This section investigates the functional properties of neurotransmitter receptors and ion channels in the vertebrate CNS utilizing electrophysiological and molecular biological techniques. Receptor selective agonists and antagonists are applied by rapid perfusion to cells and membrane patches under voltage clamp. Current work focuses on recombinant glutamate receptors expressed in transfected mammalian cells and Xenopus oocytes. Work over the past year has focussed on allosteric regulation of AMPA receptors by PEPA, a novel modulator with flop subunit selectivity. At a macroscopic level the mechanism of action of PEPA involves block of desensitization. The potency of PEPA is at least 200 times greater than that of aniracetam. In addition we have continued analysis of polyamine interactions with the ion channel of kainate receptors and have shown that spermine and spermidine are weakly permeable blockers consistent with a limiting pore size of 0.44 nm2. Kinetic analysis of polyamine block suggests a highly asymmetric barrier limiting entry of spermine into the pore, such that the voltage dependence of block arises almost entirely due changes in the unbinding rate. The role of individual residues within the pore forming region is being analysed by site directed mutagenesis. Surprisingly, the introduction of negatively charged residues at the Q/R site decreases polyamine block, in contrast to what would be expected for a cationic channel blocker. Work in progress includes an alanine scan of residues flanking the Q/R site.
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