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Block of AMPA receptor desensitization by a point mutation outside the ligand-binding domain

Published on 19.05.2004 in Journal of Neuroscience

Authors:

Maria V. Yelshansky, Alexander I. Sobolevsky, Claudia Jatzke, Lonnie P. Wollmuth

Abstract

Desensitization of ionotropic glutamate receptors (GluRs), specifically the AMPA receptor subtype, shapes the postsynaptic response at certain synapses in the brain. All known mechanisms that alter desensitization, either pharmacological or mutational, are associated with the ligand-binding domain. Here we report that substitution of a conserved positively charged arginine (R) with a negatively charged glutamate in the linker between the pore-forming M3 segment and the S2 lobe, a region outside the ligand-binding domain, blocks desensitization in homomeric AMPA receptors composed of GluR-B(i) subunits. A charge-reversing substitution of a glutamate adjacent to this conserved R enhanced desensitization, consistent with these effects attributable to electrostatics. Homologous substitutions of the conserved R in GluR-B(o), GluR-A(i) and the kainate receptor GluR-6 subunits produced comparable but less visible effects on desensitization. Subunit specificity was also apparent for accessibility of substituted cysteines in the M3-S2 linker, suggesting that this part of the channel is not structurally identical in different GluRs. Additionally, reactivity with a sulfhydryl-specific reagent was state dependent, suggesting that the conformations of the nonconducting closed and desensitized states are different at the level of the M3-S2 linker. Our results therefore represent the first identification of elements outside the ligand-binding domain affecting desensitization in non-NMDA receptor channels and suggest that electrostatic interactions involving charged residues in the M3-S2 linker influence channel gating in a subunit- and subtype-specific manner.

Effect of the R → E charge reversal in the M3–S2 linker on glutamate-activated currents in AMPAR and KAR channels. Glutamate-activated currents in outside-out patches isolated from HEK 293 cells expressing wild-type (left traces) or mutant (right traces) GluR-Ai (A), GluR-Bi (B), and GluR-6 (C) channels are shown. The mutant channels have the positively charged arginine (R) in the M3–S2 linker replaced by a glutamate (E) (R → E substitution). Currents were elicited by a 100 msec application of glutamate (3 mm; filled bar) at a holding potential of –60 mV. Current amplitudes were measured either at the peak (Ip) or near the end of the glutamate applications when they reached steady-state (Iss). Dashed lines show zero current level. The time scale is the same for all panels.