Influence of different cellular environments on [3H]DASB radioligand binding

Alterations in serotonergic neurotransmission are implicated in a variety of neurobiological disorders. One way in which serotonergic tone can be regulated is via the serotonin transporter protein (SERT). Differences in SERT binding using in vivo PET imaging have been observed between those suffering from major depression, anxiety, and obsessive compulsive disorder (OCD) compared with healthy controls (Matsumoto et al., 2010; Miller et al., 2009; Reimold et al., 2008). Additionally, alterations in SERT binding have been reported following drug use. For example, decreased SERT binding after experimental (reviewed in (Capela et al., 2009)) and recreational exposure to 3,4-methylenedioxymethamphetamine (MDMA) has been observed in numerous studies (Buchert et al., 2004; Erritzoe et al., 2011; McCann et al., 2005; Reneman et al., 2001). Whether or not the changes in SERT binding observed in these studies represents decreased SERT protein levels, changes in affinity of the SERT protein for the radioligand in question, loss of serotonergic neurons or endocytosis or downregulation of membrane bound SERT protein remains to be fully elucidated. SERT has been shown to endocytose in a clathrindependent manner to transferrin-positive endosomes (Kivell et al., 2010). MDMA and 5-HT have been shown to alter the distribution of SERT (Kittler et al., 2010; Kivell et al., 2010) between plasma membrane bound and sub-cellular compartments. Additionally, selective serotonin reuptake inhibitors have been shown to decrease SERT radioligand binding, in the absence of gene expression changes (Benmansour, 2002). The D2 receptor is known to readily internalize (Kim et al., 2001). The binding of radioligands selective for dopamine receptors have recently been suggested to be sensitive to their cellular compartments (Quelch et al., 2011a). In these studies, a decreased affinity for [H]raclopride and [H](1)PHNO was observed in endosomal conditions compared with those present at the cell surface. This decrease in affinity following endocytosis to the endosomal compartments is thought to contribute to the reduction in binding observed following pharmacological induction of dopamine release in imaging studies with these ligands. SERT is a 12 trans-membrane (TM) spanning protein; however, only 7-TM spanning GPCRs (e.g., D2) have been investigated in this in vitro model so far. Therefore, it could be speculated that the PET radioligand used for binding SERT in imaging studies, for example [C]DASB, may also exhibit a reduced ability to bind in the sub-cellular conditions compared with those at the cell membrane. In this study, we aimed to characterize the ability of [H]DASB to bind in different ionic environments, representative of the cellular compartments involved in the clathrin-dependent endocytosis pathway of SERT. Subsequently, it could be speculated that a decrease in [C]DASB radioligand binding observed in patient or drug user populations could then at least in part represent a decreased ability of the ligand to bind SERT proteins located in sub-cellular compartments as a result of drug use, disease, or pharmacotherapy.