Subunit-specific contribution of pore-forming domains to NMDA receptor channel structure and gating.

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels that contribute to fundamental physiological processes such as learning and memory and, when dysfunctional, to pathophysiological conditions such as neurodegenerative diseases, stroke, and mental illness. NMDARs are obligate heteromultimers typically composed of NR1 and NR2 subunits with the different subunits underlying the functional versatility of NMDARs. To study the contribution of the different subunits to NMDAR channel structure and gating, we compared the effects of cysteine-reactive agents on cysteines substituted in and around the M1, M3, and M4 segments of the NR1 and NR2C subunits. Based on the voltage dependence of cysteine modification, we find that, both in NR1 and NR2C, M3 appears to be the only transmembrane segment that contributes to the deep (or voltage dependent) portion of the ion channel pore. This contribution, however, is subunit specific with more positions in NR1 than in NR2C facing the central pore. Complimentarily, NR2C makes a greater contribution than NR1 to the shallow (or voltage independent) portion of the pore with more NR2C positions in pre-M1 and M3-S2 linker lining the ion-conducting pathway. Substituted cysteines in the M3 segments in NR1 and NR2C showed strong, albeit different, state-dependent reactivity, suggesting that they play central but structurally distinct roles in gating. A weaker state dependence was observed for the pre-M1 regions in both subunits. Compared to M1 and M3, the M4 segments in both NR1 and NR2C subunits had limited accessibility and the weakest state dependence, suggesting that they are peripheral to the central pore. Finally, we propose that Lurcher mutation-like effects, which were identified in and around all three transmembrane segments, occur for positions located at dynamic protein-protein or protein-lipid interfaces that have state-dependent accessibility to methanethiosulfonate (MTS) reagents and therefore can affect the equilibrium between open and closed states following reactions with MTS reagents.

State-dependent changes in the electrostatic potential in the pore of a GluR channel.

The M2 loop and the M3 segment are the major pore-lining domains in the GluR channel. These domains determine ion permeation and channel block processes and are extensively involved in gating. To study the distribution of the membrane electric potential across the GluR channel pore, we recorded from alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptors containing M2 and M3 cysteine substitutions in the GluR-A subunit and measured the voltage dependence of the modification rate of these substituted cysteines by methanethiosulfonate reagents either in the presence or absence of glutamate. In the presence of glutamate, the voltage dependence became gradually stronger for positions located deeper in the pore suggesting that the electrostatic potential drops fairly uniformly across the pore in the open state. In contrast, in the absence of glutamate, the voltage dependence was biphasic. The difference in the electrostatic potential in the presence and absence of glutamate had an apparent maximum in the middle of the extracellular vestibule. We suggest that these state-dependent changes in the membrane electric potential reflect a reorientation of the dipoles of the M2 loop alpha-helices toward and away from the center of the channel pore during gating.

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

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 outer pore of the glutamate receptor channel has 2-fold rotational symmetry.

The ligand binding domain of glutamate receptors (GluRs) has 2-fold rotational symmetry. The structure including the symmetry of the GluR ion channel remains undefined. Here we used substituted cysteines in the pore-lining M3 segment of the AMPAR GluR-A subunit and various cysteine-reactive agents to study the structure of the channel during gating. We find that cysteines substituted at A+6, located in the highly conserved SYTANLAAF motif, are grouped in pairs consistent with a 2-fold symmetry in the extracellular part of the pore. To account for this symmetry and crosslinking, we propose that the M3 segments in two neighboring GluR subunits are kinked within SYTANLAAF in opposite directions relative to the central axis of the pore. Our results extend the 2-fold rotational symmetry from the ligand binding domain to at minimum the extracellular part of the channel and suggest a model of gating movements in GluR pore-forming domains.

Different gating mechanisms in glutamate receptor and K+ channels.

The basic structural features of channel gating in glutamate receptors (GluRs) remain unknown. Here we used covalent modification of substituted cysteines and fast agonist application to study the contribution of the M3 segment in AMPA receptor GluR-A subunits to channel structure and gating. The pattern of accessibility of substituted cysteines to extracellularly applied methanethiosulfonate reagents and the rates of their modification by these reagents, measured in either the presence or absence of glutamate, indicate that M3 forms an alpha-helix that lines the pore of the channel and is involved in gating-related movements. The voltage dependence of modification rates places the tip of the M2 loop (the Q/R site) close to the middle of M3. All of these results are consistent with pore-forming domains in GluR and K+ channels having a similar structure but inverted membrane topology. Nevertheless, GluRs lack a glycine residue at a homologous structural position as the gating hinge glycine in K+ channels. Moreover, simultaneous substitution of the only two glycines in M3 of GluR-A with alanines produced channels with gating properties indistinguishable from wild type. Given the unique role of glycines in the flexibility ofalpha-helices, our results indicate that the M3 segment in GluR does not contain a glycine gating hinge and suggest that, in contrast to the homologous domain in K+ channels, M3 is rigid during gating. The different positioning and functional significance of glycines in a key structural domain may represent the basis for the distinct features of gating in GluR and K+ channels.