Gap functions channels facilitate the intercellular exchange for ions and small molecules. This intracellular communication play a significant role in embryonic and neural development. Connexin45(Cx45) is a subunit gap junction protein which is expressed in early embryos and in a number of cells of the mature brain and other organs. Cx45 forms channels with unique permeability and regulatory properties as compared to other connexins. Three groups of studies are proposed are proposed to examined Cx45 and its role in the developing nervous system.
Aim 1. Cx45 function. Since Cx45 is often co-expressed in the same cell with connexins, it may function as a """"""""regulatory"""""""" connexin by mixing with other connexins to form heteromeric hemichannels with altered channel properties (such as reduced molecular permeability). We will co-express Cx45 with Cx43 or Cx32. We will examine the transfer of microinjected dyes, propagation of intercellular calcium waves, and electrical coupling. We will examine the formation of mixed hexamers biochemically after cross- linking and sucrose gradient centrifugation.
Aim 2. Cx45 gene regulation. Genomic promoter elements and 5'-untranslated exons may contribute to the developmental and cell-type specific expression of Cx45. We will determine the structure and sequence of the chicken and mouse Cx45 genes. RNA blotting, nuclease protection, and RT- PCR will be used to determine possible differential transcription of mRNA splicing. Transfections with Cx45 gene/luciferase reporter constructs will be used to define the minimal promoter(s) for Cx45 expression and the differential expression of Cx45 mRNAs as opposed to other connexins.
Aim 3. Cx45 in development and differentiation. We will characterize the developmental time course of Cx45 expression and incorporation into gap junction plaques, its regional distribution in the early embryo, and its cellular distribution in the developing nervous and cell-type specific cultures by RNA- and immuno-blotting and immunocytochemistry. We will generate embryonic stem cells and animals that are Cx45-deficient and in which Cx45 is replaced with Cx43 to examine the consequences of Cx45- deficiency or of its replacement with another connexin with different regulatory and permeability properties.
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