Reconstituted BLM

Wang et al. reported a simple method to reconstitute membrane receptors into c-BLMs. After reconstitution, the receptor still retains its ligand activity. Furthermore, the relationship between receptor-ligand interactions and electrical properties of reconstituted BLMs... 

The effect of ATP and ATPase on the electrical properties of of reconstituted BLM. (A) Generation of membrane current upon additions of ATPase followed by ATP as a function of time. 

The mechanisms of the oscillations in biomembranes have been explained based on the gating of membrane protein called ion channel , and enormous efforts have been made to elucidate the gating process, mainly by reconstitution of channel proteins into bilayer membranes [34—36]. However, oscillations observed with artificial membranes such as thick liquid membranes, lipid-doped filter or BLM suggest that the oscillations... 

The highly complex and variable composition of natural cell membranes makes them a difficult subject for experimental studies. Artificial lipid membranes have consequently been prepared and studied for many years as models of cell membranes [1,3-7], A diverse array of geometries has been developed, including small and large unilamellar vesicles, giant lipid vesicles, lipid membranes supported on solid and polymer-coated substrates, and BLMs. These have been used to study the physical and chemical properties of lipids and lipid mixtures as well as membrane-associated proteins, including reconstituted transmembrane receptors. 

Recent studies that reported incorporation of pore-forming peptides and proteins into BLMs composed of poly(lipids) were discussed in Sect. 3. In both of these examples, a conformational change was not required for channel activity, and the bilayer was not completely polymerized. Reconstitution of TMPs into solid- and polymer-supported membranes composed solely of polymerized lipids has been reported by two groups in recent years. 

These so-called solventless BLMs [15-17] can also be formed on the end of fire-polished glass pipettes with a small tip area (tip diameter 1 fim) [19], The lipid solution in n-hexane is applied to the outside of the shaft of a pipette which is oriented vertically and penetrates the surface of the aqueous solution. The drop runs down the shaft and spreads at the air/water interface. A short exposure of the pipette tip to air and subsequent reimmersion through the lipid layer leads to the formation of a BLM at the tip of the pipette. This technique has been used to obtain a digital sensor, where off/on signals can be obtained when a single ion channel protein is reconstituted [20]. 

Charge generation, separation, and translocation are the main concern of electrochemistry. To relate these phenomena to membranes, the principal focus of research deals with the mechanisms of reactions through the membrane proper and at, as well as across, the solution-membrane interface. The electrical double layer at the interface plays a crucial role. The reconstituted planar bilayer lipid membrane (BLM) that separates two aqueous solutions was first reported in 1961. The BLM has proven to be an excellent model for biomembranes it permits charge separation and translo-... 

Similar to time-resolved spectrometry through which the transient molecular rection can be observed optically, the dynamics of modified or unmodified BLMs, or reconstituted bioreaction on a BLM, can be studied electrically by CV technology where the capacitance, resistance, membrane potential, and current peak are the fundamental parameters in determining the static or the dynamic change in the BLM system. Among these studies, the formation of a s-BLM is valuable in analyzing BLM mechanics and further electrochemical reactions thereon [34,35]. 

The interest aroused by the BLM has been mostly due to the fact that these systems form the basis for reconstitution of complex transport biomembrane systems, such as ionic channels, ATPases, the acetylcholine receptor, bacteriorhodopsins, etc. A first step in this direction was the discovery of a class of compounds that were found capable of radically affecting the electric... 

From the viewpoint of membrane electrochemistry, some of these membranes have been reconstituted and characterized elec-trochemicaUy via experimental BLMs (or planar lipid bilayer). Monographs and reviews covering many aspects of membrane research are available [1-28). 

In membrane reconstitution experiments, for example, the evidence is that intracellular signal transduction begins at membrane receptors. It should be reiterated that the research area covered in this paper is highly interdisciplinary. Emphasis has been placed on basic research. The BLM research has been benefited by a cross-fertilization of ideas among various branches of sciences. It seems likely that devices based on smart materials may... 

Purified receptor together with ATPase was reconstituted in BLM by Thompson et al. (25) for electrochemical experiments as outlined above. Figure 7.9 shows the electrochemical response of a BLM to fractions of receptor protein, ATP and naphthalene-1-acetic acid in various time sequences under an applied dc voltage of 15 mV. Only when all three components are present (in any order) is a substantial increase in current observed. Adjustment of hydronium ion concentration in solution resulted in a correlation of current at various pH values with the known receptor biochemical activity at the same values. This behaviour indicates that previous suggestions that an ATPase pump acts to translocate protons are indeed correct. From the sensor point of view, it is interesting to note that the signals exhibited a limit of detection of about 10 M NAA and were quite selective for the hormone. 

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