Block of AMPA Receptor Desensitization by a Point Mutation outside the Ligand-Binding Domain

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.

The Transmembrane AMPA Receptor Regulatory Protein γ4 Is a More Effective Modulator of AMPA Receptor Function than Stargazin (γ2)

“…Our data lead us to propose the following scheme of regulatory interaction between AMPA receptors and TARPs: until now, chiefly two regions in the AMPA receptor have been linked to the desensitization properties of the receptor, namely the ligandbinding domain (LBD) (Partin et al, 1995;Stern-Bach et al, 1998;Banke et al, 2001;Sun et al, 2002;Horning and Mayer, 2004) and the linker regions between the LBD and the pore region (Yelshansky et al, 2004). Hence, an interaction between the TARP, probably at the first extracellular loop , and one of these two regions of the AMPA receptor seems likely.…”

“…Conversely, an interaction between the TARP and the linker regions connecting the LBD and the pore region cannot be ruled out, especially because it has been shown that point mutations within a linker region influence the desensitization properties of the receptor complexes (Yelshansky et al, 2004). In this case, not only desensitization properties of the receptor complex could be altered but also the gating.…”

The Transmembrane Domain Mediates Tetramerization of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors

“…Point mutations were generated using PCR-based methods and confirmed by sequence analysis. Human embryonic kidney (HEK) 293 cells were transfected with the cDNAs using X-tremeGene 9 or X-tremeGene HP (Roche Applied Science) (20).…”

Interface Interactions Modulating Desensitization of the Kainate-Selective Ionotropic Glutamate Receptor Subunit GluR6

“…Allosteric potentiators (Jin et al, 2005) bind at the dimer interface, whereas disruptive mutations within the interface accelerate the rate of desensitization (Horning and Mayer, 2004). Kinetic (Bowie and Lange, 2002;Yelshansky et al, 2004) and electron microscopic (Tichelaar et al, 2004) studies also show that the key functional unit of AMPA receptors is a dimer. Most recently, it has been shown that NMDA subunit S1S2 domains form twofold dimers that are stabilized by mutations equivalent to R2 L483Y (Furukawa et al, 2005).…”

“…At several sites within the dimer interface in which mutations to GluR2 had promoted desensitization (Horning and Mayer, 2004), equivalent changes in GluR6 unexpectedly slowed desensitization (Fleck et al, 2003). Furthermore, the dimeric associations identified by kinetic studies in AMPA subunits were either not observed or were weaker in kainate subunits (Bowie and Lange, 2002;Yelshansky et al, 2004). Gating mechanisms also differ, with AMPA subunits gating independently and kainate subunits gating in a concerted manner .…”