Comparison of acetylcholine receptor-controlled cation flux in membrane vesicles from Torpedo californica and Electrophorus electricus : Chemical kinetic measurements in the millisecond region

Abstract

In earlier studies with the acetylcholine receptor (AcChoR) ofElectrophorus electric the rate and equilibrium constants for a model that relates the ligand binding to ion translocation were determined, and the dependence of these constants on the concentrations ofcarbamoylcholine and acetylcholine, over a 200-and 5000-fold range, respectively, could be predicted. AcChoR-controlled cation flux has now been measured in Torpedo californica vesicles by using a pulsed-quench-flow technique with a 2-msec time resolution. vesicle is the existence of a second, slower, inactivation process in T. californica with a rate coefficient, 6, of 0.12 sec'. The second process leads to undetectable flux activity during the time of observation (30 sec in 10 mM carbamoylcholine). These studies are also significant because fundamental differences may exist between the mechanism of AcChoR-controlled ion flux in synaptic (Torpedo) and conducting (E. electricus) membranes. Nachmansohn suggested (1) that the binding of acetylcholine (AcCho) to the acetylcholine receptor (AcChoR) in nerve and muscle cells induces a conformational change in the protein that results in the formation ofion-conducting channels through the membrane. The important relationship between the concentration of AcCho and the rates of AcChoR-controlled ion translocation has been investigated intensively because it determines the transfer ofinformation between cells in the nervous system. Kasai and Changeux (2) demonstrated AcChoR-controlled ion flux in vesicles prepared from Electrophorus electricus electroplax. Subsequently we developed methods for isolating and characterizing (3, 4) homogeneous vesicle preparations containing active receptors. An adaptation of rapid reaction techniques (5) allowed four phases of the process to be measured in the 2-msec to minute time region (2, 3, 6, 7). The effect of ligand concentration [5000-fold range of AcCho and 200-fold range of carbamoylcholine (CbmCho)] (2-4) on the four phases could be predicted over the entire concentration range for each ligand we investigated, and all the rate and equilibrium constants pertaining to a minimum model (Fig. LA) that relates the ligand-binding steps to ion translocation were determined (6-10).AcChoR-controlled ion translocation has been studied by using vesicles prepared from the electroplax of Torpedo (11-16). Torpedo vesicles may contain, on a weight basis, several hundred times more AcChoR than those from E. electricus, and the maximum influx rates had to be measured indirectly. Published reports differ on several important properties of the Torpedo AcChoR: maximal influx rates, the number of ions transported per AcChoR-formed channel per unit time, and the Hill coefficient (12, 16). Most strikingly, the rate of the CbmCho-induced inactivation of the AcChoR (17) appeared to be about 100 times slower than that observed with E. electricus (11,12). Here we mention some of the problems that have not been considered in previous studies using Torpedo vesicles and may account for the d...

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

“…After irreversible blockade of half of the ACh sites, the flux rate decreases, the plotted data are linear, and the apparent kd increases (6.7 s-i). Spare receptors in Torpedo vesicles have previously been cited as a problem in determination of inactivation rates (Hess et al, 1982;Heidmann et al, 1983). The strategy adopted by Hess et al (1982) was to use the agonist-induced inactivation process to reduce 86Rb+ flux rates to "measurable" levels.…”

“…In vitro studies of both ligand binding and rapid tracer ion flux techniques in AChR-rich membrane vesicles from electroplaque tissue of Torpedo species have provided estimates of slow and fast desensitization rates. Slow desensitization, which can be monitored in Torpedo AChR with manual ion flux assays (Walker et al, 1981;Walker et al, 1982) or binding experiments (Boyd and Cohen, 1980a;Neubig et al, 1982), has a maximum rate in the presence of ACh or carbamylcholine (CCh), of -0.1 s-1 (Boyd and Cohen, 1980a;Walker et al, 1981;Hess et al, 1982). Maximum rates for fast desensitization determined from rapid quenched-flow ion flux measurements range from 1.5 s-' in native Torpedo vesicles (Hess et al, 1982;Takeyasu et al, 1986) to 5.3 s-1 in reconstituted vesicles (Walker et al, 1982).…”

Acetylcholine receptor kinetics: Chemical kinetics

“…A rapid desensitization process was also observed in frog muscle cells using the single-channel current recording technique (Sakmann et al, 1980). Frog muscle and Torpedo californica receptors exhibit an additional desensitization process that occurs in the second time region (Sakmann et al, 1980;Walker et al, 1981;Feltz & Trautmann, 1982;Hess, Pasquale, Walker & McNamee, 1982), the same time region that is usually reported in electrophysiological measurements. A second slow desensitization process, which occurs in the hour time region, has recently been discovered in the E. electricus electroplax receptor (Aoshima, 1984).…”

Transmembrane flux and receptor desensitization measured with membrane vesicles. Homogeneity of vesicles investigated by computer simulation

“…1 supports the previous observations of the first order desensitization and ion flux processes Aoshima et al, 1981) and also of the slow first order ion flux process, due to receptor remaining after the rapid desensitization, with unfractionated (Hess et al, 1975) as well as purified (Hess and Andrews, 1977) vesicles. A fast phase of desensitization of cation flux mediated by acetylcholine receptor from Torpedo californica was also reported to follow first order kinetics (Walker et al, 1981;Hess et al, 1982). The lack of deviation from first order kinetics is consistent with a single receptor type and a vesicle population with a relatively narrow range of receptor concentration per internal volume [R'] consistent with the relatively narrow size dispersion.…”