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

Coverage Impacts Of Work Requirements From The Arkansas Medicaid Program

I examine changes in Medicaid coverage and insurance status surrounding a work requirement policy implemented within the Arkansas Medicaid demonstration waiver. The policy applied to able-bodied, childless adults, aged 30 to 49, not enrolled as students, and was effective from 2018 to 2019. Eligibility was conditional on policy compliance. Taking a sample from the IPUMS American Community Survey database, I use triple-differences modeling to compare Arkansans subject to the policy with unaffected Arkansans and individuals from a set of control states. I find that the policy pilot group in Arkansas was less likely to be insured or have Medicaid coverage in the two years after the work requirement took effect, compared with controls. In 2018 and 2019 respectively, I estimate increases in uninsurance for the pilot group, compared with non-pilot Arkansans, were 7.3 and 10.8 percentage points greater than those experienced by the hypothetical pilot and non-pilot groups from the control states. Similarly, I estimate declines in Medicaid coverage for pilot versus non-pilot-group Arkansans were 6.2 and 10.2 percentage points greater in magnitude, compared with the hypothetical pilot and non-pilot groups from the control states in 2018 and 2019 respectively. In tandem with a series of robustness checks, I outline how asymmetric information, unobservable government intervention, and contemporaneous policies could affect my results

Coalitional Manipulations and Immunity of the Shapley Value

We consider manipulations in the context of coalitional games, where a coalition aims to increase the total payoff of its members. An allocation rule is immune to coalitional manipulation if no coalition can benefit from internal reallocation of worth on the level of its subcoalitions (reallocation-proofness), and if no coalition benefits from a lower worth while all else remains the same (weak coalitional monotonicity). Replacing additivity in Shapley's original characterization by these requirements yields a new foundation of the Shapley value, i.e., it is the unique efficient and symmetric allocation rule that awards nothing to a null player and is immune to coalitional manipulations. We further find that for efficient allocation rules, reallocation-proofness is equivalent to constrained marginality, a weaker variant of Young's marginality axiom. Our second characterization improves upon Young's characterization by weakening the independence requirement intrinsic to marginality

Efficient extensions of communication values

We study values for transferable utility games enriched by a communication graph. The most well-known such values are component-efficient and characterized by some link-deletion property. We study efficient extensions of such values: for a given component-efficient value, we look for a value that (i) satisfies efficiency, (ii) satisfies the link-deletion property underlying the original component-efficient value, and (iii) coincides with the original component-efficient value whenever the underlying graph is connected. Béal et al. (2015) prove that the Myerson value (Myerson, 1977) admits a unique efficient extension, which has been introduced by van den Brink et al. (2012). We pursue this line of research by showing that the average tree solution (Herings et al., 2008) and the compensation solution (Béal et al., 2012a) admit similar unique efficient extensions, and that there exists no efficient extension of the position Value (Meessen, 1988; Borm et al., 1992). As byproducts, we obtain new characterizations of the average tree solution and the compensation solution, and of their efficient extensions

Efficient extension of the Myerson value

We study values for transferable utility games enriched by a communication graph (CO-games) where the graph does not necessarily affect the productivity but can influence the way the players distribute the worth generated by the grand coalition. Thus, we can envisage values that are efficient instead of values that are component efficient. For CO-games with connected graphs, efficiency and component efficiency coincide. In particular, the Myerson value (Myerson, 1977) is efficient for such games. Moreover, fairness is characteristic of the Myerson value. We identify the value that is efficient for all CO-games, coincides with the Myerson value for CO-games with connected graphs, and satisfies fairness

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

A puromycin selectable cell line for the enrichment of mouse embryonic stem cell-derived V3 interneurons

INTRODUCTION: Spinal V3 interneurons (INs) are a commissural, glutamatergic, propriospinal neuron population that holds great potential for understanding locomotion circuitry and local rewiring after spinal cord injury. Embryonic stem cells hold promise as a cell source. However, the inevitable heterogeneity resulting from differentiation protocols makes studying post-mitotic stem cell-derived neuron populations difficult because proliferative glia quickly overtake a culture. Previously, an induction protocol for V3 INs was established. However, because of the heterogeneous population resulting from the induction protocol, functional characterization of the induced cells was not possible. METHODS: A selectable murine transgenic embryonic stem cell (ESC) line (Sim1-Puro) was generated by recombineering. The expression of the puromycin resistance enzyme, puromycin N-acetyl-transferase (PAC), was knocked into the locus of a post-mitotic V3 IN marker (Sim1), allowing Sim1 gene regulatory elements to control PAC expression. The resulting cell line was characterized for Sim1 expression by in situ hybridization, for glutamatergic marker expression by immunocytochemistry and quantitative real time polymerase chain reaction (qRT-PCR), and for functional maturation by electrophysiology. RESULTS: Puromycin selection significantly enriched the population for V3 INs, allowing long-term characterization. The selected population expressed the neuronal marker β-III tubulin and the glutamatergic neuron marker VGluT2. The selected V3 INs also exhibited appropriate functional maturation, as assessed by electrophysiology, and remained glutamatergic for 2 weeks. CONCLUSION: The Sim1-Puro cell line provides a simple, high throughput method for generating large numbers of V3 INs from mouse ESCs for future in vitro and cell transplantation studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13287-015-0213-z) contains supplementary material, which is available to authorized users