The role of posttranslational modifications in the structure and function of glutamate receptors

Glutamate-activated ion channels constitute the major excitatory neurotransmitter receptor system in the brain. Glutamate receptors have been implicated in a wide variety of neurological events including synaptic plasticity and excitotoxicity in ischemia and other neurodegenerative diseases. Glutamatergic neurotransmission is essential for long-term potentiation and depression of synaptic responses, processes thought to be involved in memory acquisition and learning. The functional modulation of glutamate receptors by post-translational modifications may be an important mechanism in regulating those processes.;After optimization of glutamate receptor expression, the expression systems were used to study the roles of several post-translational modifications in the structure and function of GluR6, an ionotropic glutamate receptor of the kainate subtype. GluR6 receptors were phosphorylated in-vitro by cAMP-dependent protein kinase (PKA) but not by cGMP-dependent protein kinase. In addition, kainate-evoked currents mediated by GluR6 were potentiated by PKA. Mutagenesis of serines 684 and 666 abolished both the PKA-induced phosphorylation of GluR6 and the PKA-induced potentiation of the kainate-evoked currents mediated by GluR6. Ca;Site-directed mutagenesis of consensus sequences for asparagine-linked glycosylation at asparagines 515 and 720 in GluR6 resulted in molecular weight shifts on immunoblots indicating that those sites are glycosylated. Electrophysiological recordings of the mutant receptors demonstrated that there was no difference from wild type GluR6 in the modulation of kainate-evoked currents by Concanavalin A. Based on these results, and additional evidence for an intracellular location of the carboxyl terminus, a new 5 transmembrane domain model for the structure of GluR6 was proposed.;Three expression systems were examined for the analysis of the structure and function of glutamate receptors. These included the Xenopus oocyte system, a human embryonic kidney cell line, and the baculovirus insect cell system. Each expression system had its own distinct advantages and disadvantages.