Recent biochemical studies indicate that the serotonin transporter can form oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. For this purpose, we generated fusion proteins of hSERT and spectral variants of the green fluorescent protein (cyan and yellow fluorescent proteins, CFP and YFP, respectively). When expressed in human embryonic kidney 293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were efficiently inserted into the plasma membrane and were functionally indistinguishable from wild-type hSERT. Oligomers were visualized by fluorescence resonance energy transfer microscopy in living cells using two complementary methods, i.e. ratio imaging and donor photobleaching. Interestingly, oligomerization was not confined to hSERT; fluorescence resonance energy transfer was also observed between CFP- and YFP-labeled rat γ-aminobutyric acid transporter. The bulk of serotonin transporters was recovered as high molecular weight complexes upon gel filtration in detergent solution. In contrast, the monomers of CFP-hSERT and YFP-hSERT were essentially undetectable. This indicates that the homo-oligomeric form is the favored state of hSERT in living cells, which is not significantly affected by coincubation with transporter substrates or blockers. Based on our observations, we conclude that constitutive oligomer formation might be a general property of Na⁺/Cl⁻-dependent neurotransmitter transporters.

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 Ca2+-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.

Na⁺/Cl⁻-dependent neurotransmitter transporters form constitutive oligomers, the significance of which is not known. In soluble proteins, leucine heptad repeats drive dimerization; the rat γ-aminobutyric acid transporter GAT-1 (rGAT) contains a motif reminiscent of a leucine heptad repeat in the second transmembrane helix (TM2). We substituted leucine residues in TM2 of rGAT by alanine and tested the ability of the resulting mutants to form oligomers by three methods of Förster resonance energy transfer (FRET) microscopy. Replacement of one leucine (L97A) resulted in considerable loss of energy transfer, replacing two or more ablated it completely. Furthermore, intracellular trapping increased with the number of leucine substitutions. Only rGAT-L97A reached the cell surface to a sufficient amount such that, in intact cells, it was indistinguishable from wild type rGAT with respect to substrate transport, binding of inhibitors, and regulation by protein kinase C. However, in membrane vesicles prepared from transfected cells, all mutants were still functional. In addition, FRET was readily detected during maturation of wild type rGAT, when the bulk of the protein resided in the endoplasmic reticulum. Hence, our findings strongly argue for a role of oligomer formation during biosynthesis and subsequent delivery of the multimer from the endoplasmic reticulum to the plasma membrane.

This review provides an overview on different antibody test methods that can be applied in cases of suspected paraneoplastic neurological syndromes (PNS) and anti-neuronal autoimmune encephalitis (AIE) in order to explain their diagnostic value, describe potential pitfalls and limitations, and discuss novel approaches aimed at discovering further autoantibodies. Onconeuronal antibodies are well-established biomarkers for PNS and may serve as specific tumor markers. The recommended procedure to detect onconeuronal antibodies is a combination of indirect immunohistochemistry on fixed rodent cerebellum and confirmation of the specificity by line assays. Simplification of this approach by only using line assays with recombinant proteins bears the risk to miss antibody-positive samples. Anti-neuronal surface antibodies are sensitive and specific biomarkers for AIE. Their identification requires the use of test methods that allow the recognition of conformation dependent epitopes. These commonly include cell-based assays and tissue based assays with unfixed rodent brain tissue. Tissue based assays can detect most of the currently known neuronal surface antibodies and thus enable broad screening of biological samples. A complementary testing on live neuronal cell cultures may confirm that the antibody recognizes a surface epitope. In patients with peripheral neuropathy, the screening may be expanded to teased nerve fibers to identify antibodies against the node of Ranvier. This method helps to identify a novel subgroup of peripheral autoimmune neuropathies, resulting in improved immunotherapy of these patients. Tissue based assays are useful to discover additional autoantibody targets that play a role in diverse autoimmune neurological syndromes. Antibody screening assays represent promising avenues of research to improve the diagnostic yield of current assays for antibody-associated autoimmune encephalitis.

The human dopamine transporter (hDAT) contains an endogenous high affinity Zn²⁺ binding site with three coordinating residues on its extracellular face (His¹⁹³, His³⁷⁵, and Glu³⁹⁶). Upon binding to this site, Zn²⁺ causes inhibition of [³H]1-methyl-4-phenylpyridinium ([³H]MPP⁺) uptake. We investigated the effect of Zn²⁺ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [³H]MPP⁺. Although Zn²⁺ inhibited uptake, Zn²⁺facilitated [³H]MPP⁺ release induced by amphetamine, MPP⁺, or K⁺-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn²⁺coordinating residue His¹⁹³ to Lys (the corresponding residue in hNET) eliminated the effect of Zn²⁺ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn²⁺sensitivity to hNET. The intracellular [³H]MPP⁺ concentration was varied to generate saturation isotherms; these showed that Zn²⁺ increasedV _max for efflux (rather than K_{M-Efflux-intracellular}). Thus, blockage of inward transport by Zn²⁺ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn²⁺, because Zn²⁺ also facilitated transport reversal of DAT in rat striatal slices.