Agonist-induced changes in substituted cysteine accessibility reveal dynamic extracellular structure of M3-M4 loop of glutamate receptor GluR6

Recent evidence suggests that the transmembrane topology of ionotropic glut amate receptors differs from other members of the ligand-gated ion channel superfamily. However, the structure of the segment linking membrane domains M3 and M4 (the M3-M4 loop) remains controversial. Although various data in dicate that this loop is extracellular, other results suggest that serine r esidues in this segment are sites of phosphorylation and channel modulation by intracellular protein kinases. To reconcile these data, we hypothesized that the M3-M4 loop structure is dynamic and, more specifically that the p ortion containing putative phosphorylation sites may be translocated across the membrane to the cytoplasmic side during agonist binding. To test this hypothesis, we mutated Ser 684, a putative cAMP-dependent protein kinase si te in the kainate-type glutamate receptor GluR6, to Cys. Results of biochem ical and electrophysiological experiments are consistent with Cys 684 being accessible, in the unliganded state, from the extracellular side to modifi cation by a Cys-specific biotinylating reagent followed by streptavidin (SA ). Interestingly, our data suggest that this residue becomes inaccessible t o the extracellular biotinylating reagent during agonist binding. However, we find it unlikely that Cys 684 undergoes membrane translocation, because the addition of SA to Cys-biotinylated GluR6(S684C) has no effect on peak g lutamate-evoked current and only a small effect on macroscopic desensitizat ion. We conclude that residue 684 in GluR6 is extracellular in the receptor -channel's closed, unliganded state and does not cross the membrane after a gonist binding. However, an agonist-induced conformational change in the re ceptor substantially alters accessibility of position 684 to the extracellu lar environment.