AMPH causes DAT mediated DA efflux at least in part by increasing intracellular Ca2+
and stimulating CaMKII activity (Fog et al., 2006
; Gnegy et al., 2004
). Nonetheless, the molecular underpinnings of how CaMKII regulates DAT-mediated reverse transport of DA are not entirely defined. Here we show for the first time that: a) SYN1A directly interacts with residues in the first 33 amino acids of the DAT N-terminus (); b) AMPH increases the association of SYN1A with cell-surface DAT (); c) SYN1A promotes AMPH-induced DA efflux (–); and d) both the DAT/SYN1A interaction induced by AMPH and SYN1A regulation of AMPH-induced DA efflux are dependent on CaMKII activity ().
An increasing number of proteins have been reported to interact with DAT (reviewed by Torres, 2006
). For example Hic5 (a focal adhesion adaptor protein) and Pick1 (a PDZ domain containing protein) have been shown to interact with the DAT C-terminus. Importantly, CaMKII has been shown not only to interact with the DAT C-terminus but also to regulate AMPH-induced DA efflux (Fog et al., 2006
). RACK1 (receptor for activated C kinase) also has been shown to interact with the N-terminus of DAT. Interestingly, Lee et al
. reported that SYN1A interacts with the DAT N-terminus in a yeast two hybrid screen and also demonstrated that the full-length DAT N-terminus pulls down SYN1A from synaptosomal extracts and that DAT and SYN1A co-immunoprecipitate from synaptosomal extracts (Lee et al., 2004
). Here we define the region of interaction between DAT and SYN1A by showing with GST pull down assays that the first 33 amino acids of the DAT N-terminus are sufficient to bind recombinant SYN1A (). Since only the two recombinant proteins were present, our data demonstrate, for the first time, that the DAT/SYN1A interaction is direct.
In order to understand their functional significance it is important to determine whether interactions of associated proteins, such as SYN1A, with transporters are dynamically regulated, in which cellular compartment these interactions occur (intracellular versus plasma membrane), and whether these interactions are modulated by psychostimulants such as AMPH. We previously showed that AMPH increases NET/SYN1A interaction (Dipace et al., 2007
). Here, we show that AMPH increases DAT/SYN1A interaction in hDAT cells and in an ex vivo
preparation (striatal synaptosomes) specifically at the plasma membrane, where it could alter the functional properties of DAT. Because AMPH stimulates CaMKII activity (Fog et al., 2006
; Wei et al., 2007
), we hypothesized that the AMPH-induced increase in DAT/SYN1A association is supported by CaMKII activity. Consistent with this hypothesis, inhibition of CaMKII by small molecule or peptide inhibitors blocks the ability of AMPH to increase DAT/SYN1A association ().
For ion channels, such as the chloride selective anion channel CFTR, binding of SYN1A to the N-terminal tail regulates channel open probability (Chang et al., 2002
). Importantly, SYN1A also modulates the voltage dependence of activation of the Kv
1.2 potassium channel (Neshatian et al., 2007
) and decreases the macroscopic conductance of the Kv
1.1 potassium channel at depolarizing membrane potential (Michaelevski et al., 2007
). Intriguingly, for the Kv
1.1 channel, SYN1A increases the macroscopic conductance at resting membrane potential (Michaelevski et al., 2007
). These results suggest that SYN1A binding to membrane carriers regulates their functional conformations in a voltage dependent manner.
Other reports suggested that SYN1A interaction with GAT1 transporters inhibits the forward and reverse transport of GABA as measured by radiolabeled substrate in non-voltage clamped experiments (Deken et al., 2000
; Fan et al., 2006
; Wang et al., 2003
). For monoamine transporters, SYN1A regulates the ionic coupling of the SERT without affecting the rate of forward transport (Quick, 2003
). Consistently, SYN1A reduces the uncoupled component (channel-like activity) of the NET- mediated current (Sung et al., 2003
Considering that AMPH enhances DAT/SYN1A association we hypothesized that SYN1A binding to DAT regulates DA efflux. – demonstrate that increasing availability of intracellular SYN1A, either by overexpression or by intracellular perfusion with the whole-cell electrode, results in an increased AMPH-induced DA efflux both in hDAT cells and mouse midbrain DA neurons. DAT-mediated DA efflux has previously shown to be voltage-dependent and importantly to require depolarization (Khoshbouei et al., 2003
). Nevertheless we were able to record AMPH-induced DA efflux at a negative resting membrane potential by increasing SYN1A availability (). These results suggest that the requirement of depolarization for DA efflux is overcome by increased SYN1A interaction with DAT. We speculate that under physiological non-voltage clamped conditions endogenous SYN1A in concert with AMPH-induced depolarization (Kahlig et al., 2004
) is sufficient to sustain DA efflux. Also, an increased SYN1A interaction with DAT may alter the voltage-dependence of DA efflux promoting reverse transport at negative voltages. Therefore, modified DAT/SYN1A interactions caused by signaling pathways and/or by DAT polymorphisms could result in altered AMPH-induced DA efflux and constitutive DA efflux in the absence of AMPH (Mazei-Robison et al., 2008
The question remaining was whether this new functional role of SYN1A required CaMKII activity and thus might help to explain the requirement for CaMKII activity for AMPH-induced DA efflux (Fog et al., 2006
). shows that the ability of SYN1A to promote AMPH-induced DA efflux at a negative potential (−60 mV) was abolished by pharmacological inhibition of CaMKII. These results elucidate a new mechanism of AMPH action by which AMPH activates signaling pathways, including CaMKII, promoting DAT/SYN1A association and consequently the ability of DAT to efflux DA at resting membrane potentials. Also, these data highlight a new mechanism by which CaMKII regulates DA efflux and points both to CaMKII and SYN1A as putative possible targets for novel treatments for psychostimulant abuse.