Julius Bernhardt

Analysis of gated flux from or into sealed membrane fragments.

Physico-chemical factors that determine tracer substance flux from or into sealed vesicular structures are examined. Flux amplitudes are dependent on the average volume of a vesicle, while flux rates depend on the average number of transmembrane channels per vesicle. Gating processes leading to channel opening and/or closing affect both amplitudes and rates. Averaging over inhomogeneities in vesicle size and channel density leads to an explicit expression for time-dependent tracer content. Means for experimentally determining all variable factors in this expression are discussed.

Single channel gating events in tracer flux experiments: III. Acetylcholine receptor-controlled Li+ efflux from sealed torpedo marmorata membrane fragments

Filter assay measurements of Li+ efflux from acetylcholine receptor-containing vesicular Torpedo marmorata membrane fragments (microsacs) are presented. Techniques are introduced for: (a) inducing a complete emptying of the Li+ content of all microsacs containing one or more functionally intact receptors, and (b) for determining the distribution of internal volumes of the microsacs using filtration with membrane filters of different pore sizes. The flux amplitudes resulting for acetylcholine receptor-controlled Li+ efflux, when receptors are inhibited by alpha-bungarotoxin or inactivated by a neuroactivator-induced desensitization process, were measured. Amplitude analysis was used to determine characteristic parameters of the microsacs that may vary with the technique of preparation (e.g., the distribution in size and receptor content), as well as the mean single channel flux amplitude contribution (e-kt)infinity, which represents the mean reduction of the Li+ content of a microsac due to efflux from a single receptor-controlled channel closing due to inhibition or inactivation of the receptor. The ratio keff/ki was found to lie in the range 0.1 less than keff/ki less than 0.5, where keff and ki are, respectively, the rate constant for Li+-Na+ exchange flux and for the slow inactivation reaction mode of the acetylcholine receptor induced by carbamoylcholine at high concentrations.

Preparation of sealed Torpedo marmorata membrane fragments suitable for quantitative tracer flux studies

Sealed vesicular membrane fragments (microsacs) prepared from Torpedo electric organs have been used extensively in tracer flux experiments. Microsac suspensions with high AcChR and low AcChE content are obtained by density gradient separation of crude electric organ homogenates [I-S]. Special methods selectively yielding gradient fractions containing microsacs with functionally intact AcChR have been developed [6,7]. However, recent advances in the techniques [8-lo] and in the mathematical analysis [ 11,121 of flux measurements, raise the need for more suitable methods of preparation. selective neuroactivator-induced filling (AcCh pulses) of functional microsacs with Li’; (iii) Separation of Li’-filled and Cs’-filled microsacs using a continuous self-generating Percoll gradient; (iv) Extensive characterization of the resulting fractions on the basis of stringent criteria.

Physical factors determining gated flux from or into sealed membrane fragments.

Publisher Summary This chapter discusses the physical factors determining gated flux from or into sealed membrane fragments. The introduction of integrated flux rate coefficients into the analysis of flux measurements provides a rigorous tool for the study of gating mechanisms. The recent application of this method to the acetylcholine system has revealed that the receptor in isolated membrane fragments of Torpedo marmorata a priori exists in two conformations: an activatable structure leading to ion flow upon activator binding and an inactivated, desensitized conformation. The functionally relevant, ion-conducting structure is a transient, metastable state; in the presence of activator, the inactivated structure is the most stable state. The forward rate constant for inactivation is much larger than the backward rate constant. Inactivation occurs not only via the transient, short-lived conducting conformation but also (to about 20%) via the direct binding of activator molecules to the inactivated structure. In the study of the physical factors determining gated flux from or into sealed membrane fragments, it is found that that microsacs with different numbers of open channels having different flux rates requires the development of a detailed formalism in terms of transient tracer ion binding and specific gating processes. It turns out that the distribution of microsacs as well as that of open channels per microsac are important factors in a rigorous analysis of flux data.

Single channel gating events in tracer flux experiments: I. Theory

Tracer ion flux measurements are a commonly used method for studying ion transport through membranes of cellular systems, where the rate of ion flow is determined by gating processes which control the opening and closing of transmembrane channels. Due to recent advances in the theoretical analysis of tracer flux from or into closed membrane structures (CMS), the mechanism of gating reactions can, in principle, be derived from flux data. A physically well founded analysis is presented for the dependence of the total tracer ion content of a collection of CMS on the gating processes. For functionally uncoupled gating units a mean single channel flux contribution [equation, see text] can be defined, where k is the intrinsic single channel flux coefficient, t the time over which flux is measured, and p(tau,t) is the probability that a given channel was open for a total period tau during t. This quantity reflects the mean time course of the tracer content due to flux through a single channel. Expressions for are derived that explicitly take into account a distribution in the lifetime of open channels. On the basis of the results, kinetic and thermodynamic parameters of multiphasic gating reactions can be determined from the time course of the overall tracer content in a colleciion of CMS.

Chemical Control of Membrane Transport by the Acetylcholine Receptor System

The function of the acetylcholine receptor system in excitable membranes of nerve and muscle cells is to control the passive transport of Na+ and K+ ions, which are common carriers of bioelectric signals. Details of this gating function can only be studied by the investigation of the transport properties of membrane-bound receptors. A powerful technique at a less complex level than whole cells is the measurement of metal ion exchange fluxes induced by activator molecules in sealed, receptor-rich membrane fragments (1). The aim of these experiments is to derive from flux parameters information on the permeability control system. Recently, the basic elements of a rigorous physical chemical analysis of complex flux curves have been discussed in terms of explicit relations to receptor-specific rate constants (2,3). In particular, the ligand-dependent reduction (inactivation) of the flux (analogous to pharmacological desensitization) has been analyzed in terms of a specific reaction scheme, excluding alternatives. The results are principally the same as the data from electrophysiological studies on muscle and electroplax: the activated, ion-conducting conformation of the acetylcholine receptor is a transient, short-lived metastable state; comparable to the conducting state of the Na+ ion gating system in axonal excitable membranes (3). A variety of experimental correlations to the electrical properties of whole cells shows that molecular functional details of receptor-mediated transport can be studied with sealed membrane fragments.