STRUCTURE AND FUNCTION OF GLUTAMATE RECEPTORS

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DESCRIPTION: The proposed experiments are aimed at establishing the role of polyamines in conferring rectification to certain types of glutamate receptors. The investigator presents three specific aims relating to the general problem of glutamate receptor function and polyamine action. The first aim will determine the role of specific polyamines in conferring the observed inward rectification observed for specific types of glutamate receptors. For this purpose the investigator will block the production of specific types of polyamines in Xenopus oocytes and mammalian cells which have been transfected with RNA coding for glutamate receptors. The block will be confirmed by HPLC analysis of the polyamine levels and the functional consequences will be determined by measurement of glutamate evoked outward currents. The information provided from these experiments will demonstrate that polyamines are the endogenous source of rectification as well as identifying the specific polyamine type responsible. In a second set of experiments the investigator will determine the sensitivity of different glutamate receptor types and mutant receptor types to different polyamines. Preliminary evidence suggests that GluR4 has a higher sensitivity to spermine than GluR1 in spite of the fact that the putative pore forming regions (TM2) are identical between these two subunits. Therefore, the investigator will use the information obtained from these experiments to design GluR1 and GluR4 chimeras to locate additional pore contributing regions. In a final set of experiments the investigator will search for the bases of current rectification which is observed for Glutamate receptors. In Specific Aim 2 the investigator will determine the residues responsible for differences in permeability of AMPA and Kainate receptor channels to calcium. The investigator proposes, on the bases of homology to voltage-dependent channels, a pore lining loop outside TM2. Point mutations will be made on GluR3 and GluR6 receptor subunits at locations shown to affect properties of voltage-dependent or ligand gated channels. The permeability to divalent cations and the sensitivity to polyamines will be assessed for each mutation. In Specific Aim 3 the investigator will perform single channel studies on wild type and mutant glutamate receptors in order to ascertain the bases for outward rectification. Three different potential sources of rectification will be directly tested; rectification at the single channel level, voltage-dependent gating, and multiple conductances which are voltage-dependent. The information obtained from these aims takes on additional significance given the recent disclosure that the Glutamate tertiary structure appears distinct from other members of the ligand gated receptor family.