The long-term goal of this research is to better understand the role of glutamate transport in the physiology and pathophysiology of excitatory synapses. Although glutamate transport has been assumed to be largely a function of astrocytes, evidence from a variety of approaches suggests that neuronal glutamate uptake mechanisms are important as well, both for the physiology of excitatory transmission as well as for the pathophysiology of excitotoxicity. We studied glutamate transport in rat forebrain neurons in culture, and found that the pharmacology of glutamate transport was distinct from that of the neuronal transporter EAAC1, and resembled that of GLT1, known as an astrocyte transporter. We cloned glutamate transporters form a cDNA library made from these cultures. We found, in addition to EAAC1 and GLT1, a variant form of GLT1 that differs at the C-terminal 11 amino acids from the originally cloned GLT1. We now call the originally form """"""""GLT1a,"""""""" and the variant form cloned from the neuronal library """"""""GLT1b."""""""" Preliminary LM and EM immunocytochemical studies have shown that GLT1b is localized to neurons as well as astrocytes in the forebrain. In addition, GLT1b, but not GLT1a, has a PDZ-domain interaction consensus sequence at its C-terminal. The hypothesis motivating this project is that GLT1b is an important neuronal transporter, and that this variant form has interactions through its C-terminal with other proteins that are functionally important and not available to GLT1a.
The specific aims of this project are to: 1. Compare the expression of GLT1a & b in neuronal cultures and in the brain. The hypothesis is that GLT1b expression accounts for the widespread presence of mRNA for GLT1 in neurons in the brain. 2. Characterize interactors found in a yeast two-hybrid screen with GLT1b. The hypothesis is that GLT1b interacts with one or more PDZ domain containing proteins. 3. Characterize the effect of protein interactors on the function of GLT1a & b. The hypothesis is that the function of variant forms of GLT1 will be influenced by interactions with other proteins.
