Characterization of Curaremimetic Neurotoxin Binding Sites on Cellular Membrane Fragments Derived from the Rat Pheochromocytoma PC 12

Studies were conducted on the properties of 125I-labeled alpha-bungarotoxin binding sites on cellular membrane fragments derived from the PC12 rat pheochromocytoma. Two classes of specific toxin binding sites are present at approximately equal densities (50 fmol/mg of membrane protein) and are characterized by apparent dissociation constants of 3 and 60 nM. Nicotine and d-tubocurarine are among the most potent inhibitors of high-affinity toxin binding. The affinity of high-affinity toxin binding sites for nicotinic cholinergic agonists is reversibly or irreversibly decreased, respectively, on treatment with dithiothreitol or dithiothreitol and N-ethylmaleimide. The nicotinic receptor affinity reagent bromoacetylcholine irreversibly blocks high-affinity toxin binding to PC12 cell membranes that have been treated with dithiothreitol. Two polyclonal antisera raised against the nicotinic acetylcholine receptor from Electrophorus electricus inhibit high-affinity toxin binding. These detailed studies confirm that curaremimetic neurotoxin binding sites on the PC12 cell line are comparable to toxin binding sites from neural tissues and to nicotinic acetylcholine receptors from the periphery. Because toxin binding sites are recognized by anti-nicotinic receptor antibodies, the possibility remains that they are functionally analogous to nicotinic receptors.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

Presynaptic Cholinergic Mechanisms in the Rat Cerebellum: Evidence for Nicotinic, but Not Muscarinic Autoreceptors

The present study shows that N-[3H]methylcarbamylcholine ([3H]MCC) binds to a single population of high-affinity/low-density (KD = 5.0 nM; Bmax = 8.2 fmol/mg of protein) nicotinic binding sites in the rat cerebellum. Also, there exists a single class of high-affinity binding sites (KD = 4.8 nM; Bmax = 24.2 fmol/mg of protein) in the cerebellum for the M1 specific muscarinic ligand [3H]pirenzepine. In contrast, the M2 ligand, [3H]AF-DX 116, appears to bind to two classes of binding sites, i.e., a high-affinity (KD = 3 nM)/low-capacity (Bmax = 11.7 fmol/mg of protein) class, and a second class of lower affinity (KD = 28.4 nM) and higher capacity (Bmax = 36.3 fmol/mg of protein) sites. The putative M3 selective ligand [3H]4-diphenylacetoxy-N-methylpiperidine also binds to two distinct classes of binding sites in cerebellar homogenates, one of high affinity (KD = 0.5 nM)/low capacity (Bmax = 19.5 fmol/mg of protein) and one of low affinity (KD = 57.5 nM)/high capacity (Bmax = 140.6 fmol/mg of protein). In experiments which tested the effects of cholinergic drugs on acetylcholine release from cerebellar brain slices, the nicotinic agonist MCC enhanced spontaneous acetylcholine release in a concentration-dependent manner, and the maximal increase in acetylcholine release (59.0-68.0%) occurred at 10(-7) M. The effect of MCC to increase acetylcholine release was Ca2+-dependent and tetrodotoxin-insensitive, suggesting an action on cholinergic terminals. Also, the MCC-induced increase in acetylcholine release was effectively antagonized by dihydro-beta-erythroidine, d-tubocurarine, and kappa-bungarotoxin, but was insensitive to either atropine or alpha-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of antinicotinic acetylcholine receptor antibodies with rat brain and muscle antigenic determinants

Studies were performed to determine whether antibodies prepared against nicotinic acetylcholine receptors (nAcChoR) from electric tissue are reactive toward nAcChoR-like antigenic determinants in rat brain. Reference experiments involved the use of Torpedo electroplax and rat innervated muscle as tissue controls and an anti-alpha-bungarotoxin antiserum as a probe for curaremimetic neurotoxin binding sites. As evinced by their ability to inhibit immunoprecipitation of Torpedo nAcChoR, brain or muscle membranes specifically interact with polyclonal antisera raised against Electrophorus electroplax nAcChoR. When the extent of polyclonal anti-nAcChoR antibody binding to muscle membranes is measured by protein A binding protocols, receptor-like antigenic determinants and toxin binding sites are found to be present in approximately equal quantities. In contrast, nAcChoR-like antigenic determinants on rat brain membranes are present at concentrations in excess of those of toxin binding sites. The results are consistent with the earlier observation that some antibodies prepared against nAcChoR from peripheral tissues recognize rat brain high-affinity alpha-bungarotoxin binding sites. The results also suggest the existence of nAcChoR-like entities in brain that do not bind toxin with a high affinity.     

Effects of chronic nicotine treatment on nicotinic receptors in the rabbit urinary bladder

Nicotine induced a phasic contraction in the rabbit urinary bladder. The response was abolished by hexamethonium and partially reduced by atropine and capsaicin. Simultaneous atropine and capsaicin treatment did not abolish the contraction. These findings suggest that the response to nicotine is due to acetylcholine, tachykinins, and unknown mediator release. In contrast, nicotine-induced contraction diminished following the chronic nicotine treatment without a change of its pharmacological properties. These results suggest the possibility that chronic nicotine treatment causes a decrease in nicotinic receptor numbers. Therefore, the binding properties of (-)-[3H]nicotine on rabbit urinary detrusor muscle membrane fractions were studied to evaluate the effects of chronic nicotine treatment on nicotinic receptors. Specific (-)-[3H]nicotine binding reached saturation and Scatchard plots were curvilinear, suggesting the existence of two different affinity sites for (-)-[3H]nicotine. Dissociation constants (KD) and maximum binding sites (Bmax) were KD1 = 4.91 +/- 1.88 nM, Bmax1 = 2.42 +/- 0.22 fmol/mg protein and KD2 = 263 +/- 56 nM, Bmax2 = 25.0 +/- 4.3 fmol/mg protein. In urinary bladder membrane fractions from chronic nicotine-treated rabbits, KD and Bmax values were KD1 = 3.96 +/- 0.38 nM, Bmax1 = 1.07 +/- 0.25 fmol/mg protein and KD2 = 249 +/- 12 nM, Bmax2 = 10.8 +/- 1.5 fmol/mg protein. Dissociation constants for both sites following chronic nicotine treatment did not change but maximum binding site numbers for both sites significantly decreased (p less than 0.05). These results suggest that the decrease in contractile response evoked by nicotine after chronic nicotine treatment in rabbit urinary bladder is due to a decrease in numbers of nicotinic receptors.     

A comparison of binding properties and structure of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells

Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound 125I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: KD = 5.2 nM sites and KD = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and 125I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (KD = 1.0 nM), all binding sites were trypsin sensitive, and 125I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (KD = 3 nM) that were trypsin sensitive, and 125I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of 125I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.     

Effects of Chronic Nicotinic Ligand Exposure on Functional Activity of Nicotinic Acetylcholine Receptors Expressed by Cells of the PC12 Rat Pheochromocytoma or the TE671/RD Human Clonal Line

Studies were conducted to ascertain the temporal and dose-dependent effects of nicotinic ligand exposure on functional activity of different nicotinic acetylcholine receptor (nAChR) subtypes, as expressed by cells of the PC12 rat pheochromocytoma (ganglia-type nAChR) or the TE671/RD human (muscle-type nAChR) clonal line. Chronic (3-72-h) agonist (nicotine or carbamylcholine) treatment of cells led to a complete (TE671) or nearly complete (PC12) loss of functional nAChR responses, which is referred to as "functional inactivation." Some inactivation of nAChR function was also observed for the nicotinic ligands d-tubocurarine (d-TC), mecamylamine, and decamethonium. Half-maximal inactivation of nAChR function was observed within 3 min for TE671 cells and within 10 min for PC12 cells treated with inactivating ligands. Functional inactivation occurred with dose dependencies that could not always be reconciled with those obtained for acute agonist activation of nAChR function or for acute inhibition of those responses by d-TC, decamethonium, or mecamylamine. Treatment of TE671 or PC12 cells with the nicotinic antagonist pancuronium or alcuronium alone had no effect on levels of expression of functional nAChRs. However, evidence was obtained that either of these antagonists protected TE671 cell muscle-type nAChRs or PC12 cell ganglia-type nAChRs from functional inactivation on long-term treatment with agonists. Recovery of TE671 cell nAChR function following treatment with carbamylcholine, nicotine, or d-TC occurred with half-times of 1-3 days whether cells were maintained in situ or harvested and replated after removal of ligand. By contrast, 50% recovery of functional nAChRs on PC12 cells occurred within 2-6 h after drug removal. In either case the time course for recovery from nAChR functional inactivation is much slower than recovery from nAChR "functional desensitization," which is a reversible process that occurs on shorter-term (0-5-min) agonist exposure of cells. These results indicate that ganglia-type and muscle-type nAChRs are similar in their sensitivities to functional inactivation by nicotinic ligands but differ in their rates of recovery from and onset of those effects. The ability of drugs such as the agonists d-TC, decamethonium, and mecamylamine to induce functional inactivation may relate to their activities as partial/full agonists, channel blockers, and/or allosteric regulators. Effects of drugs such as pancuronium and alcuronium are likely to reflect simple competitive inhibition of primary ligand binding at functional activation sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solubilisation and Characterisation of a Putative Quisqualate-Type Glutamate Receptor from Chick Brain

The brains of 1-day-old chicks were shown to be a rich source of binding sites with the pharmacological characteristics expected of a quisqualate-type glutamate receptor. alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) bound with KD and Bmax values, measured at 0 degree C in the presence of the chaotrope potassium thiocyanate, of 55 nM and 2.6 pmol/mg protein. The regional localisations of [3H]AMPA and [3H]kainate binding sites were manifestly different. The membrane-bound [3H]AMPA binding sites were efficiently solubilised by N-octyl-beta-D-glucopyranoside (1%) in the presence of 0.2 M thiocyanate. In the detergent extract the affinity was 69 nM and there was an apparent increase in the number of sites (Bmax, 4.6 pmol/mg protein). The rank order of potency for competitive ligands in displacing [3H]AMPA binding was quisqualate approximately AMPA greater than 6-cyano-7-nitroquinoxaline-2,3-dione greater than L-glutamate greater than kainate and was identical for the membrane-bound and solubilised sites. Dissociation was biphasic with rate constants of 0.117 min-1 and 0.015 min-1. The association rate constants for [3H]AMPA at the solubilised sites were 1.45 x 10(6) M-1 min-1 and 6.55 x 10(6) M-1 min-1. The kinetically derived KD values were 80.7 nM and 2.3 nM. The detection of higher affinity binding sites by kinetic analysis but not by equilibrium binding may be explained by the greater sensitivity of dissociation data to small populations of high-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical characterization of two nicotinic receptors from the optic lobe of the chick

We have studied putative nicotinic acetylcholine receptors in the optic lobe of the newborn chick, using 125I-labeled alpha-bungarotoxin, a specific blocker of acetylcholine receptors in the neuromuscular junction, and [3H]acetylcholine, a ligand which in the presence of atropine selectively labels binding sites of nicotinic character in rat brain cortex (Schwartz et al., 1982). [3H]Acetylcholine binds reversibly to a single class of high affinity binding sites (KD = 2.2 X 10(-8) M) which occur at a tissue concentration of 5.7 pmol/g. A large fraction (approximately 60%) of these binding sites is solubilized by Triton X-100, sodium cholate, or the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization increases the affinity for acetylcholine and several nicotinic drugs from 1.5- to 7-fold. The acetylcholine-binding macromolecule resembles the receptor for alpha-bungarotoxin present in the same tissue with respect to subcellular distribution, hydrodynamic properties, lectin binding, and agonist affinity rank order. It differs from the toxin receptor in affinity for nicotinic antagonists, sensitivity to thermal inactivation, and regional distribution. The solubilized [3H]acetylcholine binding activity is separated from the toxin receptor by incubation with agarose-linked acetylcholine, by affinity chromatography on immobilized Naja naja siamensis alpha-toxin, and by precipitation with a monoclonal antibody to chick optic lobe toxin receptor.     

Novel Photoactivatable Agonist of the Nicotinic Acetylcholine Receptor of Potential Use for Exploring the Functional Activated State

Abstract: The nicotinic acetylcholine receptor (AChR) exhibits at least four different conformational states varying in affinity for agonists such as acetylcholine (ACh). Photoaffinity labeling has been previously used to elucidate the topography of the AChR. However, to date, the photosensitive probes used to explore the cholinergic binding site photolabeled only closed or desensitized states of the receptor. To identify the structural modifications occurring at the ACh binding site on allosteric transition associated with receptor activation, we have investigated novel photoactivatable 4-diazocyclohexa-2,5-dienone derivatives as putative cholinergic agonists. Such compounds are fairly stable in the dark and generate highly reactive carbenic species on irradiation. In binding experiments using AChRs from Torpedo marmorata, these ligands had affinities for the ACh binding site in the micromolar range and did not interact with the noncompetitive blocker site (greater than millimolar affinity). Irreversible photoinactivation of ACh binding sites was obtained with the ligand 1b (up to 42% at 500 µM) in a protectable manner. In patch-clamp studies, 1b was shown to be a functional agonist of peripheral AChR in TE 671 cells, with the interesting property of exhibiting no or very little desensitization even at high concentrations.     

Characterization of γ-Aminobutyric Acid Binding Sites on Crude Synaptic Membranes

[3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X-100 [membranes (TX)] involved two classes of GABA binding sites (KD = 38.7 and 78.0 nM) in the absence of Na+, but the high-affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD = 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high-affinity class of GABA receptor.     

Buprenorphine: High-Affinity Binding to Dorsal Spinal Cord

The binding of the mixed opiate agonist-antagonist [3H]buprenorphine was compared with [3H]naloxone and [3H]dihydromorphine binding in membranes prepared from rat whole brain and dorsal spinal cord. Scatchard analysis of binding to whole brain yielded KD values close to 1.0 nM for all three 3H-ligands studied, although [3H]buprenorphine labelled five times as many binding sites. [3H]Naloxone and [3H]dihydromorphine bound to dorsal spinal cord with approximately the same affinity as to whole brain, although both 3H-ligands labelled fewer sites in the spinal cord. In contrast, Scatchard analysis of [3H]buprenorphine binding to spinal cord yielded curvilinear Scatchard plots, suggesting the presence of a very high-affinity (KD = 0.12 nM) binding site in addition to the high-affinity site (KD = 1.0 nM) present in the brain. Studies on the displacement of [3H]buprenorphine by opiates and D-Ala2,Met5-enkephalinamide supported the presence of two binding sites for this ligand in the spinal cord.     

Design and synthesis of peptides that bind α-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor

α-Bungarotoxin (α-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind α-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the α-BTX/AChR system yielded 13-mer peptides that bind to α-BTX with high affinity and are potent inhibitors of α-BTX binding to AChR (IC₅₀ of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel β-hairpin structure when bound to α-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.     

Possible Involvement of Pertussis Toxin-Sensitive G Proteins and D2 Dopamine Receptors in the A1 Adenosine Receptor-Adenylate Cyclase System in Rat Cerebral Cortex

To identify the involvement of dopamine receptors in the transmembrane signaling of the adenosine receptor-G protein-adenylate cyclase system in the CNS, we examined the effects of pertussis toxin (islet-activating protein, IAP) and apomorphine on A1 adenosine agonist (-)N6-R-[3H]phenylisopropyladenosine ([3H]PIA) and antagonist [3H]xanthine amine congener ([3H]XAC) binding activity and adenylate cyclase activity in cerebral cortex membranes of the rat brain. Specific binding to a single class of sites for [3H]XAC with a dissociation constant (KD) of 6.0 +/- 1.3 nM was observed. The number of maximal binding sites (Bmax) was 1.21 +/- 0.13 pmol/mg protein. Studies of the inhibition of [3H]XAC binding by PIA revealed the presence of two classes of PIA binding states, a high-affinity state (KD = 2.30 +/- 1.16 nM) and a low-affinity state (KD = 1.220 +/- 230 nM). Guanosine 5'-(3-O-thio)triphosphate or IAP treatment reduced the number of the high-affinity state binding sites without altering the KD for PIA. Apomorphine (100 microM) increased the KD value 10-fold and decreased Bmax by approximately 20% for [3H]PIA. The effect of apomorphine on the KD value increase was irreversible and due to a conversion from high-affinity to low-affinity states for PIA. The effect was dose dependent and was mediated via D2 dopamine receptors, since the D2 antagonist sulpiride blocked the phenomenon. The inhibitory effect of PIA on adenylate cyclase activity was abolished by apomorphine treatment. There was no effect of apomorphine on displacement of [3H]quinuclidinyl benzilate (muscarinic ligand) binding by carbachol. These data suggest that A1 adenosine receptor binding and function are selectively modified by D2 dopaminergic agents.     

Neuronal nicotinic receptor deficits in Alzheimer patients with the Swedish amyloid precursor protein 670/671 mutation

The influence of beta-amyloid on cholinergic neurotransmission was studied by measuring alterations in nicotinic acetylcholine receptors (nAChRs) in autopsy brain tissue from subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation. Significant reductions in numbers of nAChRs were observed in various cortical regions of the Swedish 670/671 APP mutation family subjects (-73 to -87%) as well as in sporadic Alzheimer's disease (AD) cases (-37 to -57%) using the nicotinic agonists [3H]epibatidine and [3H]nicotine, which bind with high affinity to both alpha3 and alpha4 and to alpha4 nAChR subtypes, respectively. Saturation binding studies with [3H]epibatidine revealed two binding sites in the parietal cortex of AD subjects and controls. A significant decrease in Bmax (-82%) for the high-affinity site was observed in APP 670/671 subjects with no change in K(D) compared with controls (0.018 nM APP 670/671; 0.036 nM control). The highest load of neuronal plaques (NPs) was observed in the parietal cortex of APP 670/671 brains, whereas the number of [3H]nicotine binding sites was less impaired compared with other cortical brain regions. Except for a positive significant correlation between the number of [3H]nicotine binding sites and number of NPs in the parietal cortex, no strict correlation was observed between nAChR deficits and the presence of NPs and neurofibrillary tangles, suggesting that these different processes may be closely related but not strictly dependent on each other.     

Radioactive labelling of toxin I from Anemonia sulcata and binding to crayfish nerve in vitro

1. Radioactive derivatives of neurotoxin I (ATX I) from Anemonia sulcata have been synthesized: Iodination of ATX I with 125I yielded a mixture of reaction products from which monoiodo and diiodo ATX I were isolated. 2. 125I-ATX I was shown to bind to the axonal membrane from Astacus leptodactylus main walking nerve. Specificity of binding was shown by saturability of the binding sites and by competitive binding of native and radioactive toxin. 3. Astacus nerve bound 44 fmol of 125I-ATX I/mg nerve (wet weight). The axonal membrane surface of the nerve was determined to be 7800 cm2/g nerve. This amounts to a binding site density of around 35/mu2 axonal surface. Binding was not inhibited by tetrodotoxin, the blocker of the selectivity filter of voltage-dependent sodium channels. 125I-ATX I therefore may bind to the sodium channel-inactivating gate. 4. The affinity of the nerve membrane receptors for 125I-ATX I appears to be voltage-dependent: KD = 5 nM was found with whole crayfish nerves in the presence of tetrodotoxin, KD = 40nM in the absence of tetrodotoxin and an even lower affinity was obtained with axonal membrane fragments isolated from the nerve. Drugs destabilizing the membrane potential, e.g. veratridine, ouabain and sodium azide lowered the affinity or abolished binding completely.     

Interconversion between different states of affinity for acetylcholine of the cholinergic receptor protein from Torpedo marmorata.

In receptor-rich membrane fragments from Torpedo, acetylcholine binds, in the presence of 70 muM Tetram, to a homogeneous population of high-affinity sites with Kd = (3.4 +/- 0.8) x 10(08) M. Dissolution of these membrane fragments by sodium cholate causes a decrease of affinity associated with the appearance of medium-affinity (Kd approximately 10(-7) M) and low-affinity (Kd greater than or equal to 10(-6) M) sites. Dissolution by neutral detergents Triton X-100 or Emulphogene preserves the high affinity of the acetylcholine binding sites. In all the soluble states of the receptor protein, Ca2+ ions and local anaesthetics no longer enhance the affinity for acetylcholine. Elimination of sodium cholate by dilution leads to the reassociation of the receptor protein, the recovery of high-affinity sites and the control by Ca2+ ions and local anaesthetics. Purification by affinity chromatography of the receptor protein in Triton X-100 is accompanied by a conversion of a majority of the acetylcholine sites into their state of low affinity. High-affinity sites can no longer be recovered by detergent dilution from these low-affinity ones.     

Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver.

The distribution of inositol 1,4,5-trisphosphate and ryanodine binding sites between plasma membrane, microsomal, and mitochondrial fractions of rat liver were compared. IP3 bound mostly to the plasma membrane fraction (Kd = 6 nM; Bmax = 802 fmol/mg protein). Some IP3 binding sites were also present in the microsomal and mitochondrial fractions (Kd = 2.5 and 2.9 nM; Bmax = 35 and 23 fmol/mg protein respectively). The possibility that these binding sites are due to contamination of the fractions with plasma membrane cannot be excluded. Binding of IP3 to the plasma membrane was inhibited by heparin but not by either caffeine or tetracaine. High-affinity ryanodine binding sites were present mostly in the microsomal fraction (Kd = 13 nM; Bmax = 301 fmol/mg protein). Lower affinity binding sites were also found to be present in the mitochondrial and plasma membrane fractions. Binding of ryanodine to the microsomal fraction was inhibited by both caffeine and tetracaine but not by heparin. These data demonstrate that IP3 and ryanodine binding sites are present in different cellular compartments in the liver. These differences in the localization of the binding sites might be indicative of their functional differences.