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Assessment of the structure and function of kainate-type glutamate receptors GluR6 Mohammadi, Shahin

Abstract

Glutamate is the main excitatory neurotransmitter in the brain and is essential in most physiological processes. Recent evidence .indicates that the structure of ionotropic glutamate receptors (GluRs) is unique among the ligand-gated ion channel superfamily. The C-terminus of GluR is intra- rather than extracellular and the pore forming segment (M2) forms a hairpin structure in the membrane. In addition, several studies indicate that the M3-M4 loop (the segment connecting transmembrane 3 and 4) is extracellular. However, other studies suggest that residues within the M3-M4 loop are sites of phosphorylation and modulation of channel function by intracellular protein kinases, and therefore the loop is intracellular. To reconcile these conflicting data, we hypothesized that GluR may have a dynamic structure in which, upon agonist binding to and activation of the channel, a portion of the M3-M4 loop containing putative phosphorylation sites translocates across the membrane to the cytosolic side. In order to test this hypothesis, we used site directed mutagenesis to mutate a putative PKA phosphorylation site located in M3-M4 loop of GluR6 (Ser684) to Cys, allowing further modification of this residue by Cys-specific reagents. Using a combined biochemical and electrophysiological approach, we determined that in the Hgand-unbound state of GluR6(S684C), Cys-684 is accessible from the extracellular side to modification by a Cys-specific biotinylating reagent as well as by streptavidin. In addition, we found that this residue becomes inaccessible for biotinylation upon agonist binding and activation of the channel. However, our electrophysiological data indicate that translocation of C684 to the cytoplasmic side is unlikely. The results of our biochemical and electrophysiological experiments indicate that residue 684 in the M3-M4 loop is located extracellularly in the channel's ligand unbound, closed state, as well as ligand-bound, activated or desensitized state. In addition, we analyzed the role of cysteine residues in the normal function of GluR6 receptors. Cys-residues have been shown to be important in the function of several ligand-gated ion channels. Our data indicate that cysteine residues within the M3-M4 loop and N-terminal region contribute to the function and pharmacological properties of GluR6 receptors. Mutation of GluR6 Cys-719 to serine or threonine (C719S, C719T) resulted in a change in the EC50 for glutamate and slowed desensitization of the channel. Other mutations (C773S, C316S and C719S/C773S) resulted in loss of protein expression. Moreover, our data suggest that Cys-719 and Cys-65 form disulfide bonds with other cysteine residues in the M3-M4 loop and N-terminal part of the receptor. These results increase our understanding of the structure and function of glutamate receptors and may prove useful in development of drugs specific for glutamate receptor subunits.

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