Studies on the structure and molecular diversity of the gap junction

Citation

Hoh, Jan Hakan (1991) Studies on the structure and molecular diversity of the gap junction. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/8n7d-2t98. https://resolver.caltech.edu/CaltechETD:etd-06212007-075739

Abstract

An improved method for the isolation of hepatic gap junctions that substantially shortens preparation time and improves the yield of previous methods is described. The topology of the 28 kD protein component (connexin-32, Cx32) of gap junctions isolated with this method is examined using proteases and antibodies against specific peptides. These experiments are consistent with the current model for the organization of the protein in the membrane, but reveal that an unexpectedly large part of the carboxy-terminus is protected from proteolytic attack. Together with data from comparisons of the Cx32 protein sequence with other channel proteins, a modified topological model is proposed. The structure of the gap junction is further studied by atomic force microscopy. Using this new technology, high resolution images of a gap junction in phosphate buffered saline are obtained, and after "force dissection," which removes half the plaque, the extracellular domains of individual connexons in a hexagonal array with lattice constant of 9.1 nm are revealed. These are the first images of an ion channel by atomic force microscopy, and the observations open the door for a variety of new experiments not previously possible. Low stringency screening of a rat genomic library produced genomic clones for Cx32 and a new member of the gene family, connexin-31 (Cx31) or [beta]3. Cx31 has a unique distribution and is found in the eye, Harderian gland, skin, and placenta. Comparison of the Cx31 with the other known connexins, reveals unique and conserved domains in the protein sequences. This comparison is extended to a phylogenetic analysis of the entire gene family that shows two major branches of connexins that diverged 1.3-1.9 billion years ago. Comparison with other ion channels reveals a short sequence similarity between the connexins and channels such as the voltage activated K+ channel. In K+ channels the sequence has been shown to line the aqueous pore, and the model for connexin organization is modified to account for this possibility. The similarity also suggests that gap junctions are part of a superfamily of ion channels.

Thesis Files