Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIalpha stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIalpha phosphorylated serines in the distal N terminus of DAT in vitro, and mutation of these serines eliminated the stimulatory effects of CaMKIIalpha. A mutation of the DAT C terminus impairing CaMKIIalpha binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux.

Fifty years ago, increased whole-blood serotonin levels, or hyperserotonemia, first linked disrupted 5-HT homeostasis to Autism Spectrum Disorders (ASDs). The 5-HT transporter (SERT) gene (SLC6A4) has been associated with whole blood 5-HT levels and ASD susceptibility. Previously, we identified multiple gain-of-function SERT coding variants in children with ASD. Here we establish that transgenic mice expressing the most common of these variants, SERT Ala56, exhibit elevated, p38 MAPK-dependent transporter phosphorylation, enhanced 5-HT clearance rates and hyperserotonemia. These effects are accompanied by altered basal firing of raphe 5-HT neurons, as well as 5HT 1A and 5HT 2A receptor hypersensitivity. Strikingly, SERT Ala56 mice display alterations in social function, communication, and repetitive behavior. Our efforts provide strong support for the hypothesis that altered 5-HT homeostasis can impact risk for ASD traits and provide a model with construct and face validity that can support further analysis of ASD mechanisms and potentially novel treatments. development | monoamine | neurotransmitter A utism spectrum disorder (ASD) is a male-predominant disorder that is characterized by deficits in social interactions and communication, as well as repetitive behavior (1). Hyperserotonemia, or increased whole-blood serotonin [i.e., 5-hydroxytryptamine (5-HT)], is a well replicated biomarker that is present in approximately 30% of subjects with ASD (2, 3). Some data suggest an association of hyperserotonemia with stereotyped or self-injurious behavior, but results have been inconsistent (4, 5). Despite the high heritability of whole-blood 5-HT levels (6), a mechanistic connection between hyperserotonemia and specific components of the pathophysiology of ASD remains enigmatic. In blood, 5-HT is contained almost exclusively in platelets (7) that lack 5-HT biosynthetic capacity but accumulate the monoamine via the antidepressant-sensitive serotonin transporter (SERT; 5-HTT). A genome-wide study of whole-blood 5-HT as a quantitative trait found association with the SERT-encoding gene SLC6A4, as well as with ITGB3, which encodes the SERT-interacting protein integrin β3. In both cases, the strongest evidence for association was found in males (8-10). Linkage studies in ASD also implicate the 17q11.2 region containing SLC6A4, again with stronger evidence in males (11, 12).As common SLC6A4 variants are only modestly associated with ASD (13), we and our colleagues previously screened SLC6A4 for rare variants in multiplex families that demonstrate strong linkage to 17q11.2. In this effort, we identified five rare SERT coding variants, each of which confers increased 5-HT transport in transfected cells as well as in lymphoblasts derived from SERT variant-expressing probands (11,14,15). We found the most common of these variants, Ala56 (allele frequency in subjects of European ancestry of 0.5-1%), to be overtransmitted to autism probands, and to be associated with both rigid-compulsive behavior and sensory aversion (11,16). N...

A number of environmental and industrial chemicals are reported to possess androgenic or antiandrogenic activities. These androgenic endocrine disrupting chemicals may disrupt the endocrine system of humans and wildlife by mimicking or antagonizing the functions of natural hormones. The present study developed a low cost recombinant androgen receptor (AR) competitive binding assay that uses no animals. We validated the assay by comparing the protocols and results from other similar assays, such as the binding assay using prostate cytosol. We tested 202 natural, synthetic, and environmental chemicals that encompass a broad range of structural classes, including steroids, diethylstilbestrol and related chemicals, antiestrogens, flutamide derivatives, bisphenol A derivatives, alkylphenols, parabens, alkyloxyphenols, phthalates, siloxanes, phytoestrogens, DDTs, PCBs, pesticides, organophosphate insecticides, and other chemicals. Some of these chemicals are environmentally persistent and/or commercially important, but their AR binding affinities have not been previously reported. To the best of our knowledge, these results represent the largest and most diverse data set publicly available for chemical binding to the AR. Through a careful structure-activity relationship (SAR) examination of the data set in conjunction with knowledge of the recently reported ligand-AR crystal structures, we are able to define the general structural requirements for chemical binding to AR. Hydrophobic interactions are important for AR binding. The interaction between ligand and AR at the 3- and 17-positions of testosterone and R1881 found in other chemical classes are discussed in depth. The SAR studies of ligand binding characteristics for AR are compared to our previously reported results for estrogen receptor binding.