Characterization of Torpedo californica Acetylcholine Receptor: Its Subunit Composition and Ligand-binding Properties

Raftery, Michael A.; Vandlen, Richard; Reed, K.; Lee, T.

doi:10.1101/sqb.1976.040.01.021

Cited by 135 publications

(50 citation statements)

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“…Finally, the interconversion between states of increasing affinities was postulated on the basis of kinetics in the minute range done concurrently by fluorescence spectroscopy and direct ligand binding [8,19,211 and has been confirmed meanwhile [31,33].…”

Section: Reaction Mechanism and Kineticsmentioning

confidence: 99%

Fast Kinetic Studies on the Interaction of Cholinergic Agonists with the Membrane‐Bound Acetylcholine Receptor from Torpedo marmorata as Revealed by Quinacrine Fluorescence

Grünhagen

Iwatsubo

Changeux

1977

European Journal of Biochemistry

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Rapid changes of fluorescence intensity take place after fast mixing of quinacrine-labelled receptorrich membrane fragments from Torpedo marmorata with cholinergic effectors and are recorded in the time range of 1 -1000 ms, accessible to the stopped-flow technique. Agonists such as acetylcholine, phenyltrimethylammonium, carbamylcholine, suberyldicholine or choline cause the change, but not antagonists such as d-tubocurarine, flaxedil or Naja nigricollis cx-toxin. In addition, preincubation of the membrane fragments with N . nigricollis a-toxin blocks the response to agonists. Ceruleotoxin, a toxin from Bungarus ceruleus venom which blocks the postsynaptic response to acetylcholine at a site distinct from the a-toxin site, causes a decrease of the apparent amplitude of the fast signal. In the presence of 0.05 mM tetram, an acetylcholinesterase inhibitor, the kinetics of the fast response to acetylcholine do not change; in the second or minute range and in the case of acetylcholine, however, a decrease of fluorescence intensity occurs in the absence of acetylcholinesterase inhibitor showing that the fast fluorescence signal is reversible.Under the present experimental conditions the observed fast kinetics do not vary with the concentration of receptor sites or quinacrine but do vary with the concentration of agonist. The data can be analyzed in terms of the phenomenological reaction scheme A + B AB 3 A*B, when k, % k,, if the fluorescence signal monitors the isomerization from A to A*. The following values of the constants are found: for acetylcholine: k4=

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Section: Reaction Mechanism and Kineticsmentioning

confidence: 99%

Fast Kinetic Studies on the Interaction of Cholinergic Agonists with the Membrane‐Bound Acetylcholine Receptor from Torpedo marmorata as Revealed by Quinacrine Fluorescence

Grünhagen

Iwatsubo

Changeux

1977

European Journal of Biochemistry

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“…Although the acetylcholine receptor has been isolated and is being characterized in many laboratories (for a recent review see ref, 5 and also refs. [6][7][8][9], little is known, on the molecular level, about the relationship between ligand binding and receptor-mediated changes in permeability of membranes to inorganic ions. Because the receptor has no function in solution we used receptor-rich membrane vesicles (microsacs) prepared, according to the procedure of Kasai and Changeux (10), from the electric organ of Electrophorus electricus.…”

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confidence: 99%

Functional acetylcholine receptor--electroplax membrane microsacs (vesicles): purification and characterization.

Hess¹,

Andrews²

1977

Proc. Natl. Acad. Sci. U.S.A.

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Kinetic analysis of the flux of sodium ions in a heterogeneous population of acetylcholine receptor-rich microsacs (vesicles) formed by membrane fragments of electroplax indicated that functional microsacs, which on average comprise only 15% of the preparation, can be filled with 190 mM sodium chloride while nonfunctional microsacs are filled by 19Q mM cesium chloride. The functional microsacs have then been successfully separated from nonfunctional microsacs on the basis of their density differences with a continuous sucrose-190 mM cesium chloride density gradient. In the presence of acetylcholine analogs all the internal sodium ions in these microsacs rapidly exchange with external ions. The efflux of sodium ions follows a single exponential decay. The isolation of functional microsacs opens up at least two new avenues of investigation of the Molecular mechanism of reep-mediated processes. The first deals with the efficiency of the process, and the second with the characterization of membrane components important in this process. The conclusions reached so far are: (i) The efficiency of the receptor-mediated process that allows inorganic ions to equilibrate .across the membranes of the microsacs can adequately account for electrophysiological results obtained with muscle and nerve cells. (ii) In the receptor-rich heterogeneous population of microsacs the concentration of receptor sites in functional and nonfunctional microsacs is about the same and is therefore not the only factor determining functionality. Significant differences between functional and nonfunctional microsacs have been found so far in the concentrations of acetylcholinesterase and Na+-K+ ATPase.Initiation of increased ion flow across neural membranes by the combination of acetylcholine or its congeners with the membrane-bound acetylcholine receptor plays an important role in the generation (1) (11,12), the mechanism by which receptor ligands bind to the membrane-bound receptor was studied with these membrane preparations (13). We showed that there are two types of receptor binding sites, one for activators and one for inhibitors of neural excitability (12,14). Using a fluorescent lanthanide, terbium, to probe the calcium-and activatorbinding sites of the purified receptor prepared from Torpedo ocellata, we obtained additional evidence (15, 16) for different receptor binding sites for activators and inhibitors. In addition, we demonstrated a half-of-the-sites relationship between inhibitors and activators (13). On the basis of kinetic data, we developed a relatively simple two-state model for the membrane-bound receptor that takes into account both equilibrium and kinetic measurements of receptor-ligand interaction (14).Recently we investigated the kinetics of sodium-22 ion flux using the same membrane microsac preparation (17, 18). We found that sodium-22 efflux from 85% of the microsacs exhibited complex kinetics and was not affected by receptor ligands. Development of a kinetic method (17) allowed us to measure the efflux from the f...

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“…With receptor-rich membrane fragments, an overall equilibrium constant of K = 10-40 nM either with positive cooperativity (Weber & Changeux, 1974b;Damle et al, 1976;Schiebler et al, 1977;Eldefrawi et al, 1978;Neubig & Cohen, 1979;Fels et al, 1982) or without cooperativity (Raftery et al, 1975;Sugiyama & Changeux, 1975;Blanchard et al, 1982) has been reported. While some laboratories report 1 of these high-affinity AcCh binding sites per a-toxin site, others have found a ratio of 0.5 [see reviews by Changeux (1981) and Taylor et al (1983)l.…”

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confidence: 99%

Long-lived metastable state and hysteresis in the binding of acetylcholine to Torpedo californica acetylcholine receptor

Chang¹,

Bock²,

Neumann³

1984

Biochemistry

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Studies of the binding of [3H]acetylcholine to receptor-rich membranes of Torpedo californica electric organ under conditions that normally iead to a state of equilibrium did not give rise to equilibrium binding curves. Instead, the acetylcholine receptor was found to develop very long lived metastable states resulting in hysteresis in binding. Under conditions where the concentration of free [3H]acetylcholine is both less than 0.1 NM and smaller or comparable to the total receptor concentration, the degree of binding of acetylcholine depends on the rate, Le., the mode, of increasing the acetylcholine concentration (rapid mixing vs. dialysis). The equilibrium positive cooperativity in high-affinity acetylcholine binding previously inferred from the data is deceiving; the curvature in Scatchard representations is a consequence of long-lived nonequilibrium distributions between high-affinity and lower affinity receptor conformers. By manipulation of the experimental conditions, true equilibrium binding, resulting in a linear Scatchard binding curve, was obtained and yielded the apparent equilibrium constant, K = 5 f 1 nM at 4 OC.The stoichiometry of the high-affinity site associated with this K value was found to be one acetylcholine per receptor monomer ( M , 250 000) when carefully standardized [3H]-acetylcholine analyzed for both radiopurity and acetylcholine concentration was used. While our fresh membrane fragments prepared in the presence of 4 mM Ca2+ revealed up to twice as many '251-a-bungarotoxin sites in 0.1% nonionic detergent relative to those assayed in the absence of detergent, nonionic %e binding of acetylcholine (AcCh)' (A in equations and schemes) by the acetylcholine receptor (AcChR; R in equations and schemes) is a key reaction in the chemical control of the bioelectric excitation of cholinergic membranes [for monographs, see Nachmansohn (1959) and Katz (1 969)]. The nicotinic AcChR protein of fish electric organs, which regulates the rapid Na+-K+ fluxes through the electroplax membranes, exhibits major conformational variability consistent with its functional properties [for recent reviews, see Karlin (1980), Changeux (1 98 l), Adams (198 l), Conti-Tronconi & Raftery (1982) and Taylor et al. (1983)l. Electrophysiological data from frog neuromuscular junctions, which in their electrical-chemical behavior are similar to that of fsh electric organs, suggested that the AcChR channel coexists in at least two conformational states prior to any AcCh binding: an activatable resting state (RJ and an inactivated desensitized state (Rh) with a binding affinity for AcCh that is higher than that 0006-2960/84/0423-4546$01.50/0 detergent treatment of membrane fragments did not result in any change in total available acetylcholine binding sites. Therefore, we favor the interpretation either that the detergent loosens the membrane and exposes a second sterically hindered a-toxin binding site or that it increases its affinity. Our [3H]acetylcholine binding data with solubilized and purified acetylchol...

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Characterization of Torpedo californica Acetylcholine Receptor: Its Subunit Composition and Ligand-binding Properties

Cited by 135 publications

References 1 publication

Fast Kinetic Studies on the Interaction of Cholinergic Agonists with the Membrane‐Bound Acetylcholine Receptor from Torpedo marmorata as Revealed by Quinacrine Fluorescence

Fast Kinetic Studies on the Interaction of Cholinergic Agonists with the Membrane‐Bound Acetylcholine Receptor from Torpedo marmorata as Revealed by Quinacrine Fluorescence

Functional acetylcholine receptor--electroplax membrane microsacs (vesicles): purification and characterization.

Long-lived metastable state and hysteresis in the binding of acetylcholine to Torpedo californica acetylcholine receptor

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