Creatine and its phosphorylated derivative creatine phosphate (PC) play an important role in the maintenance of intracellular adenosine triphosphate (ATP). Creatine is selectively accumulated from the extracellular space through the action of a sodium-coupled high-affinity creatine transporter (CREAT) located in the plasma membranes of highly metabolic cells in brain, muscle, and other peripheral tissues. After accumulation into cells, creatine is converted to phosphocreatine by creatine kinase. Phosphocreatine serves as a source of high-energy phosphates required for ATP homeostasis. Recent cloning studies reveal that CREAT is structurally related to the norepinephrine/gamma-aminobutyric acid transporter gene family. Sites of accumulation of creatine are poorly defined and the localization, developmental expression, and regulation of CREAT remains undetermined. This proposal outlines studies directed towards the regional and cellular localization of the sodium-dependent creatine transporter gene product in mammalian brain. The anatomical and cellular distribution of the creatine transporter will be determined by tissue immunohistochemical analysis employing novel fusion protein-specific polyclonal antisera. Subsequently, regional and cellular localization of CREAT mRNAs in rat brain will be determined through in situ hybridization studies employing di xigenin- and isotope-labeled riboprobes derived from recently cloned CREAT cDNA templates. Combination of the proposed immunohistochemical and in situ localization studies will provide a detailed characterization of CREAT gene expression in the mammalian brain. Moreover, completion of the proposed studies will provide the applicant with extensive training in molecular neurobiology to be applied in future studies of brain energy metabolism upon return to the Republic of Georgia.
