Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP 2 ) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP 2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP 2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP 2 -synthesis. We tested the effects of the verified scarcity of PIP 2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP 2 in pull-down assays. On decreased PIP 2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamineinduced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP 2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP 2 . Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP 2 -dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP 2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.phosphoinositide | reuptake | release | mass spectrometry | amperometry

Background: DAT activity is regulated by protein kinases. Results: We identify Thr 53 as a DAT phosphorylation site in rat striatum by mass spectrometry and a phospho-specific antibody; Thr 53 mutation reduced dopamine influx and ablated transporter-mediated efflux. Conclusion: Phosphorylation of DAT Thr 53 is involved in transport activity. Significance: These results identify Thr 53 phosphorylation of DAT in vivo and elucidate associated functional properties.

No abstract

Mesial temporal lobe epilepsy (MTLE), the most common form of epilepsy, is characterised by cytoarchitectural abnormalities including neuronal cell loss and reactive gliosis in hippocampus. Determination of aberrant cytoskeleton protein expression by proteomics techniques may help to understand pathomechanism that is still elusive. We searched for differential expression of hippocampal proteins by an analytical method based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry unambiguously identifying 77 proteins analysed in eight control and eight MTLE hippocampi. Proteins were quantified and we observed 18 proteins that were altered in MTLE. Cytoskeleton proteins tubulin alpha-1 chain, beta-tubulin, profilin II, neuronal tropomodulin were significantly reduced and one actin spot was missing, whereas ezrin and vinculin were significantly increased in MTLE. Proteins of several classes as e.g. antioxidant proteins (peroxiredoxins 3 and 6), chaperons (T-complex protein 1-alpha, stress-induced-phosphoprotein 1), signaling protein MAP kinase kinase 1, synaptosomal proteins (synaptotagmin I, alpha-synuclein), NAD-dependent deacetylase sirtuin-2 and 26S protease regulatory subunit 7 protein, neuronal-specific septin 3 were altered in MTLE. Taken together, the findings may represent or lead to cytoskeletal impairment; aberrant antioxidant proteins, chaperons, MAP kinase kinase 1 and NAD-dependent deacetylase sirtuin-2 may have been involved in pathogenetic mechanisms and altered synaptosomal protein expression possibly reflects synaptic impairment in MTLE.

Kv1.2 ␣-subunits are components of low-threshold, rapidly activating voltage-gated potassium (Kv) channels in mammalian neurons. Expression and localization of Kv channels is regulated by trafficking signals encoded in their primary structure. Kv1.2 is unique in lacking strong trafficking signals and in exhibiting dramatic cell-specific differences in trafficking, which is suggestive of conditional trafficking signals. Here we show that a cluster of cytoplasmic C-terminal phosphorylation sites regulates Kv1.2 trafficking. Using tandem MS to analyze Kv1.2 purified from rat, human, and mouse brain, we identified in each sample in vivo phosphoserine (pS) phosphorylation sites at pS434, pS440, and pS441, as well as doubly phosphorylated pS440/pS441. We also found these sites, as well as pS449, on recombinant Kv1.2 expressed in heterologous cells. We found that phosphorylation at pS440/pS441 is present only on the post-endoplasmic reticulum (ER)/cell surface pool of Kv1.2 and is not detectable on newly synthesized and ER-localized Kv1.2, on which we did observe pS449 phosphorylation. Elimination of PS440/PS441 phosphorylation by mutation reduces cell-surface expression efficiency and functional expression of homomeric Kv1.2 channels. Interestingly, mutation of S449 reduces phosphorylation at pS440/pS441 and also decreases Kv1.2 cell-surface expression efficiency and functional expression. These mutations also suppress trafficking of Kv1.2/ Kv1.4 heteromeric channels, suggesting that incorporation of Kv1.2 into heteromeric complexes confers conditional phosphorylation-dependent trafficking to diverse Kv channel complexes. These data support Kv1.2 phosphorylation at these clustered C-terminal sites as playing an important role in regulating trafficking of Kv1.2-containing Kv channels.ion channel ͉ mass spectrometry ͉ proteomics ͉ brain ͉ phosphospecific V oltage-gated potassium, or Kv, channels that form as tetramers of Kv1 ␣-subunits play important and diverse roles in regulating excitability of axons, nerve terminals, and dendrites of mammalian neurons (1). The major Kv1 ␣-subunits in mammalian brain, Kv1.1, Kv1.2, and Kv1.4, are found predominantly in heterotetrameric channel complexes (2-4). Homotetrameric Kv1.2 channels form low-threshold, sustained-or delayed-rectifier Kv channels (5) and, when coassembled with other Kv1 ␣-subunits, can generate a diverse array of channel subtypes (6). In mammalian brain, Kv1.2 is found prominently along unmyelinated axons, in nerve terminals and/or in preterminal axon segments, at axon initial segments, and at juxtaparanodes of myelinated axons, but it is also present in the dendrites of some neurons (7). The physiological importance of Kv1.2 is underscored by the recent finding that Kv1.2 knockout mice have enhanced seizure susceptibility and die in the third postnatal week (8).Biosynthetic trafficking of Kv1 channels from their site of translation in the rough endoplasmic reticulum (ER) to the cell surface is governed by a set of intrinsic targeting motifs encoded in primary s...