Petra Scholze

Glycine transporters : essential regulators of synaptic transmission

Glycine is a major inhibitory neurotransmitter in the mammalian CNS (central nervous system). Glycinergic neurotransmission is terminated by the uptake of glycine into glycinergic nerve terminals and neighbouring glial cells. This uptake process is mediated by specific Na(+)/Cl(-)-dependent GlyTs (glycine transporters), GlyT1 and GlyT2. GlyT1, in addition, is thought to regulate the concentration of glycine at excitatory synapses containing NMDARs (N-methyl-D-aspartate receptors), which require glycine as a co-agonist. We have analysed the physiological roles and regulation of GlyT1 and GlyT2 by generating transporter-deficient mice and searching for interacting proteins. Our genetic results indicate that at glycinergic synapses, the glial transporter GlyT1 catalyses the removal of glycine from the synaptic cleft, whereas GlyT2 is required for the re-uptake of glycine into nerve terminals, thereby allowing for neurotransmitter reloading of synaptic vesicles. Both GlyT1 and GlyT2 are essential for CNS function, as revealed by the lethal phenotypes of the respective knockout mice. Mice expressing only a single GlyT1 allele are phenotypically normal but may have enhanced NMDAR function. GlyT2 is highly enriched at glycinergic nerve terminals, and Ca(2+)-triggered exocytosis and internalization are thought to regulate GlyT2 numbers in the pre-synaptic plasma membrane. We have identified different interacting proteins that may play a role in GlyT2 trafficking and/or pre-synaptic localization.

Serotonin-transporter mediated efflux: A pharmacological analysis of amphetamines and non-amphetamines

The physiological function of neurotransmitter transporter proteins like the serotonin transporter (SERT) is reuptake of neurotransmitter that terminates synaptic serotoninergic transmission. SERT can operate in reverse direction and be induced by SERT substrates including 5-HT, tyramine and the positively charged methyl-phenylpyridinium (MPP(+)), as well as the amphetamine derivatives para-chloroamphetamine (pCA) and methylene-dioxy-methamphetamine (MDMA). These substrates also induce inwardly directed sodium currents that are predominantly carried by sodium ions. Efflux via SERT depends on this sodium flux that is believed to be a prerequisite for outward transport. However, in recent studies, it has been suggested that substrates may be distinct in their properties to induce efflux. Therefore, the aim of the present study was a pharmacological characterization of different SERT substrates in uptake experiments, their abilities to induce transporter-mediated efflux and currents. In conclusion, the rank order of affinities in uptake and electrophysiological experiments correlate well, while the potencies of the amphetamine derivatives for the induction of efflux are clearly higher than those of the other substrates. These discrepancies can be only explained by mechanisms that can be induced by amphetamines. Therefore, based on our pharmacological observations, we conclude that amphetamines distinctly differ from non-amphetamine SERT substrates.

Two Discontinuous Segments in the Carboxyl Terminus Are Required for Membrane Targeting of the Rat γ-Aminobutyric Acid Transporter-1 (GAT1)

Like all members of the Na+/Cl--dependent neurotransmitter transporter family, the rat γ-aminobutyric acid transporter-1 (GAT1) is sorted and targeted to specialized domains of the cell surface. Here we identify two discontinuous signals in the carboxyl terminus of GAT1 that cooperate to drive surface expression. This conclusion is based on the following observations. Upon deletion of the last 37 amino acids, the resulting GAT1-Δ37 remained trapped in the endoplasmic reticulum. The presence of 10 additional residues (GAT1-Δ27) sufficed to support the interaction with the coat protein complex II component Sec24D; surface expression of GAT1-Δ27 reached 50% of the wild type level. Additional extensions up to the position -3 (GAT1-Δ3) did not further enhance surface expression. Thus the last three amino acids (AYI) comprise a second distal signal. The sequence AYI is reminiscent of a type II PDZ-binding motif; accordingly substituting Glu for Ile abrogated the effect of this motif. Neither the AYI motif nor the last 10 residues rescued the protein from intracellular retention when grafted onto GAT1-Δ37 and GAT1-Δ32; the AYI motif was dispensable for targeting of GAT1 to the growth cone of differentiating PC12 cells. We therefore conclude that the two segments act in a hierarchical manner such that the proximal motif (569VMI571) supports endoplasmic reticulum export of the protein and the distal AYI motif places GAT1 under the control of the exocyst.

The neuronal glycine transporter 2 interacts with the PDZ domain protein syntenin-1

The glycine transporter subtype 2 (GlyT2) is localized at glycinergic axon terminals where it mediates the re-uptake of glycine from the extracellular space. In this study, we used the yeast two-hybrid system to search for proteins that interact with the cytoplasmic carboxy terminal tail region of GlyT2. Screening of a rat brain cDNA library identified the PDZ domain protein syntenin-1 as an intracellular binding partner of GlyT2. In pull-down experiments, the interaction between GlyT2 and syntenin-1 was found to involve the C-terminal amino acid residues of GlyT2 and the PDZ2 domain of syntenin-1. Syntenin-1 is widely expressed in brain and co-localizes with GlyT2 in brainstem sections. Furthermore, syntenin-1 binds syntaxin 1A, which is known to regulate the plasma membrane insertion of GlyT2. Thus, syntenin-1 may be an in vivo binding partner of GlyT2 that regulates its trafficking and/or presynaptic localization in glycinergic neurons.

Characterization of Carrier‐Mediated Efflux in Human Embryonic Kidney 293 Cells Stably Expressing the Rat Serotonin Transporter: A Superfusion Study

Human embryonic kidney 293 cells stably transfected with the rat plasmalemmal serotonin transporter (rSERT) were incubated with 5‐[3H]hydroxytryptamine ([3H]5‐HT) and superfused. Substrates of the rSERT, such as p‐chloroamphetamine (PCA) or methylenedioxymethamphetamine, concentration‐dependently increased basal efflux of [3H]5‐HT. 5‐HT reuptake blockers (e.g., imipramine, citalopram) also caused an enhancement of [3H]5‐HT efflux, reaching about half the maximal effect of the rSERT substrates. In uptake experiments, both groups of substances concentration‐dependently inhibited 5‐HT uptake. EC50 values obtained in superfusion experiments significantly correlated with IC50 values from uptake studies (r2 = 0.92). Addition of the Na+,K+‐ATPase inhibitor ouabain (100 μM) to or the omission of K+ from the superfusion buffer accelerated basal efflux. The effect of PCA (10 μM) was markedly enhanced by both measures, whereas the effect of uptake inhibitors remained unchanged. When [3H]MPP+, a substrate with low affinity for the rSERT, was used instead of [3H]5‐HT for labeling the cells, uptake inhibitors failed to augment efflux. By contrast, PCA accelerated [3H]MPP+ efflux, and its effect was strongly enhanced in the presence of ouabain. The results suggest that the [3H]5‐HT efflux caused by substrates of rSERT is carrier‐mediated, whereas efflux induced by uptake inhibitors is a consequence of interrupted high‐affinity reuptake that is ongoing even under superfusion conditions.

Biochemical and functional properties of distinct nicotinic acetylcholine receptors in the superior cervical ganglion of mice with targeted deletions of nAChR subunit genes

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in ganglia of the autonomic nervous system. Here, we determined the subunit composition of hetero‐pentameric nAChRs in the mouse superior cervical ganglion (SCG), the function of distinct receptors (obtained by deletions of nAChR subunit genes) and mechanisms at the level of nAChRs that might compensate for the loss of subunits. As shown by immunoprecipitation and Western blots, wild‐type (WT) mice expressed: α3β4 (55%), α3β4α5 (24%) and α3β4β2 (21%) nAChRs. nAChRs in β4 knockout (KO) mice were reduced to < 15% of controls and no longer contained the α5 subunit. Compound action potentials, recorded from the postganglionic (internal carotid) nerve and induced by preganglionic nerve stimulation, did not differ between α5β4 KO and WT mice, suggesting that the reduced number of receptors in the KO mice did not impair transganglionic transmission. Deletions of α5 or β2 did not affect the overall number of receptors and we found no evidence that the two subunits substitute for each other. In addition, dual KOs allowed us to study the functional properties of distinct α3β4 and α3β2 receptors that have previously only been investigated in heterologous expression systems. The two receptors strikingly differed in the decay of macroscopic currents, the efficacy of cytisine, and their responses to the α‐conotoxins AuIB and MII. Our data, based on biochemical and functional experiments and several mouse KO models, clarify and significantly extend previous observations on the function of nAChRs in heterologous systems and the SCG.

Chronic exposure to manganese decreases striatal dopamine turnover in human alpha-synuclein transgenic mice

Interaction of genetic and environmental factors is likely involved in Parkinsons disease (PD). Mutations and multiplications of alpha-synuclein (α-syn) cause familial PD, and chronic manganese (Mn) exposure can produce an encephalopathy with signs of parkinsonism. We exposed male transgenic C57BL/6J mice expressing human α-syn or the A53T/A30P doubly mutated human α-syn under the tyrosine hydroxylase promoter and non-transgenic littermates to MnCl₂-enriched (1%) or control food, starting at the age of 4 months. Locomotor activity was increased by Mn without significant effect of the transgenes. Mice were sacrificed at the age of 7 or 20 months. Striatal Mn was significantly increased about three-fold in those exposed to MnCl₂. The number of tyrosine hydroxylase positive substantia nigra compacta neurons was significantly reduced in 20 months old mice (-10%), but Mn or transgenes were ineffective (three-way ANOVA with the factors gene, Mn and age). In 7 months old mice, striatal homovanillic acid (HVA)/dopamine (DA) ratios and aspartate levels were significantly increased in control mice with human α-syn as compared to non-transgenic controls (+17 and +11%, respectively); after Mn exposure both parameters were significantly reduced (-16 and -13%, respectively) in human α-syn mice, but unchanged in non-transgenic animals and mice with mutated α-syn (two-way ANOVA with factors gene and Mn). None of the parameters were changed in the 20 months old mice. Single HVA/DA ratios and single aspartate levels significantly correlated across all treatment groups suggesting a causal relationship between the rate of striatal DA metabolism and aspartate release. In conclusion, under our experimental conditions, Mn and human α-syn, wild-type and doubly mutated, did not interact to induce PD-like neurodegenerative changes. However, Mn significantly and selectively interacted with human wild-type α-syn on indices of striatal DA neurotransmission, the neurotransmitter most relevant to PD.

Affinity of various ligands for GABAA receptors containing α4β3γ2, α4γ2, or α1β3γ2 subunits

Abstract The potency of 30 benzodiazepine binding site ligands from 14 different structural classes for inhibition of [3H]Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate) binding to human embryonic kidney (HEK) 293 cells transiently transfected with α4β3γ2S or α1β3γ2S subunits of GABAA receptors was investigated. Most of these compounds were unable to significantly inhibit [3H]Ro 15-4513 binding to α4β3γ2S receptors under conditions where they potently inhibited binding to α1β3γ2S receptors. Nevertheless, compounds from four different structural classes were identified which exhibited a high affinity for α4β3γ2S receptors. Variation of the structure of these compounds could lead to new ligands selectively interacting with α4β3γ2S receptors. Compounds interacting with α4β3γ2S receptors were also able to inhibit [3H]Ro 15-4513 binding to receptors consisting of α4γ2S subunits with comparable potency. These results support the conclusion that the α subunit is a major determinant of the benzodiazepine binding site properties of GABAA receptors containing α and γ subunits.

Catecholamine outflow from mouse and rat brain slice preparations evoked by nicotinic acetylcholine receptor activation and electrical field stimulation

Mice with targeted deletions of neuronal nicotinic acetylcholine receptor (nAChR) subunit genes are valuable models to study nAChR function such as catecholamine outflow by presynaptic receptor activation. Contrary to the rat, our present knowledge on presynaptic nAChRs in mice primarily relies on observations made with synaptosomes. We have now used brain slices to investigate nicotine‐induced catecholamine outflow in wild type (WT) and nAChR (β2 and α5) knockout mice for a comparison with rat brain slice preparations.

Extensive Heterogeneity of Recombinant γ-Aminobutyric AcidA Receptors Expressed in α4β3γ2-Transfected Human Embryonic Kidney 293 Cells

Abstract Human embryonic kidney 293 cells transiently transfected with α 4 -, β 3 - and γ 2 -subunits of γ-aminobutyric acid A (GABA A ) receptors from the rat exhibited specific high affinity binding sites for [ 3 H]muscimol, [ 3 H]Ro 15-4513 and [ 35 S] t -butylbicyclophosphorothionate (TBPS). B max values obtained, however, were dramatically different for these compounds. In addition, GABA was able to inhibit only 20% of specific [ 35 S]TBPS binding to membranes from α 4 β 3 γ 2 -transfected cells. In order to investigate possible receptor heterogeneity, receptors were extracted from α 4 β 3 γ 2 -transfected cells and were fractionated by chromatography on an anti-γ 2 -, followed by an anti-α 4 - and an anti-β 3 -immunoaffinity column. Western blot analysis of the column eluates indicated the separate existence of GABA A receptors consisting of α 4 β 3 γ 2 -, α 4 β 3 - or β 3 -subunits in α 4 β 3 γ 2 -transfected cells. This, and the finding that only α 4 β 3 γ 2 - but not α 4 β 3 - or β 3 -receptors possess high affinity binding sites for all three radiolabeled ligands investigated, combined with the observation that [ 35 S]TBPS binding to receptors consisting of β 3 -subunits cannot be inhibited by GABA, can explain most of the binding data obtained. The present results suggest an inefficient assembly of γ 2 - with α 4 - and/or β 3 - subunits under the conditions used, and indicate that recombinant receptors expressed in HEK cells are not necessarily homogeneous. Copyright