Amira T. Eldefrawi

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[125I]bungarotoxin but not [3H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [3H]acetylcholine, [3H]decamethonium, [3H]nicotine, [14C]dimethyl-d-tubocurarine, and α-[125I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing. The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (KD = 0.02 μM) and 5.1 nmoles a lower affinity (KD = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.

Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studied by use of a fluorescent lanthanide.

Abstract The acetylcholine receptor isolated from Torpedo ocellata binds about 10 moles of a fluorescent lanthanide, terbium, per mole α-bungarotoxin-binding site, a process which is accompanied by a fluorescence enhancement (λexcitation 295 nm, λemission 546 nm) which allows detection of receptor-Tb 3+ complexes at μM concentrations. In presence of calcium two types of terbium-binding site are revealed, both with terbium dissociation constants of 18 ± 0.5 μM. About 60% of the sites bind calcium with an apparent dissociation constant of 1.1 ± 0.1 mM. Sites which interact with calcium also interact with activators of neural transmission, carbamylcholine and decamethonium, but not with the inhibitors, d-tubocurarine and α-bungarotoxin. Whether the displacement of calcium by chemical mediators is directly responsible for activator-induced changes in ion permeability of neural membranes is an important question raised by our experiments. The results show that fluorescent lanthanides can be an important tool in such studies.

Properties of Lubrol-solubilized acetylcholine receptor from Torpedo electroplax

Abstract The efficiencies of two nonionic and four anionic detergents in solubilizing acetylcholine receptors (AChR) (as detected by ACh-binding) from electric tissue of Torpedo were compared. Lubrol WX solubilized the highest concentration of active AChR/mg protein and sodium dodecyl sulfate (SDS) abolished ACh-binding; however, removal of the latter detergent resulted in a small recovery of ACh binding. The Lubrol-solubilized AChR was similar to the particulate AChR in binding ACh at two sites reversibly, with high affinities, and in showing autoinhibition at ACh concentrations higher than 1 μ m . Also, binding of ACh was abolished by boiling and was reduced by pretreatment with trypsin, pronase, and phospholipases. However, “kinetics” of ACh binding to solubilized AChR were changed: there were increases in the affinity of the high-affinity site and in the ratio of the concentrations of the high- to low-affinity site, and the Hill coefficient was reduced to 0.66. Its nicotinic nature was demonstrated by blockade of ACh binding by 13 nicotinic drugs including α-bungarotoxin and cobrotoxin. Anticholinesterases were also blockers, but 21 noncholinergic drugs had no effect. Ultrafiltration and gel chromatography were used to separate the ACh-binding macromolecules. Sepharose 6B gave partial separation of AChR from AChE. The molecular weight of the smallest functional AChR was between 50,000 and 100,000 daltons, and had a high tendency to aggregate. The majority of Lubrol-solubilized AChR had molecular weights above 300,000 daltons.

Acetylcholine Binding to Torpedo Electroplax: Relationship to Acetylcholine Receptors

Binding of [3H]acetylcholine to a particulate fraction of Torpedo electroplax was measured by equilibrium dialysis. Two high-affinity sites present on phospholipoproteins bound acetylcholine reversibly, and binding was blocked by nicotinic drugs. Characteristics of this binding suggest that these phospholipoproteins may be acetylcholine receptors.

Binding of calcium and zinc to the acetylcholine receptor purified from Torpedo Californica

Abstract Binding of [65Zn++] and [45Ca++] to the acetylcholine (ACh)-receptor, purified from the Torpedo electric organ, was studied by equilibrium dialysis. Whereas [65Zn++] bound to 56 nmoles of sites per mg protein with a dissociation constant of 2.5 × 10−6M, no binding of [45Ca++] at concentrations up to 10−3M could be detected with this method. However, the binding of [acetyl-3H]choline to the receptor was blocked equally by very high Zn++ or Ca++ concentrations, and the Ki for this low affinity binding was 7 × 10−3M. The high affinity binding of [65Zn++] to the receptor was blocked best by Cd++ then Co++ and Mn++, but least by Mg++ and Ca++. When the purified ACh-receptor itself was analyzed for the presence of cations by atomic absorption, it was discovered that 4.7% of its weight was due to bound Ca++ that could not be removed even by extensive dialysis. When Ca++-free solutions (containing 1 mM EDTA) were used during purification, 0.6% of the molecular weight of the receptor was still due to bound Ca++. This was equivalent to 15 moles of Ca++ for each mole of ACh bound at saturation. It is suggested that the source of this Ca++ is endogenous, and that it is tightly bound to the ACh-receptor molecule.

Identification of a calcium-binding subunit of the acetylcholine receptor

Abstract Four subunits of the acetylcholine receptor molecule, obtained from the electric organ of Torpedo ocellata , have been isolated using polyacrylamide gel electrophoresis, and assayed by titration with a fluorescent lanthanide, terbium, and by affinity-labeling with p -( N -maleimido)benzyl [trimethyl-3H] ammonium iodide. The site with which the activator-analogue affinity label reacts, as well as the terbium-binding sites, are mainly associated with the smallest of the subunits of an apparent molecular weight of 40,000. Calcium competes with terbium for these binding sites. The affinity for terbium is the same in the intact molecule as in the subunit (KTb ⋍ 19 ± 1 μM), but the affinity for calcium decreases by a factor of 4 (KCa ⋍ 4 mM) in the subunit. Hydrolysis of the receptor, catalyzed by trypsin and chymotrypsin, to peptides with an apparent molecular weight of 8000 or less, does not affect the terbium-binding sites. These experiments indicate that the binding sites for neural activators and for calcium are associated with the same subunit, and that the terbium- and calcium-binding sites reflect structural properties of the polypeptide chain rather than the three-dimensional structure of the protein.

The Acetylcholine Receptor and Its Interactions with Insecticides

The beginning of this century saw the development of two important principles of neurobiology: that the nerve cell (neuron) is the building unit of the nervous system and that neurons usually communicate with one another by chemicals. Direct electrical communication between neurons is rare, because even at their closest point of approach they are insulated from each other by a gap (about 200–250 A) filled with extracellular fluid. When an electrical signal arrives at the end of a neuron, packets of a small molecule, called neurotransmitter, are released from the neuron into the gap. These chemicals act as messengers and interact with a component of the membrane of the receiving cell, whether it is another neuron, muscle, or gland. This component is called the receptor and the point of communication between the cells is called the synapse. Different chemicals have been identified as neurotransmitters, and one of these is acetylcholine (ACh). The synapse in this case is said to be cholinergic (Fig. 1). Such synapses are located in the animal brain, ganglia, and nerve-gland junctions, and also in nerve-muscle junctions in vertebrates.

BINDING OF CHOLINERGIC LIGANDS TO ELECTROPLAXES AND BRAIN TISSUES

Publisher Summary This chapter highlights acetylcholine receptors (ACR) of different kinds and describes the molecular basis of their actions. There has been progress toward identification of ACR in subcellular preparations from four tissues. As receptor is only unambiguously identified in intact cells, a number of criteria are to be met to believe that ACR is present in any given subcellular preparations. These criteria include the ability to bind the right drugs with appropriate affinity; the appropriate reversibility and quantity of such binding; and the location of such binding in suitable tissues and in suitable locations within those tissues. The tissues involved are electroplax from Torpedo and Electrophorus , and heads from houseflies, Musca domestica . The chapter reports on findings with fractions from whole brain of rats. The radioactive ligands employed are all familiar except for muscarone, which is the keto analog of muscarine, and is highly stimulatory on both the nicotinic and muscarinic variety of ACR.