Ultrastructure of Patch Clamped Membranes
Frederick Sachs Principal Investigator
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The patch clamp has revolutionized the study of electrophysiology, but little is currently known about the structure of patches. Knowledge of structure is important for proper interpretation of single channel data, especially data from mechanosensitive ion channels, where the cytoskeleton plays a major role in activating the channel. By combining electrophysiology, light and electron microscopy of patches, general techniques for understanding patch structure will be developed. These techniques will aid in determining the structural alterations that occur in the preparation of a patch for electron microscopy and the structure of the interface between the membrane and pipette. Images of "living" patches will be made using optical microscopy with high magnification video. To observe the ultrastructure, a device will be built to rapidly freeze the patch after light microscopic examination. After freeze drying, patches will be examined as whole mounts in the high voltage electron microscope. Comparison of patch images will be made with light and electron microscopy, using two dimensional projection images, stereo pairs, and complete three dimensional image sets. Techniques for freeze fracturing, etching and replicating fragments of pipette tips will be developed to study the interaction that occurs between the membrane and the wall of the pipette. Examination of several types of cells will aid in determining which structures are general and which structures represent tissue-specific differences between patches. Since the patch clamp and the electron microscope have the potential for describing single molecules, cross correlating results from both techniques will lead to a new level of structure-function relationships.