An N-terminal threonine mutation produces an efflux-favorable, sodium-primed conformation of the human dopamine transporter.

The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr62 revealed T62D-human (h)DAT partitions in a predominately efflux-preferring conformation. Compared with wild-type (WT), T62D-hDAT exhibits reduced [(3)H]DA uptake and enhanced baseline DA efflux, whereas T62A-hDAT and WT-hDAT function in an influx-preferring conformation. We now interrogate the basis of the mutants' altered function with respect to membrane conductance and Na(+) sensitivity. The hDAT constructs were expressed in Xenopus oocytes to investigate if heightened membrane potential would explain the efflux characteristics of T62D-hDAT. In the absence of substrate, all constructs displayed identical resting membrane potentials. Substrate-induced inward currents were present in oocytes expressing WT- and T62A-hDAT but not T62D-hDAT, suggesting equal bidirectional ion flow through T62D-hDAT. Utilization of the fluorescent DAT substrate ASP(+) [4-(4-(dimethylamino)styryl)-N-methylpyridinium] revealed that T62D-hDAT accumulates substrate in human embryonic kidney (HEK)-293 cells when the substrate is not subject to efflux. Extracellular sodium (Na(+) e) replacement was used to evaluate sodium gradient requirements for DAT transport functions. The EC50 for Na(+) e stimulation of [(3)H]DA uptake was identical in all constructs expressed in HEK-293 cells. As expected, decreasing [Na(+)]e stimulated [(3)H]DA efflux in WT- and T62A-hDAT cells. Conversely, the elevated [(3)H]DA efflux in T62D-hDAT cells was independent of Na(+) e and commensurate with [(3)H]DA efflux attained in WT-hDAT cells, either by removal of Na(+) e or by application of amphetamine. We conclude that T62D-hDAT represents an efflux-willing, Na(+)-primed orientation-possibly representing an experimental model of the conformational impact of amphetamine exposure to hDAT.

The impact of phonological neighborhood density on typical and atypical emerging lexicons*.

According to the Extended Statistical Learning account (ExSL; Stokes, Kern & dos Santos, 2012) late talkers (LTs) continue to use neighborhood density (ND) as a cue for word learning when their peers no longer use a density learning mechanism. In the current article, LTs expressive (active) lexicon ND values differed from those of their age-matched, but not language-matched, TD peers, a finding that provided support for the ExSL account. Stokes (2010) claimed that LTs had difficulty abstracting sparse words, but not dense, from the ambient language. If true, then LTs' receptive (passive), as well as active lexicons should be comprised of words of high ND. However, in the current research only active lexicons were of high ND. LTs' expressive lexicons may be small not because of an abstraction deficit, but because they are unable to develop sufficiently strong phonological representations to support word production.

Protein kinase Cß is a modulator of the dopamine D2 autoreceptor-activated trafficking of the dopamine transporter.

The strength and duration of extracellular dopamine concentrations are regulated by the presynaptic dopamine transporter (DAT) and dopamine D2 autoreceptors (D2autoRs). There is a functional interaction between these two proteins. Activation of D2autoRs increases DAT trafficking to the surface whereas disruption of this interaction compromises activities of both proteins and alters dopaminergic transmission. Previously we reported that DAT expression and activity are subject to modulation by protein kinase Cß (PKCß). Here, we further demonstrate that PKCß is integral for the interaction between DAT and D2autoR. Inhibition or absence of PKCß abolished the communication between DAT and D2autoR. In mouse striatal synaptosomes and transfected N2A cells, the D2autoR-stimulated membrane insertion of DAT was abolished by PKCß inhibition. Moreover, D2autoR-stimulated DAT trafficking is mediated by a PKCß-extracellular signal-regulated kinase signaling cascade where PKCß is upstream of extracellular signal-regulated kinase. The increased surface DAT expression upon D2autoR activation resulted from enhanced DAT recycling as opposed to reduced internalization. Further, PKCß promoted accelerated DAT recycling. Our study demonstrates that PKCß critically regulates D2autoR-activated DAT trafficking and dopaminergic signaling. PKCß is a potential drug target for correcting abnormal extracellular dopamine levels in diseases such as drug addiction and schizophrenia.