Chimeric glutamate receptor subunits reveal the transmembrane domain is sufficient for NMDA receptor pore properties but some positive allosteric modulators require additional domains

NMDA receptors are ligand-gated ion channels that underlie transmission at excitatory synapses and play an important role in regulating synaptic strength and stability. Functional NMDA receptors require two copies of the GluN1 subunit coassembled with GluN2 (and/or GluN3) subunits into a heteromeric tetramer. A diverse array of allosteric modulators can upregulate or downregulate NMDA receptor activity. These modulators include both synthetic compounds and endogenous modulators, such as cis-unsaturated fatty acids, 24(S)-hydroxycholesterol, and various neurosteroids. To evaluate the structural requirements for the formation and allosteric modulation of NMDA receptor pores, we have replaced portions of the rat GluN1, GluN2A, and GluN2B subunits with homologous segments from the rat GluK2 kainate receptor subunit. Our results with these chimeric constructs show that the NMDA receptor transmembrane domain is sufficient to account for most pore properties, but that regulation by some allosteric modulators requires additional cytoplasmic or extracellular domains. SIGNIFICANCE STATEMENT Glutamate receptors mediate excitatory synaptic transmission by forming cation channels through the membrane that open upon glutamate binding. Although many compounds have been identified that regulate glutamate receptor activity, in most cases the detailed mechanisms that underlie modulation are poorly understood. To identify what parts of the receptor are essential for pore formation and sensitivity to allosteric modulators, we generated chimeric subunits that combined segments from NMDA and kainate receptors, subtypes with distinct pharmacological profiles. Surprisingly, our results identify separate domain requirements for allosteric potentiation of NMDA receptor pores by pregnenolone sulfate, 24(S)-hydroxycholesterol, and docosahexaenoic acid, three endogenous modulators derived from membrane constituents. Understanding where and how these compounds act on NMDA receptors should aid in designing better therapeutic agents

Cadmium activates AMPA and NMDA receptors with M3 helix cysteine substitutions

AMPA and NMDA receptors are ligand-gated ion channels that depolarize postsynaptic neurons when activated by the neurotransmitter L-glutamate. Changes in the distribution and activity of these receptors underlie learning and memory, but excessive change is associated with an array of neurological disorders, including cognitive impairment, developmental delay, and epilepsy. All of the ionotropic glutamate receptors (iGluRs) exhibit similar tetrameric architecture, transmembrane topology, and basic framework for activation; conformational changes induced by extracellular agonist binding deform and splay open the inner helix bundle crossing that occludes ion flux through the channel. NMDA receptors require agonist binding to all four subunits, whereas AMPA and closely related kainate receptors can open with less than complete occupancy. In addition to conventional activation by agonist binding, we recently identified two locations along the inner helix of the GluK2 kainate receptor subunit where cysteine (Cys) substitution yields channels that are opened by exposure to cadmium ions, independent of agonist site occupancy. Here, we generate AMPA and NMDA receptor subunits with homologous Cys substitutions and demonstrate similar activation of the mutant receptors by Cd. Coexpression of the auxiliary subunit stargazin enhanced Cd potency for activation of Cys-substituted GluA1 and altered occlusion upon treatment with sulfhydryl-reactive MTS reagents. Mutant NMDA receptors displayed voltage-dependent Mg block of currents activated by agonist and/or Cd as well as asymmetry between Cd effects on Cys-substituted GluN1 versus GluN2 subunits. In addition, Cd activation of each Cys-substituted iGluR was inhibited by protons. These results, together with our earlier work on GluK2, reveal a novel mechanism shared among the three different iGluR subtypes for prying open the gate that controls ion entry into the pore

Radial symmetry in a chimeric glutamate receptor pore

Ionotropic glutamate receptors comprise two conformationally different A/C and B/D subunit pairs. Closed channels exhibit 4-fold radial symmetry in the transmembrane domain (TMD) but transition to 2-fold dimer-of-dimers symmetry for extracellular ligand binding and N-terminal domains. Here, to evaluate symmetry in open pores we analyzed interaction between the Q/R editing site near the pore loop apex and the transmembrane M3 helix of kainate receptor subunit GluK2. Chimaeric subunits that combined the GluK2 TMD with extracellular segments from NMDA receptors, which are obligate heteromers, yielded channels made up of A/C and B/D subunit pairs with distinct substitutions along M3 and/or Q/R site editing status, in an otherwise identical homotetrameric TMD. Our results indicate that Q/R site interaction with M3 occurs within individual subunits and is essentially the same for both A/C and B/D subunit conformations, suggesting that 4-fold pore symmetry persists in the open state

Amino Acid Substitutions in the Pore Helix of GluR6 Control Inhibition by Membrane Fatty Acids

RNA editing at the Q/R site in the GluR5 and GluR6 subunits of neuronal kainate receptors regulates channel inhibition by lipid-derived modulators including the cis-unsaturated fatty acids arachidonic acid and docosahexaenoic acid. Kainate receptor channels in which all of the subunits are in the edited (R) form exhibit strong inhibition by these compounds, whereas wild-type receptors that include a glutamine (Q) at the Q/R site in one or more subunits are resistant to inhibition. In the present study, we have performed an arginine scan of residues in the pore loop of the GluR6(Q) subunit. Amino acids within the range from −19 to +7 of the Q/R site of GluR6(Q) were individually mutated to arginine and the mutant cDNAs were expressed as homomeric channels in HEK 293 cells. All but one of the single arginine substitution mutants yielded functional channels. Only weak inhibition, typical of wild-type GluR6(Q) channels, was observed for substitutions +1 to +6 downstream of the Q/R site. However, arginine substitution at several locations upstream of the Q/R site resulted in homomeric channels exhibiting strong inhibition by fatty acids, which is characteristic of homomeric GluR6(R) channels. Based on homology with the pore loop of potassium channels, locations at which R substitution induces susceptibility to fatty acid inhibition face away from the cytoplasm toward the M1 and M3 helices and surrounding lipids

Equity and Discrimination in NCAA athletic Departments: Perceptions of Senior Women Administrators

Perceptions of Senior Woman Administrators (SWAs) were sought regarding areas of equity and discrimination in NCAA Athletic Departments. A five point agree/disagree Likert-scale survey was electronically mailed to all NCAA Senior Woman Administrators (SWAs) throughout the United States. Of the 841 surveys mailed, 406 were returned for a 48.3% return rate. To determine differences in the distribution of Likert-scale questions by demographic variable the Kruskal-Wallis rank test was used at a 0.05 nominal significance level. Significant differences with regards to Likert-scale items of agreement/disagreement were found between the following SWA demographics: marital status, reporting structure, and highest level of education completed. Significant differences between demographic variables were noted for eight of the twelve areas of equity and discrimination. Overall, the top three discrimination factors were: family responsibilities disadvantage women more than men, women are paid less than men for comparable positions, and it is more difficult for women to advance than men

Title IX Compliance in NCAA Athletic Departments: Perceptions of Senior Woman Administrators

Perceptions of Senior Woman Administrators (SWAs) were sought regarding the equal provision of 13 Title IX compliance areas in women’s athletic programs as compared to men’s. A five point agree/disagree Likert‐scale survey was electronically mailed to all SWAs at National Collegiate Athletic Association (NCAA) membership institutions throughout the United States. Of the 841 surveys mailed, 406 were returned for a 48.3% return rate. The SWAs disagreed or strongly disagreed at the highest rates that the following five Title IX compliance areas were being provided for equally in the women’s programs when compared to the men’s: publicity (31.0%), locker room facilities (27.1%), coaching (20.0%), recruitment of student‐athletes (15.4%), and equipment and supplies (14.7%). Significant differences between Likert‐scale items of agreement/disagreement were found among the following SWA demographics: marital status, NCAA Division, years of experience, and reporting structure. The SWAs agreed or strongly agreed that the 13 Title IX compliance areas were being provided for equally in the women’s programs when compared to the men’s at the following rates: housing & dining facilities (84.4%), medical & training facilities (84.3%), scheduling of games (81.1%), travel & per diem allowances (80.5%), practice facilities (78.7%), competitive facilities (78.4%), equipment and supplies (77.7%), support services (76.0%), tutoring (74.3%), recruitment of student‐athletes (73.2%), coaching (70.3%), locker room facilities (63.2%), and publicity (55.3%)

Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles

RNA editing at the Q/R site near the apex of the pore loop of AMPA and kainate receptors controls a diverse array of channel properties, including ion selectivity and unitary conductance and susceptibility to inhibition by polyamines and cis-unsaturated fatty acids, as well as subunit assembly into tetramers and regulation by auxiliary subunits. How these different aspects of channel function are all determined by a single amino acid substitution remains poorly understood; however, several lines of evidence suggest that interaction between the pore helix (M2) and adjacent segments of the transmembrane inner (M3) and outer (M1) helices may be involved. In the present study, we have used double mutant cycle analysis to test for energetic coupling between the Q/R site residue and amino acid side chains along the M3 helix. Our results demonstrate interaction with several M3 locations and particularly strong coupling to substitution for L614 at the level of the central cavity. In this location, replacement with smaller side chains completely and selectively reverses the effect of fatty acids on gating of edited channels, converting strong inhibition of wild-type GluK2(R) to nearly 10-fold potentiation of GluK2(R) L614A

Fatty acid modulation and polyamine block of GluK2 kainate receptors analyzed by scanning mutagenesis

RNA editing of kainate receptor subunits at the Q/R site determines their susceptibility to inhibition by cis-unsaturated fatty acids as well as block by cytoplasmic polyamines. Channels comprised of unedited (Q) subunits are strongly blocked by polyamines, but insensitive to fatty acids, such as arachidonic acid (AA) and docosahexaenoic acid (DHA), whereas homomeric edited (R) channels resist polyamine block but are inhibited by AA and DHA. In the present study, we have analyzed fatty acid modulation of whole-cell currents mediated by homomeric recombinant GluK2 (formerly GluR6) channels with individual residues in the pore-loop, M1 and M3 transmembrane helices replaced by scanning mutagenesis. Our results define three abutting surfaces along the M1, M2, and M3 helices where gain-of-function substitutions render GluK2(Q) channels susceptible to fatty acid inhibition. In addition, we identify four locations in the M3 helix (F611, L614, S618, and T621) at the level of the central cavity where Arg substitution increases relative permeability to chloride and eliminates polyamine block. Remarkably, for two of these positions, L614R and S618R, exposure to fatty acids reduces the apparent chloride permeability and potentiates whole-cell currents ∼5 and 2.5-fold, respectively. Together, our results suggest that AA and DHA alter the orientation of M3 in the open state, depending on contacts at the interface between M1, M2, and M3. Moreover, our results demonstrate the importance of side chains within the central cavity in determining ionic selectivity and block by cytoplasmic polyamines despite the inverted orientation of GluK2 as compared with potassium channels and other pore-loop family members

Another intermediate mass black hole in a starburst galaxy?: The luminous X-ray source in NGC 3628 reappears

In a 52 ks-long Chandra ACIS-S observation of the nearby starburst galaxy NGC 3628, obtained to study the starburst-driven outflow from this galaxy, we have detected a very luminous (L_X = 1.1e40 erg/s in the 0.3-8.0 keV energy band) point source located at least 20 arcsec (~970 pc) from the nucleus of the galaxy. No radio, optical or near-IR counterpart to this source has been found. This is most probably the reappearance of the strongly-variable X-ray-luminous source discovered by Dahlem et al (1995), which faded by a factor >27 between December 1991 and March 1994 (at which point it had faded below the detection limit in a ROSAT HRI observation). This source is clearly a member of an enigmatic class of X-ray sources that are considerably more luminous than conventional X-ray binaries but less luminous than AGN, and which are not found at the dynamical center of the host galaxy. The Chandra spectrum is best-fit by an absorbed power law model with a photon index of Gamma = 1.8+/-0.2, similar to that seen in Galactic BH binary candidates in their hard state. Bremsstrahlung models or multi-color disk models (the favored spectral model for objects in this class based on ASCA observations) can provide statistically acceptable fits only if the data at energies E > 5 keV is ignored. This is one of the first X-ray spectra of such an object that is unambiguously that of the source alone, free from the spectral contamination by X-ray emission from the rest of the galaxy that affects previous spectral studies of these objects using ASCA.Comment: Accepted for publication in the Ap