9318259 Bookman Support from the NSF is requested for the development of new instrumentation to measure rapid changes in the capacitance of biological membranes with high resolution. These measurements of changes in capacitance will permit the investigation of a number of biologically important phenomena. The principal use of this instrument is likely to be the detection of increases in cell surface area arising from the fusion of one or more secretory vesicles with the cell's plasma membrane during the process of exocytosis. The study of secretory vesicle fusion events and the quantitative characterization of the kinetics of secretion made possible by this instrument can improve our understanding of a fundamental process in cell biology. An additional component of membrane capacitance arises from the mobility of charged groups on proteins lying within the electric field of the membrane. Recent results show that the detection of such charge movements from voltage-dependent ion channels as a capacitance signal can provide new information about ion channel gating and its modification by toxins. Thus it is expected that the proposed instrument can help to establish new methods for studying conformational changes of membrane proteins. The project consists of the development of software to implement a stimulus synthesizer, the development of improved and user-tunable algorithms for measuring membrane capacitance, their implementation in software, development of new analysis and data base tools for the data produced by this instrument, and documentation of the instrument for both users and programmers. The fundamental assumptions behind this instrument include: 1) that the cell under study can be represented by an equivalent electrical circuit, and 2) that a cell under voltage clamp can be driven by a band-limited, arbitrary-frequency voltage stimulus and the resulting current can be faithfully sampled with high resolution. Based on these assumptions, the inst rument will be able to combine traditional transient membrane depolarization with more spectrally complicated waveforms in order to study processes activated by the cell's electrical activity. This proposed instrument will attempt to resolve changes in membrane capacitance that are on the order of 10-1s Farads and to resolve such changes occurring in a 1kHz bandwidth. This instrument is an example of a new class of instrumentation made possible by developments in three areas of technology: 1) high speed general purpose microprocessors, 2) fast, high resolution A/D and D/A converters, and 3) special purpose digital signal processing algorithms. A key feature of this project is the reliance upon hardware components that already exist in most laboratories, i.e., a personal computer and a data acquisition interface. With the increasing power of the scientist's data acquisition computer, new types of measurements become possible solely by the addition of software. With its implementation of appropriate algorithms, the proposed instrument will provide information that is unattainable with any commercial instrument and make possible new types of experiments to study membrane processes. A particular advantage is that the proposed instrument can be widely and rapidly disseminated to the research community electronically (i.e., through Internet)) at no additional capital cost to the nation's laboratories.
