The dopamine transporter(DAT) plays a key role in clearing extracellular dopamine(DA) from the synapse. Moreover DAT is a target for the action of widely abused psychostimulants such as cocaine and amphetamine(AMPH). AMPH is a substrate for the DAT and promotes the reversal of transport and thus release of DA in the synapse. This release is believed to be critical for the addictive properties of AMPH. In a search for proteins associated with the DAT we used the DAT C-terminus as bait in a yeast two-hybrid screen. From this we isolated a clone encoding a part of the sequence for Ca2+/Calmodulin dependent kinase II- (CaMKII- ). A direct interaction between CaMKII- and DAT C-terminus was verified by the ability of C-terminal DAT GST fusion proteins to pull down both purified CaMKII- and CaMKII- from rat brain extracts. The ability of CaMKII- to exist in a complex with DAT was supported by co-immunoprecipitation experiments. Also, their co-existence was supported by immunostanings of midbrain dopaminergic neurons. Using outside-out patches from heterologous cells stably expressing DAT or from dopaminergic neurons we were able to obtain evidence that addition of activated but not heat-inactivated CaMKII- to the patch pipette enhanced AMPH stimulated efflux of DA. This effect was markedly attenuated by mutation of N-terminal serines. We hypothesize that in the presence of AMPH, association of CaMKII with the C-terminus of DAT facilitates phosphorylation of N-terminal serines that in turn promotes reverse transport of DA. J.U.F. and H.K. contributed equally.
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Society for Neuroscience 35th annual meeting, 2005