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Background Monoamine transporters such as the dopamine transporter (DAT) and the serotonin transporter (SERT) mediate the reuptake of previously released monoamines dopamine (DA) and serotonin from the synaptic cleft; thereby, these transporters regulate the monoamine content available for synaptic transmission. Certain stimuli, such as changes in ionic composition of the extracellular fluid or psychostimulants (e.g. amphetamines) are able to induce outward transport and thus increase extracellular monoamine concentrations. Influx and efflux of substrate are thought to be asymmetrical processes regulated by intracellular kinases. It has been demonstrated that removal of N-terminal serines ablates amphetamine-induced reverse transport in the DAT. Furthermore, the Ca/calmodulin-dependent protein kinase II a (aCaMKII) can bind to the DAT C-terminus and phosphorylate N-terminal serines. Pharmacological inhibition of aCaMKII dramatically reduces amphetamine-induced efflux both in cells stably transfected with the human DAT as well as in rat striatal slices. Here, we test whether aCaMKII-regulation of amphetamineinduced reverse transport of monoamines is affected in mice with mutations in the aCaMKII gene.

Background The organic cation transporter 3 (OCT3) shows a broad expression pattern in the nervous system and has been detected in neurons and glial cells. Here, OCT3 serves as an additional re-uptake system for neurotransmitters, such as dopamine, serotonin and norepinephrine and therefore OCT3 mediated uptake has been termed “uptake 2”. Methods We measured OCT3 mediated uptake of the fluorescent OCT3 substrate 4-Di-1-ASP (4-(-4(dimethylamino(styryl)-N-methylpyridinium (ASP) in real-time in HEK293 cells stably expressing the human and the mouse isoforms of OCT3. Data obtained on OCT3 mediated uptake of fluorescent ASP were compared to uptake of tritiated 1-methyl-4-phenylpyridinium (MPP). Results We show that ASP is selectively taken up via OCT3 in real time and this allows for sensitive assessment of transport via OCT3. Hence, we analyzed the mode of action of several OCT3 substrates and transport inhibitors, such as the stress hormone corticosterone and decynium-22. All results obtained from ASP uptake studies are comparable with data obtained from uptake studies with tritiated MPP and in line with previously published reports. Finally, we tested if OCT-mediated uptake is sensitive to phosphorylation and found that the protein kinase C inhibitor GF109203X inhibited uptake. Discussion The use of ASP as substrate for OCT3 provides the opportunity to analyze OCT3 mediated transport with a high temporal resolution and is comparable to OCT3 mediated uptake of tritiated MPP. Uptake inhibition by corticosterone was comparable using either ASP or MPP and similar to inhibition of protein kinase C.

Background Neuronal functions, such as excitability or endoand exocytosis, require phosphatidylinositol-4,5-bisphosphate (PIP2) since ion channels and other proteins involved in these processes are regulated by PIP2. Monoamine transporters control neurotransmission by removing monoamines from the extracellular space. They also display channel properties, but their regulation by PIP2 has not been reported. The psychostimulant amphetamine acts on monoamine transporters to stimulate transportermediated currents and efflux and thereby increases the levels of extracellular monoamines.

Most cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y(1), P2Y(2), P2Y(12), P2Y(13), P2X(2), A(1), A(2A) receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y(1), P2Y(12), P2Y(13), A(1), A(2A), and P2X(2) receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not NTPDase2. The specificity of prototypic interactions (P2Y(1)/P2Y(1), A(2A)/P2Y(1), A(2A)/P2Y(12)) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network.