PHYSIOLOGY OF RECONSTITUTED CONNEXIN CHANNELS

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The gap junction channel forms a dynamically regulated aqueous pathway that mediates direct transfer of ions and small molecules between cells. The channel is composed of two integral membrane structures (each in one plasma membrane) formed of connexin protein. The intercellular communication mediated by junctional channels is considered crucial for normal development and mature function of many tissues. The channels are permeable to all known cytoplasmic second messengers. Therefore, information about how the channel functions, how it is gated, what goes through it, and how these properties can be modulated is of medical, biological and biophysical importance. The gap junction channel is difficult to study in situ because it is inaccessible for the experimental techniques and manipulations commonly applied to other channels - both ends of the channel are inside of cells. Access to the pore is via cytoplasm, so it is difficult to distinguish factors that act directly on the channel from those that act on it via intermediate cellular components. The long-term objective of this proposal is to understand the physiology and bio;physics of the gap junction channel. the approach is to incorporate the channel-forming structure into single phospholipid membranes where its properties can be studied. This proposal is to study the ion channels formed by connexin32, which forms gap junction channels between cells in vivo. Specifically, it is proposed to (1) describe and explore the permeation, gating and modulation of connexin32 channels in single phospholipid bilayers, (2) explore the effects of phosphorylation by protein kinases on the properties of connexin32 channels, and (3) explore the functional roles of specific domains of the connexin molecules. By studying the physiology of connexin channels in an experimentally accessible system, one hopes to understand the regulation of the protein that mediates junctional communication. Coupling by way of gap junction is so widespread that elucidation of this process will undoubtedly have profound effects in many areas of cellular and developmental biology.