Trafficking and ethanol-induced inhibition of AMPA receptors

AMPA receptors are an important class of ionotropic glutamate receptors which participate in fast excitatory synaptic transmission in most brain areas. They have a pivotal role in adjustment of cell membrane excitability as their cell membrane expression levels is altered in brain physiology such as in learning and memory formation. AMPA receptor function and trafficking is regulated by several proteins, such as transmembrane AMPA receptor regulatory proteins (TARPs). NMDA-type glutamate receptors are important target molecules of ethanol. The role of AMPA receptors in the actions of ethanol has not been clarified as thoroughly. Furthermore, the regulation of AMPA receptor synthesis and their possible adaptation in neurons with altered inhibitory mechanisms are poorly understood. In this thesis work AMPA receptor pharmacology, trafficking and synaptic localization was studied using patch-clamp electrophysiology. Both native and recombinant AMPA receptors were studied. Hippocampal slices from transgenic Thy1alfa6 mice with altered inhibition were used to study adaptation of AMPA receptors. Ethanol was found to inhibit AMPA receptor function by increasing desensitization of the receptor, as the steady-state current was inhibited more than the peak current. Ethanol inhibition was reduced when cyclothiazide was used to block desensitization and when non-desensitizing mutant receptors were studied. Ethanol also increased the rate of desensitization, which was increased further by the coexpression of TARP-proteins. We found that the agonist binding capability is important for trafficking AMPA receptors from endoplasmic reticulum to the cell membrane. TARP rescues the surface expression of non-binding AMPA receptor mutants in HEK293 cells, but not in native neurons. Studies with Thy1alfa6 mice revealed that decreased inhibition decrease AMPA receptor mediated excitation keeping the neurotransmission in balance. Thy1alfa6 mice also had lower sensitivity to electroshock convulsions, presumably due to the decreased AMPA receptor function. The results suggest that during alcohol intoxication ethanol may inhibit AMPA receptors by increasing the rate and the extent of desensitization. TARPs appear to enhance ethanol inhibition. TARPs also participate in trafficking of AMPA receptors upon their synthesis in the cell. AMPA receptors mediate also long-term adaptation to altered neuronal excitability, which adds to their well-known role in synaptic plasticity.FM Tommi Möykkysen väitöstutkimuksessa tutkittiin aivojen yleisimmän kiihdyttävän välittäjäaineen, glutamaatin, AMPA-tyyppisten reseptorien kuljetusta ja ilmentymistä solukalvolla sekä alkoholin (etanolin) vaikutuksia kyseisiin reseptoreihin. AMPA reseptorit ovat keskeisiä hermosolujen toiminnassa, koska ne välittävät sähkösignaalien siirtymistä hermosolulta toiselle kaikkialla aivoissa. Niiden määrän ja ominaisuuksien säätely on tärkeää normaaleissa fysiologisissa tilanteissa, kuten muistin ja oppimisen yhteydessä. Tämän lisäksi AMPA reseptorien toiminnan on havaittu muuttuneen myös joissakin tautitiloissa ja huumausaineiden käytön yhteydessä. Hermosoluilla tehdyissä kokeissa etanolin todettiin lisäävän AMPA reseptoreiden inaktivaatiota, eli ei-aktiivista tilaa, mikä luonnollisesti estää reseptorien toimintaa. HEK-solulinjalla tehdyissä lisäkokeissa etanolin havaittiin nopeuttavan inaktiiviseen tilaan siirtymistä. Kokeissa havaittiin myös, että TARP-proteiinit nopeuttivat entisestään inaktiiviseen tilaan siirtymistä. Reseptorien solukalvoilmentymistä koskevissa tutkimuksissa osoitettiin, että reseptorien kyky sitoa välittäjäaine glutamaattia tarkastetaan solun sisällä ennen kuin ne kuljetetaan lopulliseen määränpäähänsä, solukalvolle. Vähentyneen estävän viestinvälityksen puolestaan todettiin vähentävän AMPA reseptorivälitteistä kiihdyttävää viestinvälitystä, mikä viittaa siihen, että hermosolut pitävät yllä tasapainotilaa hermoviestinvälityksessä. Etanolin on todettu vaikuttavat moniin aivojen välittäjäainesysteemeihin ja proteiineihin, ja onkin ollut vaikeaa osoittaa mitkä näistä vaikutuksista ovat tärkeitä humalatilassa ja alkoholin aiheuttamissa terveyshaitoissa. Glutamaattireseptorien on pitkään tiedetty olevan etanolin kohdemolekyylejä, mutta näistä vain NMDA reseptoreita on pidetty kliinisesti tärkeänä. Tämä väitöstutkimus paljastaa osaltaan miten etanoli estää AMPA reseptorien toimintaa ja paljastaa ne olosuhteet missä etanoli vaikuttaa myös AMPA reseptoreihin. Tutkimustulosten perusteella voidaan päätellä, että etanoli vaikuttaa AMPA reseptoreihin niillä aivoalueilla, missä inaktivaatiolla on merkitystä reseptoreiden toiminnassa. Tulokset osoittavat lisäksi, että AMPA reseptoreiden kanssa yhteydessä olevat TARP-proteiinit vaikuttavat reseptorien etanoliherkkyyteen. Tuloksista voivat osaltaan auttaa alkoholin terveyshaittojen selvittämisessä ja torjumisessa

L-Cysteine Containing Vitamin Supplement Which Prevents or Alleviates Alcohol-related Hangover Symptoms : Nausea, Headache, Stress and Anxiety

Correction ALCOHOL AND ALCOHOLISM Volume: 55 Issue: 6 Pages: 705-705 DOI: 10.1093/alcalc/agaa088 Published: NOV 2020Aims: Alcohol-related hangover symptoms: nausea, headache, stress and anxiety cause globally considerable amount of health problems and economic losses. Many of these harmful effects are produced by alcohol and its metabolite, acetaldehyde, which also is a common ingredient in alcohol beverages. The aim of the present study is to investigate the effect of the amino acid L-cysteine on the alcohol/acetaldehyde related aftereffects. Methods: Voluntary healthy participants were recruited through advertisements. Volunteers had to have experience of hangover and/or headache. The hangover study was randomized, double-blind and placebo-controlled. Nineteen males randomly swallowed placebo and L-cysteine tablets. The alcohol dose was 1.5 g/kg, which was consumed during 3 h. Results: The primary results based on correlational analysis showed that L-cysteine prevents or alleviates hangover, nausea, headache, stress and anxiety. For hangover, nausea and headache the results were apparent with the L-cysteine dose of 1200 mg and for stress and anxiety already with the dose of 600 mg. Conclusions: L-cysteine would reduce the need of drinking the next day with no or less hangover symptoms: nausea, headache, stress and anxiety. Altogether, these effects of L-cysteine are unique and seem to have a future in preventing or alleviating these harmful symptoms as well as reducing the risk of alcohol addiction.Peer reviewe

Autoinactivation of the Stargazin–AMPA Receptor Complex: Subunit-Dependency and Independence from Physical Dissociation

are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steadystate responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This ‘‘autoinactivation’’ has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1–A4 AMPA receptors (all flip isoform) expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.Peer reviewe

Importance of GluA1 Subunit-Containing AMPA Glutamate Receptors for Morphine State-Dependency

Peer reviewe

Aggregation Limits Surface Expression of Homomeric GluA3 Receptors

AMPA receptors are glutamate-gated cation channels assembled from GluA1-4 subunits and have properties that are strongly dependent on the subunit composition. The subunits have different propensities to form homomeric or various heteromeric receptors expressed on cell surface, but the underlying mechanisms are still poorly understood. Here, we examined the biochemical basis for the poor ability of GluA3 subunits to form homomeric receptors, linked previously to two amino acid residues, Y454 and R461, in its ligand-binding domain (LBD). Surface expression of GluA3 was improved by co-assembly with GluA2 but not with stargazin, a trafficking chaperone and modulator of AMPA receptors. The secretion efficiency of GluA2 and GluA3 LBDs paralleled the transport difference between the respective full-length receptors and was similarly dependent on Y454/R461, but not on LBD stability. In comparison to GluA2, GluA3 homomeric receptors showed a strong and Y454/R461-dependent tendency to aggregate both in the macroscopic scale measured as lower solubility in nonionic detergent and in the microscopic scale evident as the preponderance of hydrodynamically large structures in density gradient centrifugation and native gel electrophoresis. We conclude that the impaired surface expression of homomeric GluA3 receptors is caused by nonproductive assembly and aggregation to which LBD residues Y454 and R461 strongly contribute. This aggregation inhibits the entry of newly synthesized GluA3 receptors to the secretory pathway

Ethanol increases desensitization of recombinant GluR-D AMPA receptor and TARP combinations

Glutamate receptors are important target molecules of the acute effect of ethanol. We studied ethanol sensitivity of homomeric GluR-D receptors expressed in human embryonic kidney 293 cells and examined whether recently discovered transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory proteins (TARPs) affect ethanol sensitivity. Coexpression of the TARPs, stargazin, and γ4 increased the time constant (τ-value) of current decay in the presence of agonist, thus slowing the onset of desensitization and increasing the steady-state current. Ethanol produced less inhibition of the peak current than the steady-state current for all types of the GluR-D receptors. In addition, ethanol concentration-dependently accelerated the rate of desensitization, measured as the τ-value of fast decay of peak current. This effect was enhanced with coexpression of TARPs. The recovery from desensitization was slowed down by coexpression of γ4 but ethanol did not affect this process in any GluR-D combination. The results support the idea that increased desensitization is an important mechanism in the ethanol inhibition of AMPA receptors and indicate that coexpression of TARPs can alter this effect of ethanol

Functional changes of AMPA responses in human induced pluripotent stem cell-derived neural progenitors in fragile X syndrome

International audienceAltered neuronal network formation and function involving dysregulated excitatory and inhibitory circuits are associated with fragile X syndrome (FXS). We examined functional maturation of the excitatory transmission system in FXS by investigating the response of FXS patient-derived neural progenitor cells to the glutamate analog (AMPA). Neural progenitors derived from induced pluripotent stem cell (iPSC) lines generated from boys with FXS had augmented intracellular Ca 2+ responses to AMPA and kainate that were mediated by Ca 2+-permeable AMPA receptors (CP-AMPARs) lacking the GluA2 subunit. Together with the enhanced differentiation of glutamate-responsive cells, the proportion of CP-AMPAR and N-methyl-d-aspartate (NMDA) receptor-coexpressing cells was increased in human FXS progenitors. Differentiation of cells lacking GluA2 was also increased and paralleled the increased inward rectification in neural progenitors derived from Fmr1-knockout mice (the FXS mouse model). Human FXS progenitors had increased the expression of the precursor and mature forms of miR-181a, a microRNA that represses translation of the transcript encoding GluA2. Blocking GluA2-lacking, CP-AMPARs reduced the neurite length of human iPSC-derived control progenitors and further reduced the shortened length of neurites in human FXS progenitors, supporting the contribution of CP-AMPARs to the regulation of progenitor differentiation. Furthermore , we observed reduced expression of Gria2 (the GluA2-encoding gene) in the frontal lobe of FXS mice, consistent with functional changes of AMPARs in FXS. Increased Ca 2+ influx through CP-AMPARs may increase the vulnerability and affect the differentiation and migration of distinct cell populations, which may interfere with normal circuit formation in FXS