Cardiac gap junctions electrically couple adjacent myocardial cells to mediate action potential propagation. These membrane channels are therefore intimately involved in normal conduction as well as arrhythmias. In the last funding cycle, electron cryo-microscopy and image analysis of two-dimension (2D) crystals enabled us for the first time to directly visualize transmembrane alpha-helices in a recombinant gap junction membrane channel. We now plan to pursue the three- dimensional (3D) structure analysis of the recombinant gap junction channels in the open and closed states, as well as pursue higher resolution crystallization trials: Aim 1: Determine the 3D structure of recombinant gap junction channels: A 3D map derived by image analysis of tilted 2D crystals should reveal important details about the external shape of the channel, internal contours that define the aqueous pathway, folding of the extracellular domains, as well as the secondary structure of the transmembrane domains. We plan to assign the transmembrane domains using undecagold reagents covalently attached to specific cysteine residues. Aim 2: Examine structural changes associated with channel gating. We recently discovered that oleamide, a sleep inducing lipid, blocked dye transfer between coupled BHK cells. A projection map at 7A resolution of oleamide-treated gap junction 2D crystals contained significant shifts in density compared with the untreated crystals. 3D electron cryo-crystallography will now be applied as in Aim 1 to examine changes in the shape and secondary structure associated with oleamide- induced channel gating. pH induced gating of gap junction channels involves the cytoplasmic C-tail. Hence, we plan to also examine the structure of the full length connexin. These crystals will be particularly difficult to examine in 3D since they will be approximately 250A thick. Therefore, we also plan to separately pursue expression, purification and structural characterization of the C-tail. Aim 3: Perform model building using constraints provided by the connexin amino acid sequences and the 3D density map. The resolution of our crystals may not allow us to visualize amino acid side chains or even the connectivity between transmembrane domains. To place constraints on possible folding models, we plan to scrutinize connexin amino acid sequences to extract the conserved and divergent features. The goal is to generate chemically reasonable atomic models for the gap junction channel by combining features of the amino acid sequences and the geometric constraints provided by 3D maps. Aim 4: Characterize the structure of expressed and reconstituted gap junctions. The expression levels of alpha1 connexin in BHK cells is not high enough to feasibly attempt purification and reconstitution into lipid bilayers. However, we have successfully expressed alpha1 connexin to higher levels in SF9 cells using a baculovirus vector. The total is to solubilize and purify milligram quantities of protein with the intent of growing high resolution 2D crystals by reconstitution into lipid bilayers. The structural description of gap junction membrane channels revealed by our studies will be fundamental for understanding the molecular basis of current flow in the heart and may provide clues for the rational design of strategies to treat arrhythmias.