Molecular and Cellular Structure of Gap Junctions

University Of California-San Diego, La Jolla CA

Investigators

Abstract

The gated connexin channels found in gap junctions regulate communication between tissue cells. These membrane channels are critical for integrating and regulating basic cell processes such as metabolic cooperation, ionic transmission, differentiation and hormonal regulation. Breakdown in gap junctional communication can lead to developmental defects, abnormal cell proliferation and failure of tissue regulation as well as physical or developmental diseases. The channels consist of a dimer of two hexamers each called a connexon, of the constituent protein, connexin. These channels are unique in that when the two hexamers pair up, they self-assemble into distinct cellular junctional areas that are morphologically distinct. The connexins are a family of related proteins sharing a common folding motif. Different connexins are found in the same tissue and different connexins can be localized to the same gap junctional plaques. In the current structural model of the protein, the C and N termini are located on the cytoplasmic side of the membrane and cross the membrane four times in a helical segments. Mutagenesis studies have indicated that the extracellular domains may contain b sheet structures. The overall goal of this research is to image at the electron microscopic level the molecular structure of gap junction proteins as well as their arrangements within and between cells. This project is divided into two parts. Both address organizational principles that determine not only the molecular structure of connexins but also how these connexins are organized and interact with other cellular components. This is, in essence, the cellular structure of connexins. The goals of this project are: (1) To significantly improve the resolution of the present 3d molecular structure of the Cx26 channel: Connexin26 (Cx26) is the smallest of the gap junction family. This connexin isoform has the potential for molecular structure determination since it has been hypothesized that the smaller the carboxy terminus, the tighter the packing and less disorder within the connexon unit. Using the large gap junction two-dimensional crystals obtainable from these HeLa Cx26 transfectants, computational methods will be developed and applied to increase the resolution of the reconstructions. An approach using single particle/correlation averaging in combination with a crystallographic analysis will be used in order to circumvent the lattice disorder inherent in these crystals. Development and application of this methodology will be useful not only for Cx26 crystals but also for application to other connexin crystals. (2) To continue to specifically label gap junction proteins in situ using photooxidation of either genetically tagged connexins or specific antibodies and then determine their 3D spatial arrangements and interactions with other cellular components using fluorescence photooxidation and electron tomography. The potential impact of this project is to advance the present knowledge of connexin channel structure, cellular organization, assembly and disassembly. Results from these experiments would impact the interpretation of other channel structures and understanding of how membrane proteins traffic. These results should also help to understand the molecular basis of connexin-related diseases.