Eric S. Nisenbaum

New Transmembrane AMPA Receptor Regulatory Protein Isoform, γ-7, Differentially Regulates AMPA Receptors

AMPA-type glutamate receptors (GluRs) mediate most excitatory signaling in the brain and are composed of GluR principal subunits and transmembrane AMPA receptor regulatory protein (TARP) auxiliary subunits. Previous studies identified four mammalian TARPs, γ-2 (or stargazin), γ-3, γ-4, and γ-8, that control AMPA receptor trafficking, gating, and pharmacology. Here, we explore roles for the homologous γ-5 and γ-7 proteins, which were previously suggested not to serve as TARPs. Western blotting reveals high levels of γ-5 and γ-7 in the cerebellum, where γ-7 is enriched in Purkinje neurons in the molecular layer and glomerular synapses in the granule cell layer. Immunoprecipitation proteomics shows that cerebellar γ-7 avidly and selectively binds to AMPA receptor GluR subunits and also binds to the AMPA receptor clustering protein, postsynaptic density-95 (PSD-95). Furthermore, γ-7 occurs together with PSD-95 and AMPA receptor subunits in purified postsynaptic densities. In heterologous cells, γ-7 but not γ-5 greatly enhances AMPA receptor glutamate-evoked currents and modulates channel gating. In granule cells from stargazer mice, transfection of γ-7 but not γ-5 increases AMPA receptor-mediated currents. Compared with stargazin, γ-7 differentially modulates AMPA receptor glutamate affinity and kainate efficacy. These studies define γ-7 as a new member of the TARP family that can differentially influence AMPA receptors in cerebellar neurons.

Long-term effects of dopamine-depleting brain lesions on spontaneous activity of type II striatal neurons: Relation to behavioral recovery

The long-term effects of dopamine (DA)-depleting brain lesions on behavior and spontaneous activity of Type II striatal neurons were measured in rats after intraventricular injection of the neurotoxin 6-hydroxydopamine (6-OHDA). Spontaneous firing rates were increased relative to control values when recorded 4-8 days or 4-6 weeks postlesion in animals displaying aphagia, adipsia and akinesia. In contrast, spontaneous activity was not increased when recorded 4-6 weeks after the lesion in animals that had recovered from behavioral deficits. Other animals that had recovered from the effects of an earlier 6-OHDA treatment were given either a second injection of 6-OHDA or a systemic injection of haloperidol, a DA receptor antagonist. In both groups, discharge rates were elevated relative to control levels in association with a reinstatement of behavioral deficits. These results demonstrate that behavioral recovery after large DA-depleting brain lesions is associated with a return of spontaneous activity of striatal neurons to normal levels, and suggest that both behavioral and electrophysiological measures are dependent on the functioning of residual elements of the DA system.

Functionally distinct subpopulations of striatal neurons are differentially regulated by gabaergic and dopaminergic inputs—II. In vitro analysis

In the companion report [Nisenbaum and Berger (1992) Neuroscience 48, 561-578] the contrasting paired impulse responses to stimulation of the corticostriatal pathway which define the Type I and Type II subpopulations of striatal neurons were shown to reflect differential regulation by GABAergic and dopaminergic inputs. More specifically, the decreased probability of spike discharge (inhibition) to long interstimulus intervals (60-260 ms) characteristic of Type I neurons was found to be dependent on dopaminergic input via D1 receptor activation, whereas the inhibition to short interstimulus intervals (10-20 ms) distinctive of Type II neurons was found to be mediated by GABAergic input acting through GABAA receptor stimulation. The present experiments have further investigated the contribution of GABAergic and dopaminergic feedforward and/or feedback circuits to the functional identities of Type I and Type II neurons using an in vitro corticostriatal slice preparation. In this preparation, the cortical afferents to the striatum are preserved, allowing for activation of striatal cells in a manner similar to that used in vivo; however, all axons arising from midbrain and brainstem structures including the substantia nigra are transected, and intrastriatal GABAergic pathways are reduced. Consistent with the predicted effect of disrupting these two neurotransmitter pathways, the paired impulse responses of striatal neurons recorded in vitro were not similar to the responses of either Type I or Type II neurons recorded in vivo. Indeed, the paired impulse profiles of striatal neurons recorded in vitro were relatively homogeneous in that virtually all cells displayed an increased probability of spike discharge (facilitation) to the second impulse of all interstimulus intervals (10-500ms) tested. Low concentrations of allosteric agonists for the GABAA receptor, pregnanolone (5 microM) and pentobarbital (50 microM), selectively inhibited spike discharge in response to short interstimulus intervals (10-20 ms) for approximately 40% of the neurons sampled, but produced no change in facilitation to longer interstimulus intervals (30-500 ms). The agonist-induced inhibition to short interstimulus intervals was blocked by bicuculline (10-20 microM), and was not mimicked by the GABAB receptor agonist, baclofen (1-5 microM). In addition, application of dopamine (5-10 microM) or the D1 receptor agonist, SKF38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; 5 microM), inhibited spike discharge to longer interstimulus intervals (40-500 ms) for approximately 10% of striatal cells recorded. The inhibition to longer interstimulus intervals was blocked by the D1 receptor antagonist, SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin+ ++-7-ol], but not the D2 antagonist, sulpiride.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

Cerebellar motor coordination and cerebellar Purkinje cell synaptic function require metabotropic glutamate receptor 1 (mGluR1, Grm1). We used an unbiased proteomic approach to identify protein partners for mGluR1 in cerebellum and discovered glutamate receptor δ2 (GluRδ2, Grid2, GluΔ2) and protein kinase Cγ (PKCγ) as major interactors. We also found canonical transient receptor potential 3 (TRPC3), which is also needed for mGluR1-dependent slow EPSCs and motor coordination and associates with mGluR1, GluRδ2, and PKCγ. Mutation of GluRδ2 changes subcellular fractionation of mGluR1 and TRPC3 to increase their surface expression. Fitting with this, mGluR1-evoked inward currents are increased in GluRδ2 mutant mice. Moreover, loss of GluRδ2 disrupts the time course of mGluR1-dependent synaptic transmission at parallel fiber–Purkinje cells synapses. Thus, GluRδ2 is part of the mGluR1 signaling complex needed for cerebellar synaptic function and motor coordination, explaining the shared cerebellar motor phenotype that manifests in mutants of the mGluR1 and GluRδ2 signaling pathways.

Molecular determinants responsible for differences in desensitization kinetics of AMPA receptor splice variants.

Flip (i) and flop (o) alternatively spliced variants of the four glutamate AMPA receptor subunits (GluR1-4) are differentially expressed in the CNS and can display distinct rates of desensitization that contribute to the heterogeneity of native AMPA receptor-dependent synaptic responses. In the present study, we initially compared the kinetics of desensitization in response to fast application of glutamate (1 mm) for the eight different homomeric recombinant human AMPA receptors (hGluR1-4i and o) heterologously expressed in mammalian cells. Consistent with previous reports on recombinant rat AMPA receptors, the time constants of desensitization between human GluR1i and GluR1o receptors were the same, whereas the flip isoforms for GluR2-4 receptors exhibited significantly slower rates of desensitization compared with the flop isoforms. To identify the molecular determinants responsible for these functional differences, the effects of exchanging amino acid residues in the flip-flop cassette of GluR2i and GluR2o were investigated. Three amino acid residues in the flip-flop region (Thr765, Pro766, and Ser775 in flip and Asn765, Ala766, and Asn775 in flop) were identified that contribute to splice-variant differences in the rate of desensitization. Recent structural data show that these three residues are located on helix J, which forms part of the intradimer interface of AMPA receptor ligand-binding cores, and that the stability of this interface may regulate desensitization. The present results suggest that these three residues may confer differences in flip and flop receptor desensitization rates by directly and/or indirectly influencing the stability of the interface between adjacent subunits.

Tetrodotoxin-resistant sodium channels in sensory neurons generate slow resurgent currents that are enhanced by inflammatory mediators.

Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the β4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel β4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the β4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.

Spontaneous activity of type II but not type I striatal neurons is correlated with recovery of behavioral function after dopamine-depleting brain lesions.

The relation between the spontaneous firing of Type I striatal neurons and recovery of behavioral function after near-total dopamine depletions of the rat striatum was investigated. The results demonstrate that the activity of Type I neurons remains elevated in recovered animals, which contrasts with our previous finding that the firing rates of Type II striatal neurons return to normal levels in association with behavioral recovery.

Protein kinase C enhances human sodium channel hNav1.7 resurgent currents via a serine residue in the domain III–IV linker

Resurgent sodium currents likely play a role in modulating neuronal excitability. Here we studied whether protein kinase C (PKC) activation can increase resurgent currents produced by the human sodium channel hNav1.7. We found that a PKC agonist significantly enhanced hNav1.7‐mediated resurgent currents and this was prevented by PKC antagonists. The enhancing effects were replicated by two phosphorylation‐mimicking mutations and were prevented by a phosphorylation‐deficient mutation at a conserved PKC phosphorylation site (Serine 1479). Our results suggest that PKC can increase sodium resurgent currents through phosphorylation of a conserved Serine residue located in the domain III–IV linker of sodium channels.

Structural Determinants of the γ-8 TARP Dependent AMPA Receptor Antagonist

The forebrain specific AMPA receptor antagonist, LY3130481/CERC-611, which selectively antagonizes the AMPA receptors associated with TARP γ-8, an auxiliary subunit enriched in the forebrain, has potent antiepileptic activities without motor side effects. We designated the compounds with such activities as γ-8 TARP dependent AMPA receptor antagonists (γ-8 TDAAs). In this work, we further investigated the mechanisms of action using a radiolabeled γ-8 TDAA and ternary structural modeling with mutational validations to characterize the LY3130481 binding to γ-8. The radioligand binding to the cells heterologously expressing GluA1 and/or γ-8 revealed that γ-8 TDAAs binds to γ-8 alone without AMPA receptors. Homology modeling of γ-8, based on the crystal structures of a distant TARP homologue, murine claudin 19, in conjunction with knowledge of two γ-8 residues previously identified as critical for the LY3130481 TARP-dependent selectivity provided the basis for a binding mode prediction. This allowed further rational mutational studies for characterization of the structural determinants in TARP γ-8 for LY3130481 activities, both thermodynamically as well as kinetically.

Methods for Evaluation of Positive Allosteric Modulators of Glutamate AMPA Receptors

Hypofunctioning of glutamate synaptic transmission in the central nervous system (CNS) has been proposed as a factor that may contribute to cognitive deficits associated with various neurological and psychiatric disorders. Positive allosteric modulation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) subtype of glutamate receptors has been proposed as a novel therapeutic approach, because these receptors mediate the majority of rapid excitatory neurotransmission and are intimately involved in long-term changes in synaptic plasticity thought to underlie mnemonic processing. By definition, positive allosteric modulators do not affect AMPA receptor activity alone but can markedly enhance ion flux through the ion channel pore in the presence of bound agonist. Despite this commonality, positive allosteric modulators can be segregated on the basis of the preferential effects on AMPA receptor subunits, their alternatively spliced variants and/or their biophysical mechanism of action. This chapter provides a detailed description of the methodologies used to evaluate the potency/efficacy and biophysical mechanism of action of positive allosteric modulators of AMPA receptors.