News and Publications

Communication Between Cells Via Gap Junctions

N.B. Gilula, N.M. Kumar, N. Unwin, D. Chow, M. Falk, X. Gong, X. Guan

The gap junction contains channels for the transmission of information, in the form of small molecules, from cell to cell. These junctions are responsible for synchronizing the activities of cells in multicellular tissues. We are studying several problems related to the gap junction channel. These include its structure, the genetic control of its expression, and the function of cell-cell communication during development and differentiation.

STRUCTURE-FUNCTION RELATIONSHIPS OF THE GAP JUNCTION CHANNEL

Progress in determining the 3-dimensional structure of the gap junction channel has been facilitated by using recombinant gap junction channels in BHK cells and by the development of methods for isolating the gap junction plaques as crystals that are then analyzed with cryo-electron microscopy. In collaboration with V. Unger and M. Yeager, Department of Cell Biology, a projection analysis of the gap junction channel has been achieved at 7 Å. This study provided the first definition of the organization of the connexin protein into a connexon at this level of resolution. An extension of this analysis with tilt images yielded a 3-dimensional reconstruction of this projection map at 7 Å. The structure analysis is now being completed on an uncoupled channel (oleamide treated) that can provide some insight into the mechanism that controls the permeability of the channel.

We are also exploiting the ability to prepare and isolate gap junction connexons in milligram quantities to generate 3-dimensional crystals for x-ray diffraction analysis in collaboration with I. Wilson, Department of Molecular Biology. We have isolated milligram quantities of the purified connexons containing either ß₁- or ß₂-connexin proteins. Concentrated preparations of the connexons are being used to generate 3-dimensional crystals of the material. In addition, monovalent Fab fragments have been prepared for antigenic determinants on the ß₁ and ß₂ proteins, and these Fab fragments are being used for cocrystallization with the isolated connexons.

During the past year, we defined the principles involved in the assembly of connexin proteins into the oligomeric unit of structure and function, the connexon. Using both in vitro (cell-free translation) and intact cellular systems with baculovirus infection, we found that gap junction proteins are assembled into oligomers with a connexin selectivity based on connexin classes. For example, hetero-oligomers are formed quite readily both in vitro and in vivo between different connexins of the same class. ß-Connexins can form hetero-oligomers with other ß-connexins but not with -connexins, and vice versa. Thus, the ability to form hetero-oligomers can provide a basis for generating gap junction channels with diverse physiologic properties. At the same time, the selectivity of connexins from the same class for hetero-oligomeric formation may restrict or specify the type of association that can result in an oligomeric assembly.

The structure-function analysis of gap junction channels has been facilitated by the discovery of a class of natural products, fatty acid amides, that effectively and reversibly block the conductance of the channels. One of the molecules of this class, oleamide, induces sleep in animals and humans. We applied oleamide to cells in culture and found that it effectively blocks the conductance of gap junction channels reversibly, and in cells that form channels with different connexins. In addition, oleamide blocks channel activity in glial cells without any detectable effect on the cell-to-cell transmission of the "calcium wave." We used this compound to obtain an embryonic arrest of Caenorhabditis elegans. Consequently, strategies involving this approach are being applied to isolate gap junction mutants.

GAP JUNCTIONAL COMMUNICATION DURING DEVELOPMENT

During the past year, most of our efforts on the contribution of gap junctional communication in development and differentiation have focused on the contribution of gap junction genes during embryogenesis and organogenesis in mice. We found that the targeted disruption of the gene for the connexin ₃, which has restricted use in mice, preferentially in the lens, causes the development of cataracts. The cataracts that develop at an early stage in these mice are strikingly similar to the age-dependent cataracts that develop in humans. The cataracts in mice form in the presence of the gene for another connexin, ₈, which is still used by the lens fiber cells. This ₃-targeted disruption provides an important animal model that can be used to understand the mechanism for generating age-dependent cataracts in humans and, ultimately, should make it possible to determine the specific molecular signal that is normally transmitted through gap junctional communication to retain normal lens transparency.

PUBLICATIONS

Boger, D.L., Patterson, J.E., Guan, X., Cravatt, B.F., Lerner, R.A., Gilula, N.B. Chemical requirements for inhibition of gap junction communication by the biologically active lipid oleamide. Proc. Natl. Acad. Sci. U.S.A. 95:4810, 1998.

Falk, M.M., Gilula, N.B. Connexin membrane protein biosynthesis is influenced by polypeptide positioning within the translocon and signal peptidase access. J. Biol. Chem. 273:7856, 1998.

Gong, X., Le, E., Klier, F.G., Huang, Q., Wu, Y., Lei, H., Kumar, N.M., Horwitz, J., Gilula, N.B. Disruption of ₃ connexin gene leads to proteolysis and cataractogenesis in mice. Cell 91:833, 1997.

Guan, X., Cravatt, B.F., Ehring, G.R., Hall, J.E., Boger, D.L., Lerner, R.A., Gilula, N.B. The sleep-inducing lipid oleamide deconvolutes gap junction communication and calcium wave transmission in glial cells. J. Cell Biol. 139:1785, 1997.

Risek, B., Pozzi, A., Gilula, N.B. Modulation of gap junction expression during transient hyperplasia of rat epidermis. J. Cell Sci. 111:1395, 1998.

Thomas, E.A., Cravatt, B.F., Danielson, P.E., Gilula, N.B., Sutcliffe, J.G. Fatty acid amide hydrolase, the degradative enzyme for anandamide and oleamide, has selective distribution in neurons within the rat central nervous system. J. Neurosci. Res. 50:1047, 1997.