Excitable acetylcholine receptor rich membrane fragments from Torpedo marmorata have been used to measure, in parallel, (1) the permeability response to the fluorescent cholinergic agonist Dns-C,-Cho (in the 0.1 pM to' millimolar concentration range) characterized by both the initial rate of Li+ transport and the rate of channel closure using the rapid-mixing quench-flow technique and (2) the kinetics of interaction of Dns-C,-Cho with the acetylcholine receptor sites using the rapid-mixing stopped-flow technique. Analysis of the kinetics of Dns-C6-Cho binding in the millisecond to minute time scale leads to the identification of at least three conformational states of the acetylcholine receptor: a "low-affinity" one (-50 pM) that can be interconverted in A t the neuromuscular junction and at the electromotor synapse, the neurotransmitter acetylcholine (AcCh)' elicits two distinct classes of reactions. Applied as a brief (millisecond) pulse of high concentration (fraction of millimolar), AcCh causes the rapid opening-or "activation"-of transmembrane ion channels selective for cations (Katz, 1966). When maintained for a longer period of time (fraction of a second or minutes), in contact with the postsynaptic membrane, and at a sufficient concentration (approximately micromolar), AcCh initiates a complex time-dependent decrease of the permeability response-or "desensitization". Both classes of reactions have been kinetically analyzed by various electrophysiological techniques [reviews in Adams (1 98 l), Feltz &Trautmann (1982), andNeher et al. (1983)l. The opening of the ion channel by AcCh takes place in the fraction of millisecond to millisecond time scale and in an "all-or-none" manner at the molecular level (Katz & Miledi, 1970; Munchen, FRG (J.B. and E.N.). Received April 7,1983. This work was supported by grants from the Muscular Dystrophy Association of America, the Fondation de France, the CollEge de France, the MinistZre de la Recherche et de l'hdustrie, the Centre National de la Recherche Scientifique, the Institut National de la Santi et de la Recherche MBdicale, and the Commissariat i I'Energie Atomique. We gratefully acknowledge an EMBO short-term stipend (to J.B.) and the support of the Deutsche Forschungsgemeinschaft (Grant 227 to E.N.).

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A detailed kinetic analysis is presented for activator-receptor-mediated efflux of tracer substances from vesicular membrane systems in general and from sealed fragments of excitable membranes in particular. Rate constants and amplitudes, as the primary measurable quantities of the efflux kinetics, are expressed in terms of fundamental properties of vesicular membrane systems containing receptors of chemical gating systems. The expernmental determination and theoretical analysis of single contributions to a complex receptor-controlled efflux has been treated for the acetylcholine receptor system; also the effect of "pharmacological densensitization" on efflux is explicitly formulated. The dependence of the measured efflux parameters on the concentration of activators can be used to derive the kinetic and thermodynamic constants for receptor activation and inactivation processes; a general kinetic scheme and two limiting cases are analyzed. The efflux of UlNa from "excitable microsacs" of Toredo marmorata is treated as an example, and the power of the rigorous analytical method is demonstrated. In particular, the analysis of efflux amplitudes from only a few data points offers an alternative to the longer lasting measurements for obtaining efflux curves when a safety factor is involved, as in the case of tracer ions like 22Na. Many readily prepared from fish electric organs. A filtration assay method may be used to determine the kinetics of tracer ion efflux from microsacs loaded with tracer species-(2-5). The utility of the method lies in the fact that ligand-induced receptor activation leads to an opening of transmembrane channels, which promotes tracer efflux. Receptor activator binding is thus explicitly related to efflux behavior. The measured curves represent cpm due to tracer in microsacs plotted against time, as a function of activator concentration.Analysis of these curves is complicated by the presence of several superimposed efflux processes, of which only one is of interest (4). Furthermore, with T. marmorata membrane fragments, the influence of ligand-induced receptor inactivation, frequently referred to as "desensitization" (6, 7), has to be contended with. Changeux and coworkers have used a phenomenological excitability parameter, defined in terms of representative decay times, to analyze the ligand concentration dependence of their data. They obtained dose-response constants representing apparent dissociation constants for the ligand-receptor complex. Using a modified experimental procedure, Hess and coworkers (4) were able to obtain single exponential decay curves for efflux. Ligand dissociation constants were determined by fitting the rate constants for efflux to an expression involving the fraction of occupied receptors as a function of ligand concentration. For microsacs from electric eel tissue, Hess et al. cited no evidence that receptor inactivation influences efflux.In the following, a general kinetic analysis of tracer efflux will be presented. It will be shown that such divers...

We have performed calculations of the glycine zwitterion surrounded by water molecules with the help of the mutually consistent field (MCF) method and perturbation theoretical expressions. Two different models for the hydration shell have been chosen, the glycine.6HzO and glycine. 12H20 complexes, representing the most probable first and second solvation shell, respectively. To calculate the exchange and charge transfer energy contributions we have applied approximative expressions derived from perturbation theory for weakly overlapping subunits. For the sake of comparison we also calculated the interaction energy in the supermolecule approach for the smaller of the two solvation complexes. Furthermore, we have investigated the part of the potential energy surface which is determined by varying the lengths of the hydrogen bonds between glycine and water in the complex glycine. 12H20 using the electrostatic approach. The exchange energy contribution to the interaction energy for different points on the surface was approximated with the help of an analytical expression fitted to three directly calculated points. For the charge transfer energy a polynomial expansion of second order was established on the basis of five values, computed with the aid of the perturbation theoretical expression. To get a more detailed insight in the relatively strong hydrogen bonds between the water molecules and the ionic hydrophilic parts of glycine ab initio model studies on NH2.3H20 and HCOO-.3H20 systems are reported.

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