This section of the Program Project will examine the distribution, synthesis, and regulation of gap junctions in the nervous system. The brain contains multiple gap junction proteins which are identical or highly homologous to the three connexins found in liver and heart. Specific antibodies and cDNA probes to the liver and heart proteins will be used to characterize the biogenesis of gap junctions in neural tissues in vitro and in vivo, and to determine whether there are unique brain-specific gap junction transcripts or proteins. Regulation of gap junction biosynthesis and of intercellular communication will be examined in parallel in neural tissues to help determine how channel function and metabolism are coordinated. Specifically we plan to: 1. Determine which gap junction genes are expressed in the brain and define the cellular and anatomic distribution of each connexin. 2. Examine the regulation of gap junction metabolism and function in neuronal, glial,and leptomeningeal cultures. 3. Examine the cellular distribution, synthesis, and turnover of connexins in cultured neurons and glia. Metabolic labeling of connexins with radioactive amino acids and temporal analysis of the appearance of the proteins will help define rates of synthesis and turnover, and the extent to which they are modifiable. Examination of phosphorylation of the proteins and correlation with changes in coupling of cells will help define how gap junctions in the brain are regulated. 4. Define factors which influence coupling between cultured cells. The second messenger systems which mediate these regulatory effects will be examined in detail. 5. Define effects on gap junction mRNA and protein of those factors which are found to influence coupling between cells. These studies will help determine how transcriptional, translational, and posttranslational mechanisms regulate gap junctional communication. 6. Characterize biophysical properties of neuronal, glial, and leptomeningeal gap junctions at macroscopic and single channel levels. The overall goals of this phase of the Program Project are to define the role of gap junctions in brain function and to elucidate mechanisms by which altered intercellular communication may contribute to disorders of the nervous system including epilepsy, post-ischemic and post-traumatic degeneration, and developmental abnormalities of the brain.
