Biomedical subjects

Michelle S Mazei-Robison

Publications and source records attributed to Michelle S Mazei-Robison.

3 recordsLinked to original sources

PubMed2005

Expression studies of naturally occurring human dopamine transporter variants identifies a novel state of transporter inactivation associated with Val382Ala.

Multiple, rare, human dopamine (DA) transporter (hDAT, SLC6A3) coding variants have been described, though to date they have been incompletely characterized. Here we present studies analyzing the function and regulation of five naturally occurring coding variants, V55A, R237Q, V382A, A559V and E602G, expressed in COS-7 and SH-SY5Y cells. All variants, except V382A, exhibited levels of surface protein expression and DA transport activity comparable to hDAT. V382A, divergent at the most highly conserved residue among reported hDAT variants, exhibited significantly diminished surface expression, likely derived from inefficient plasma membrane delivery. Moreover, a greater expression of V382A protein was required to achieve comparable levels of transport to hDAT, consistent with a loss of transport function. V382A displayed a decrease in sensitivity to phorbol ester (PMA)-induced internalization, as well as an altered substrate selectivity for DA versus norepinephrine (NE). Analysis of PMA-induced V382A internalization revealed a trafficking-independent action of PMA, consistent with the existence of a surface-localized, transport-inactive state.

Alanine↗

PubMed2005

Sequence variation in the human dopamine transporter gene in children with attention deficit hyperactivity disorder.

The activity of the presynaptic dopamine (DA) transporter (DAT) is critical in mediating the magnitude and duration of dopaminergic signaling in the brain. Multiple genetic studies have found an association between attention deficit hyperactivity disorder (ADHD) and a variable number tandem repeat (VNTR) in the 3'-untranslated region (3'VNTR) of the hDAT gene (SLC6A3), however none of these studies examined the hDAT coding region for polymorphisms. Thus, we sought evidence of polymorphisms in hDAT, focusing on the coding region and splice junctions, utilizing genomic DNA from children diagnosed with ADHD. Two separate ADHD cohorts (N=70 and N=42) were screened and sampled for both status of the 3'VNTR and for common/novel genomic variants. We found evidence of increased DAT variation in African-American subjects as well as in predominantely hyperactive-impulsive probands. Cumulatively, multiple hDAT sequence variants were identified, including five novel variants, as well as one nonsynonymous single nucleotide polymorphism (SNP), converting Ala559 to Val (A559V). A559V was identified in two Caucasian male siblings with ADHD and both subjects were homozygous for the ADHD-associated, 10-repeat 3'VNTR allele. Interestingly, the A559V variant was previously identified in a subject with bipolar disorder [. Molecular Psychiatry 5, 275], a psychiatric disorder that has a significant number of overlapping symptoms with ADHD.

Adolescent↗

PubMed2005

Single nucleotide polymorphisms in the human norepinephrine transporter gene affect expression, trafficking, antidepressant interaction, and protein kinase C regulation.

The role of norepinephrine (NE) in attention, memory, affect, stress, heart rate, and blood pressure implicates NE in psychiatric and cardiovascular disease. The norepinephrine transporter (NET) mediates reuptake of released catecholamines, thus playing a role in the limitation of signaling strength in the central and peripheral nervous systems. Nonsynonymous single nucleotide polymorphisms (SNPs) in the human NET (hNET) gene that influence transporter function can contribute to disease, such as the nonfunctional transporter, A457P, identified in orthostatic intolerance. Here, we examine additional amino acid variants that have been identified but not characterized in populations that include cardiovascular phenotypes. Variant hNETs were expressed in COS-7 cells and were assayed for protein expression and trafficking using cell-surface biotinylation and Western blot analysis, transport of radiolabeled substrate, antagonist interaction, and regulation through protein kinase C (PKC)-linked pathways by the phorbol ester beta-phorbol-12-myristate-13-acetate. We observed functional perturbations in 6 of the 10 mutants studied. Several variants were defective in trafficking and transport, with the most dramatic effect observed for A369P, which was completely devoid of the fully glycosylated form of transporter protein, was retained intracellularly, and lacked any transport activity. Furthermore, A369P and another trafficking variant, N292T, impeded surface expression of hNET when coexpressed. F528C demonstrated increased transport and, remarkably, exhibited both insensitivity to down-regulation by PKC and a decrease in potency for the tricyclic antidepressant desipramine. These findings reveal functional deficits that are likely to compromise NE signaling in SNP carriers in the population and identify key regions of NET contributing to transporter biosynthesis, activity, and regulation.

Animals↗