the identity of their 3'-terminal coding sequence (lsp or orf316). In conclusion, the ileS-lsp operon contains five genes, x-ileS-lsp-orf149-orf316, whose transcription probably is dependent upon promoter(s) located upstream of or within gene x. The next gene downstream, orf304, apparently does not reside in the operon.

Amino Acyl-tRNA Synthetases↗

PubMed1987

Is agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action?

Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring 86Rb+ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: integrated net ion efflux (in 10 s) from untreated vesicles, integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with alpha-bungarotoxin, and initial rates of efflux (5-100 ms) from vesicles that were partially blocked with alpha-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10(8)-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum 86Rb+ efflux by 50% (KB values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, KB values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times KB did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. We conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.

Acetylcholine↗

PubMed1987

Lipid-dependent differential effects of stereoisomers of anesthetic alcohols.

The cis- and trans-alkenols are equally potent general anesthetics but, respectively, lower and raise the gel-to-liquid crystalline phase transition temperature of saturated phosphatidylcholines (Pringle, M.J. and Miller, K.W. (1978) Biochem. Biophys. Res. Commun. 85, 1191-1198). Here we show that although this differential effect is somewhat reduced when a double bond is introduced into the sn-2 position of phosphatidylcholine, it is abolished when the ethanolamine head group is substituted for the choline head group in dimyristoyl lipids at neutral pH. At high pH, however, dimyristoylphosphatidylethanolamine assumes a negative charge, and its phase transition temperature drops to a value close to that for the corresponding phosphatidylcholine. Under these conditions the differential effect of the alkenol isomers is restored; the cis-alkenol lowers, while the trans-alkenol raises, the phase transition temperature of deprotonated dimyristoylphosphatidylethanolamine. Thus, the differential effects of cis- and trans-alkenols on the gel-to-liquid crystalline phase transition are dependent on the physical chemical characteristics of the polar region of the perturbed lipid species, but only weakly on that of the acyl region.

Alcohols↗

PubMed1987

Two pools of cholesterol in acetylcholine receptor-rich membranes from Torpedo.

The acetylcholine receptor (AChR)-containing electroplax membranes from Torpedo californica have a relatively high cholesterol content. Reconstitution studies suggest that this cholesterol may be important in preserving or modulating the function of the acetylcholine receptor-channel complex. We have manipulated cholesterol levels in intact Torpedo AChR-rich membrane fragments using small, unilamellar phosphatidylcholine liposomes. Conditions have been established that allow further subfractionation of sucrose gradient purified Torpedo electroplax membranes into AChR-rich and ATPase-rich populations and that, at the same time, achieve cholesterol depletion without phospholipid back exchange or fusion. The incubation of membranes with excess liposomes could only achieve about a 50% reduction in the molar ratio of cholesterol to phospholipid. In no case was the number of cholesterol molecules per AChR oligomer reduced below 36. The remaining cholesterol could not be depleted either by longer incubations or by multiple, sequential depletions. Cholesterol depletion was accompanied by a significant increase in bulk membrane fluidity as measured by electron spin resonance spectroscopy, but the equilibrium binding parameters of acetylcholine to its receptor were unaltered. This suggests strongly that there exist two pools of cholesterol in the AChR-rich Torpedo electroplax membrane: an easily depleted fraction that influences bulk fluidity, and a tightly-bound fraction perhaps surrounding the AChR oligomer.

Animals↗