Cardiac gap junctions electrically couple adjacent myocardial cells to mediate action potential propagation. These ion channels are therefore intimately involved in normal condition as well as arrhythmias. Our long term goal is to determine the structure and dynamics of cardiac gap junction channels to understand the molecular basis for gating. We previously used electron image analysis of 2-d membrane crystals to show that the channels formed by a hexameric cluster of highly asymmetric M= 43kD subunits (axial ratio 4-5:1) termed alpha1 connexin or connexin 43. Protease cleavage and immunolabeling with 7 site-directed peptide antibodies defined the membrane topology: alpha1 connexin has a 5.1kD loop and a 13kD carboxy-tail accessible on the cytoplasmic membrane face, external to the transmembrane channel.
The aims of this proposal are as follows:
Aim 1 : Examine molecular structure and conformational dynamics. The external shape of the hexameric oligomer and internal contours of the ion channel will be defined by 3-dimensional image reconstruction of 2-d membrane crystals titled in the electron microscope. Cryo-electron microscopy and differential contrast provided by cationic and anionic negative stains will selectively probe the protein-water and proteins- lipid boundaries. Atomic force microscopy coupled with selective protease cleavage will be used to determine the orthogonal height of the cytoplasmic domains for the lipid bilayer. Labeling of 2-d crystals by Fab fragments directed against the C-terminus will assess whether the cytoplasmic domain has a well ordered conformation. CD spectroscopy coupled with selective protease cleavage will allow determination of the 2 structure in the cytoplasmic, extracellular and transmembrane domains of alpha connexin.
Aim 2 : Examine structural changes associated with channel gating. The methods developed in Aim 1 will be used to examine changes in the shape and 2 structure associated with pH and Ca++ dependent channel in the binding of peptide antibodies directed to specific cytoplasmic sites and variations in the accessibility of the cytoplasmic domains to protease cleavage will be used to map conformational changes during gating.
Aim 3 : Characterize the structure of expressed alpha, connexin. alpha connexin has been expressed in SF9 cells using a baculovirus vector, with the goal of obtaining milligram quantities of protein which will greatly expedite all of our studies. The experimental approaches outlined in Aims 1 and 2 will be applied to expressed material to examine whether the structural features and conformational changes of native alpha, be used to verify that expressed alpha1 connexin. For instance, site-specific peptide antibody labeling will be used to verify that expressed al connexin has the same topology as the native protein. Our long term goal is to pursue systematic crystallization studies with expressed protein to obtain 2-d and/or 3-d crystals suitable for high resolution structure analysis. The structural description of alpha1 connexin ion channels revealed by our studies will be fundamental for a complete understanding of the molecular basis of current flow in the heart an may provide clues for the rational design of strategies to treat arrhythmias.
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