Derek Bowie

Control of GluR1 AMPA Receptor Function by cAMP-Dependent Protein Kinase

Modulation of postsynaptic AMPA receptors in the brain by phosphorylation may play a role in the expression of synaptic plasticity at central excitatory synapses. It is known from biochemical studies that GluR1 AMPA receptor subunits can be phosphorylated within their C terminal by cAMP-dependent protein kinase A (PKA), which is colocalized with the phosphatase calcineurin (i.e., phosphatase 2B). We have examined the effect of PKA and calcineurin on the time course, peak open probability (PO,PEAK), and single-channel properties of glutamateevoked responses for neuronal AMPA receptors and homomeric GluR1(flip) receptors recorded in outside-out patches. Inclusion of purified catalytic subunit Cα-PKA in the pipette solution increased neuronal AMPA receptorPO,PEAK (0.92) compared with recordings made with calcineurin included in the pipette (PO,PEAK 0.39). Similarly, Cα-PKA increased PO,PEAK for recombinant GluR1 receptors (0.78) compared with patches excised from cells cotransfected with a cDNA encoding the PKA peptide inhibitor PKI (PO,PEAK 0.50) or patches with calcineurin included in the pipette (PO,PEAK 0.42). Neither PKA nor calcineurin altered the amplitude of single-channel subconductance levels, weighted mean unitary current, mean channel open period, burst length, or macroscopic response waveform for recombinant GluR1 receptors. Substitution of an amino acid at the PKA phosphorylation site (S845A) on GluR1 eliminated the PKA-induced increase in PO,PEAK, whereas the mutation of a Ca2+,calmodulin-dependent kinase II and PKC phosphorylation site (S831A) was without effect. These results suggest that AMPA receptor peak response open probability can be increased by PKA through phosphorylation of GluR1 Ser845.

Inward rectification of both AMPA and kainate subtype glutamate receptors generated by polyamine-mediated ion channel block

CA2+-permeable glutamate receptors assembled from subunits containing a GLN residue at the RNA editing site in membrane domain 2 show strong inward rectification. In HEK 293 cells transfected with the kainate receptor subunit GluR6(Q), inward rectification is lost in outside-out patches, suggesting a role for diffusible, cytoplasmic factors. Inclusion of different polyamines in the internal solution restored inward rectification, whereas Mg2+ (1 mM) was inactive. Spermidine (Kd[0 mV] = 5.5 microM) was of higher affinity than spermidine (Kd[0 mV] = 25.4 microM) or putrescine (Kd[0 mV] = 1.2 mM). AMPA receptors assembled from GluRA(flip) showed even higher affinity for spermine (Kd[0 mV] = 1.5 microM). Analysis of the voltage dependence of whole-cell responses predicted intracellular free spermine and spermidine concentrations of 51 and 153 muM, respectively.

Structural determinants of allosteric regulation in alternatively spliced AMPA receptors

The flip and flop splice variants of AMPA receptors show strikingly different sensitivity to allosteric regulation by cyclothiazide; heteromers assembled from GluR-A and GluR-B also exhibit splice variant-dependent differences in efficacy for activation by glutamate and kainate. The sensitivity for attenuation of desensitization by cyclothiazide for homomeric GluR-A was solely dependent upon exchange of Ser-750 (flip) and Asn-750 (flop), and was unaffected by mutagenesis of other divergent residues. In contrast, substantial alteration of the relative efficacy of glutamate versus kainate required mutation of multiple residues in the flip/flop region. Modulation by cyclothiazide was abolished by mutation of Ser-750 to Gin, the residue found at the homologous site in kainate-preferring subunits, whereas introduction of Ser at this site in GluR6 imparted sensitivity to cyclothiazide.

Ionotropic glutamate receptors & CNS disorders.

Disorders of the central nervous system (CNS) are complex disease states that represent a major challenge for modern medicine. Although aetilogy is often unknown, it is established that multiple factors such as defects in genetics and/or epigenetics, the environment as well as imbalance in neurotransmitter receptor systems are all at play in determining an individuals susceptibility to disease. Gene therapy is currently not available and therefore, most conditions are treated with pharmacological agents that modify neurotransmitter receptor signaling. Here, I provide a review of ionotropic glutamate receptors (iGluRs) and the roles they fulfill in numerous CNS disorders. Specifically, I argue that our understanding of iGluRs has reached a critical turning point to permit, for the first time, a comprehensive re-evaluation of their role in the cause of disease. I illustrate this by highlighting how defects in AMPA receptor (AMPAR) trafficking are important to fragile X mental retardation and ectopic expression of kainate receptor (KAR) synapses contributes to the pathology of temporal lobe epilepsy. Finally, I discuss how parallel advances in studies of other neurotransmitter systems may allow pharmacologists to work towards a cure for many CNS disorders rather than developing drugs to treat their symptoms.

Excitotoxic Death of Retinal Neurons In Vivo Occurs via a Non-Cell-Autonomous Mechanism

The central hypothesis of excitotoxicity is that excessive stimulation of neuronal NMDA-sensitive glutamate receptors is harmful to neurons and contributes to a variety of neurological disorders. Glial cells have been proposed to participate in excitotoxic neuronal loss, but their precise role is defined poorly. In this in vivo study, we show that NMDA induces profound nuclear factor κB (NF-κB) activation in Müller glia but not in retinal neurons. Intriguingly, NMDA-induced death of retinal neurons is effectively blocked by inhibitors of NF-κB activity. We demonstrate that tumor necrosis factor α (TNFα) protein produced in Müller glial cells via an NMDA-induced NF-κB-dependent pathway plays a crucial role in excitotoxic loss of retinal neurons. This cell loss occurs mainly through a TNFα-dependent increase in Ca2+-permeable AMPA receptors on susceptible neurons. Thus, our data reveal a novel non-cell-autonomous mechanism by which glial cells can profoundly exacerbate neuronal death following excitotoxic injury.

Permeation and block of rat glur6 glutamate receptor channels by internal and external polyamines

1 Polyamine block of rat GluR6(Q) glutamate receptor channels was studied in outside‐out patches from transiently transfected HEK 293 cells. With symmetrical 150 mm Na+ and 30 μm internal spermine there was biphasic voltage dependence with 95% block at +40 mV but only 20% block at +140mV. Dose–inhibition analysis for external spermine also revealed biphasic block; the Ka at +40 mV (54 μm) was lower than at +80 (167μm) and –80 mV (78 μm). 2 For internal polyamines relief from block was most pronounced for spermine, weaker for N‐(4‐hydroxyphenylpropanoyl)‐spermine (PPS), and virtually absent for philanthotoxin 343 (PhTX 343), suggesting that permeation of polyamines varies with cross‐sectional width (spermine, 0.44 nm; PPS, 0.70 nm; PhTX 343, 0.75 nm). 3 With putrescine, spermidine, or spermine as sole external cations, inward currents at –120 mV confirmed permeation of polyamines. For bi‐ionic conditions with 90 mm polyamine and 150 mm Na+i reversal potentials were –12.4 mV for putrescine (permeability ratio relative to Na+, PPut/PNa= 0.42) and –32.7 mV for spermidine (PSpd/PNa= 0.07). Currents carried by spermine were too small to analyse accurately in the majority of patches. 4 Increasing [Na+]i from 44 to 330 mm had no effect on the potential for 50% block (V½) by 30 μm internal spermine; however, relief from block at positive membrane potentials increased with [Na+]i. In contrast, raising [Na+]o from 44 to 330 mm resulted in a depolarizing shift in V½, indicating a strong interaction between internal polyamines and external per meant ions. 5 The Woodhull infinite barrier model of ion channel block adequately described the action of spermine at membrane potentials insufficient to produce relief from block. For 30 μm internal spermine such analysis gave Kd(0)= 2.5 μm, z θ= 1.97; block by 30 μm external spermine was weaker and less voltage dependent (Kd(0)= 37.8 μm and zδ= 0.55); δ and θ are electrical distances measured from the outside and inside, respectively. 6 Fits of the Woodhull equation for a permeable blocker adequately described both onset and relief from block by spermine over a wide range of membrane potentials. However, the rate constants and zδ values estimated for block by internal spermine predicted much stronger external block than was measured experimentally, and vice versa. 7 An Eyring rate theory model with two energy wells and three barriers explained qualitatively many characteristic features of the action of polyamines on GluPvs, including biphasic I–V relationships, weaker block by external than internal spermine and low permeability.

External anions and cations distinguish between AMPA and kainate receptor gating mechanisms

Experiments were designed to examine if ion‐flow through α‐amino‐3‐hydroxy‐5‐methyl‐isoxazolepropionic acid (AMPA) or kainate receptors interferes with protein structures associated with the gating machinery. Gating was studied using ultra‐fast drug perfusion of outside‐out patches containing rat GluR‐A or GluR6 subunits excised from transfected human embryonic kidney cells. Deactivation rates of GluR6 kainate receptors observed following brief l‐glutamate (10 mm Glu, 1 ms) applications differed by two to threefold in high (405 mm symmetrical Na+, τdecay= 2.7 ms at −100 mV) and low ionic strength (55 mm, τdecay= 1.1 ms) solutions. In comparison, GluR‐A AMPA receptors were much less sensitive. Ion effects on GluR6 receptors did not reflect surface potential screening or ion‐agonist competition at the agonist‐binding site since deactivation rates were slower in high ionic strength solutions. Moreover, the apparent agonist affinity did not decrease with increasing ionic strength (e.g. 55 mm, EC50= 110 μmvs. 405 mm, EC50= 61 μm). GluR6 responses were strongly dependent on ions present on the external, but not the internal, side of the plasma membrane. Decay kinetics was regulated by the type of ion present suggesting that the chemical nature of the solution, not its ionic strength, governed channel behaviour. Both external anions and cations modulated the amplitude and decay kinetics of GluR6 responses in a concomitant manner. AMPA receptor responses recorded in identical ionic conditions did not exhibit this behaviour. These results identify a novel mechanism that distinguishes AMPA and kainate receptors. External ions regulate the gating machinery of kainate receptors through an allosteric mechanism that involves both anions and cations.

Allosteric regulation and spatial distribution of kainate receptors bound to ancillary proteins.

A diverse range of accessory proteins regulates the behaviour of most ligand‐ and voltage‐gated ion channels. For glutamate receptor 6 (GluR6) kainate receptors, two unrelated proteins, concanavalin‐A (Con‐A) and postsynaptic density protein 95 (PSD‐95), bind to extra‐ and intracellular domains, respectively, but are reported to exert similar effects on GluR6 desensitization behaviour. We have tested the hypothesis that distinct allosteric binding sites control GluR6 receptors via a common transduction pathway. Rapid agonist application to excised patches revealed that neither Con‐A nor PSD‐95 affect the onset of desensitization. The rate of desensitization elicited by 10 mm l‐glutamate was similar in control (τfast= 5.5 ± 0.4 ms), Con‐A‐treated patches (τfast= 6.1 ± 0.5 ms) and patches containing PSD‐95 and GluR6 receptors (τfast= 4.7 ± 0.6 ms). Likewise, the time course of recovery from GluR6 desensitization was similar in both control and Con‐A conditions, whereas PSD‐95 accelerated recovery almost twofold. Peak and steady‐state (SS) dose‐response relationships to glutamate were unchanged by lectin treatment (e.g. control, EC50(SS)= 31 ± 28 μmvs Con‐A, EC50(SS)= 45 ± 9 μm, n= 6), suggesting that Con‐A does not convert non‐conducting channels with high agonist affinity into an open conformation. Instead, we demonstrate that the effects of Con‐A on macroscopic responses reflect a shift in the relative contribution of different open states of the channel. In contrast, the effect of PSD‐95 on recovery behaviour suggests that the association between kainate receptors and cytoskeletal proteins regulates signalling at glutamatergic synapses. Our results show that Con‐A and PSD‐95 regulate kainate receptors via distinct allosteric mechanisms targeting selective molecular steps in the transduction pathway.

Redefining the classification of AMPA‐selective ionotropic glutamate receptors

Abstract  AMPA‐type ionotropic glutamate receptors (iGluRs) represent the major excitatory neurotransmitter receptor in the developing and adult vertebrate CNS. They are crucial for the normal hardwiring of glutamatergic circuits but also fine tune synaptic strength by cycling into and out of synapses during periods of sustained patterned activity or altered homeostasis. AMPARs are grouped into two functionally distinct tetrameric assemblies based on the inclusion or exclusion of the GluA2 receptor subunit. GluA2‐containing receptors are thought to be the most abundant AMPAR in the CNS, typified by their small unitary events, Ca2+ impermeability and insensitivity to polyamine block. In contrast, GluA2‐lacking AMPARs exhibit large unitary conductance, marked divalent permeability and nano‐ to micromolar polyamine affinity. Here, I review evidence for the existence of a third class of AMPAR which, though similarly Ca2+ permeable, is characterized by its near‐insensitivity to internal and external channel block by polyamines. This novel class of AMPAR is most notably found at multivesicular release synapses found in the avian auditory brainstem and mammalian retina. Curiously, these synapses lack NMDA‐type iGluRs, which are conventionally associated with controlling AMPAR insertion. The lack of NMDARs suggests that a different set of rules may govern AMPAR cycling at these synapses. AMPARs with similar functional profiles are also found on some glial cells suggesting they may have a more widespread distribution in the mammalian CNS. I conclude by noting that modest changes to the ion‐permeation pathway might be sufficient to retain divalent permeability whilst eliminating polyamine sensitivity. Consequently, this emerging AMPAR subclass need not be assembled from novel subunits, yet to be cloned, but could simply occur by varying the stoichiometry of existing proteins.

Automating single subunit counting of membrane proteins in mammalian cells.

Background: Although powerful, single subunit counting is time-consuming, prone to user bias, and largely restricted to Xenopus expression. Results: PIF is an automated analysis program that identifies subunit stoichiometry of any fluorescently tagged membrane protein from TIRF recordings. Conclusion: PIF is accurate to more than 90% even in noisy data typical for mammalian expression system. Significance: The PIF approach is generalizable to any membrane protein and TIRF microscope. Elucidating subunit stoichiometry of neurotransmitter receptors is preferably carried out in a mammalian expression system where the rules of native protein assembly are strictly obeyed. Although successful in Xenopus oocytes, single subunit counting, manually counting photobleaching steps of GFP-tagged subunits, has been hindered in mammalian cells by high background fluorescence, poor control of expression, and low GFP maturation efficiency. Here, we present a fully automated single-molecule fluorescence counting method that separates tagged proteins on the plasma membrane from background fluorescence and contaminant proteins in the cytosol or the endoplasmic reticulum and determines the protein stoichiometry. Lower GFP maturation rates observed in cells cultured at 37 °C were partly offset using a monomeric version of superfolder GFP. We were able to correctly identify the stoichiometry of GluK2 and α1 glycine receptors. Our approach permits the elucidation of stoichiometry for a wide variety of plasma membrane proteins in mammalian cells with any commercially available TIRF microscope.