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R D Blakely

Publications and source records attributed to R D Blakely.

At least 19 recordsLinked to original sources
PubMed2006

Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice.

Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.

Acetylcholine↗
PubMed2006

Norepinephrine transporter-deficient mice respond to anxiety producing and fearful environments with bradycardia and hypotension.

The study of anxiety and fear involves complex interrelationships between psychiatry and the autonomic nervous system. Altered noradrenergic signaling is linked to certain types of depression and anxiety disorders, and treatment often includes specific transporter blockade. The norepinephrine transporter is crucial in limiting catecholaminergic signaling. Norepinephrine transporter-deficient mice have increased circulating catecholamines and elevated heart rate and blood pressure. We hypothesized, therefore, that reduced norepinephrine clearance would heighten the autonomic cardiovascular response to anxiety and fear. In separate experiments, norepinephrine transporter-deficient (norepinephrine transporter-/-) mice underwent tactile startle and trace fear conditioning to measure hemodynamic responses. A dramatic tachycardia was observed in norepinephrine transporter-/- mice compared with controls following both airpuff or footshock stimuli, and pressure changes were also greater. Interestingly, in contrast to normally elevated home cage levels in norepinephrine transporter-deficient mice, prestimulus heart rate and blood pressure were actually higher in norepinephrine transporter+/+ animals throughout behavioral testing. Upon placement in the behavioral chamber, norepinephrine transporter-deficient mice demonstrated a notable bradycardia and depressor effect that was more pronounced in females. Power spectral analysis indicated an increase in low frequency oscillations of heart rate variability; in mice, suggesting increased parasympathetic tone. Finally, norepinephrine transporter-/- mice exhibited sexual dimorphism in freeze behavior, which was greatest in females. Therefore, while reduced catecholamine clearance amplifies immediate cardiovascular responses to anxiety- or fear-inducing stimuli in norepinephrine transporter-/- mice, norepinephrine transporter deficiency apparently prevents protracted hemodynamic escalation in a fearful environment. Conceivably, chronic norepinephrine transporter blockade with transporter-specific drugs might attenuate recognition of autonomic and somatic distress signals in individuals with anxiety disorders, possibly lessening their behavioral reactivity, and reducing the cardiovascular risk factors associated with persistent emotional arousal.

Animals↗
PubMed2006

ADHD and the dopamine transporter: are there reasons to pay attention?

The catecholamine dopamine (DA) plays an important role as a neurotransmitter in the brain in circuits linked to motor function, reward, and cognition. The presynaptic DA transporter (DAT) inactivates DA following release and provides a route for non-exocytotic DA release (efflux) triggered by amphetamines. The synaptic role of DATs first established through antagonist studies and more recently validated through mouse gene-knockout experiments, raises questions as to whether altered DAT structure or regulation support clinical disorders linked to compromised DA signaling, including drug abuse, schizophrenia, and attention deficit hyperactivity disorder (ADHD). As ADHD appears to have highly heritable components and the most commonly prescribed therapeutics for ADHD target DAT, studies ranging from brain imaging to genomic and genetic analyses have begun to probe the DAT gene and its protein for possible contributions to the disorder and/or its treatment. In this review, after a brief overview of ADHD prevalence and diagnostic criteria, we examine the rationale and experimental findings surrounding a role for human DAT in ADHD. Based on the available evidence from our lab and labs of workers in the field, we suggest that although a common variant within the human DAT (hDAT) gene (SLC6A3) is unlikely to play a major role in the ADHD, contributions of hDAT to risk maybe most evident in phenotypic subgroups. The in vitro and in vivo validation of functional variants, pursued for contributions to endophenotypes in a within family approach, may help elucidate DAT and DA contributions to ADHD and its treatment.

Adult↗
PubMed2006

The high-affinity choline transporter: a critical protein for sustaining cholinergic signaling as revealed in studies of genetically altered mice.

In cholinergic neurons, the presynaptic choline transporter (CHT) mediates high-affinity choline uptake (HACU) as the rate-limiting step in acetylcholine (ACh) synthesis. It has previously been shown that HACU is increased by behaviorally and pharmacologically-induced activity of cholinergic neurons in vivo, but the molecular mechanisms of this change in CHT function and regulation have only recently begun to be elucidated. The recent cloning of CHT has led to the generation of new valuable tools, including specific anti-CHT antibodies and a CHT knockout mouse. These new reagents have allowed researchers to investigate the possibility of a presynaptic, CHT-mediated, molecular plasticity mechanism, regulated by and necessary for sustained in vivo cholinergic activity. Studies in various mouse models of cholinergic dysfunction, including acetylcholinesterase (AChE) transgenic and knockout mice, choline acetyltransferase (ChAT) heterozygote mice, muscarinic (mAChR) and nicotinic (mAChR) receptor knockout mice, as well as CHT knockout and heterozygote mice, have revealed new information about the role of CHT expression and regulation in response to long-term alterations in cholinergic neurotransmission. These mouse models highlight the capacity of CHT to provide for functional compensation in states of cholinergic dysfunction. A better understanding of modes of CHT regulation should allow for experimental manipulation of cholinergic signaling in vivo with potential utility in human disorders of known cholinergic dysfunction such as Alzheimer's disease, Parkinson's disease, schizophrenia, Huntington's disease, and dysautonomia.

Acetylcholinesterase↗
PubMed2005

Developmental expression of the high affinity choline transporter in cholinergic sympathetic neurons.

Choline uptake by the high affinity choline transporter (CHT) is the rate-limiting step in acetylcholine synthesis. Induction of CHT is therefore a critical step in cholinergic differentiation, and we examined the developmental expression of CHT in cholinergic sympathetic neurons that innervate rodent sweat glands. During postnatal development the earliest sympathetic axons in the rear footpads are noradrenergic, containing intense tyrosine hydroxylase immunoreactivity and lacking CHT-immunoreactivity (CHT-IR). By postnatal day 7 (P7) in mouse, and P10 in rat, weak CHT-IR appeared in axons associated with the sweat gland anlagen. CHT staining intensity increased during the following weeks in conjunction with plexus arborization and gland maturation. The pattern of CHT-immunoreactivity (CHT-IR) in the sweat gland innervation was similar to staining for the vesicular acetylcholine transporter and vasoactive intestinal peptide. Immunoblots of tissue from sympathectomized rats confirmed that most of the CHT in footpad was contained in sympathetic neurons. Although CHT expression has been reported in noradrenergic sympathetic neurons of the superior cervical ganglion, these data indicate that in the sympathetic neurons projecting to sweat glands CHT is present at detectable levels only after association with the glands.

Animals↗
PubMed2004

Novel fluorescence-based approaches for the study of biogenic amine transporter localization, activity, and regulation.

Pre-synaptic norepinephrine (NE) and dopamine (DA) transporters (NET and DAT) terminate catecholamine synaptic transmission through reuptake of released neurotransmitter. Recent studies reveal that NET and DAT are tightly regulated by receptor and second messenger-linked signaling pathways. Common approaches for studying these transporters involve use of radiolabeled substrates or antagonists, methods possessing limited spatial resolution and that bear limited opportunities for repeated monitoring of living preparations. To circumvent these issues, we have explored two novel assay platforms that permit temporally resolved quantitation of transport activity and transporter protein localization. To monitor the binding and transport function of NET and DAT in real-time, we have investigated the uptake of the fluorescent organic compound 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (ASP+). We have extended our previous single cell level application of this substrate to monitor transport activity via high-throughput assay platforms. Compared to radiotracer uptake methods, acquisition of ASP+ fluorescence is non-isotopic and allows for continuous, repeated transport measurements on both transfected and native preparations. Secondly, we have extended our application of small-molecule-conjugated fluorescent CdSe/ZnS nanocrystals, or quantum dots (Qdots), to utilize antibody and peptide ligands that can identify surface expressed transporters, receptors and other membrane proteins in living cell systems. Unlike typical organic fluorophores, Qdots are highly resistant to bleaching and can be conjugated to multiple ligands. They can also be illuminated by conventional light sources, yet produce narrow, gaussian emission spectra compatible with multiple target visualization (multiplexing). Together, these approaches offer novel opportunities to investigate changes in transporter function and distribution in real-time with superior spatial and temporal resolution.

Antibodies↗
PubMed2004

Linkage and association analysis at the serotonin transporter (SLC6A4) locus in a rigid-compulsive subset of autism.

Autism is a complex genetic neurodevelopmental disorder in which affected individuals display deficits in language, social relationships, and patterns of compulsive and stereotyped behaviors and rigidity. Linkage analysis in our dataset of 57 New England and 80 AGRE multiplex autism families reveals a multipoint heterogeneity LOD (HLOD) score of 2.74 at D17S1871 in 17q11.2. Analysis of phenotypic subsets shows an increased HLOD of 3.62 in families with compulsive behaviors and rigidity. The serotonin transporter locus (SLC6A4) maps nearby and is considered a functional candidate gene in autism and obsessive-compulsive disorder. We genotyped an insertion/deletion polymorphism in the promoter (5-HTTLPR), and seven single nucleotide polymorphisms (SNPs) across the 38-kb transcriptional unit. Transmission disequilibrium (TD) analysis reveals nominal association at a SNP in intron 5 (P = 0.02) as well as 5-HTTLPR (P = 0.01), corresponding to over-transmission of the short allele. TD analysis in the rigid-compulsive subset shows no evidence for association. Intermarker linkage disequilibrium was determined. All SNPs define a single haplotype block, while 5-HTTLPR lies 5' to this block. Three SNPs are sufficient to detect all common alleles (> or =5%) in this > 26-kb block. Analysis of haplotypes for these markers demonstrates no evidence for association to autism. These data indicate that a common allele within the coding region of SLC6A4 is not responsible for the observed linkage. However, the presence of heterogeneous disease variants within the block or the existence of a common disease-associated allele either upstream or downstream of this block is possible. In fact, such variants may well account for linkage to 17q11.2 in our families.

Autistic Disorder↗
PubMed2002

Monoamine transporter gene structure and polymorphisms in relation to psychiatric and other complex disorders.

The norepinephrine, dopamine and serotonin transporters (NET, DAT and SERT, respectively), limit cellular signaling by recapturing released neurotransmitter, and serve as targets for antidepressants and drugs of abuse, emphasizing the integral role these molecules play in neurotransmission and pathology. This has compelled researchers to search for polymorphisms in monoamine (MA) transporter genes. Studies support linkage and association of MA transporter genetic variation in psychiatric and other complex disorders. Understanding the contribution of MA transporter polymorphisms to human behavior, disease susceptibility and response to pharmacotherapies will involve further progress in linkage and association that will be aided by both definition of highly selective phenotypes and utilization of a large number of polymorphic markers. The relationship of polymorphisms to alterations in transport capacity, likely a complex interaction, involving genetic background, disease state, and medication, will elucidate the means by which MA transporter genetic variability contributes to our individuality.

Biogenic Monoamines↗
PubMed2001

Physiological genomics of antidepressant targets: keeping the periphery in mind.

The plasma membrane transporters that clear extracellular serotonin (5-HT) and norepinephrine (NE), serotonin transporters (SERTs) and NE transporters (NETs), have received considerable attention over the past four decades because of their roles in amine neurotransmitter inactivation. In addition, they interact with many centrally active drugs, including multiple classes of antidepressants such as the serotonin-selective reuptake inhibitors, typified by fluoxetine (Prozac), and the more recently developed norepinephrine-selective transporter antagonists, such as reboxetine. The therapeutic utility of these agents supports biogenic amine theories of affective disorders and raises the question as to whether SERT and NET exhibit a functional genetic variation that could influence risk for behavioral disorders. Although evidence exists that a promoter polymorphism in SERT may influence behavioral states, this contention is not without complexity and its mechanism of action remains poorly understood. The identification of coding variants of NETs and SERTs would offer important opportunities to connect genotype to phenotype. However, given the limited frequency of transporter coding variations evident to date in general population surveys or in psychiatric genetic studies, the identification of informative functional variants of transporters will likely require refined phenotypes. In this regard, NET and SERT play critical roles in cardiovascular and gastrointestinal physiology, respectively. This perspective reviews recent human and mouse studies that suggest how peripheral autonomic phenotypes, linked to genetic disruption of NET and SERT function, can aid in the phenotypic segregation needed for advanced theories of biogenic amine dysfunction and pharmacogenetics.

Antidepressive Agents↗
PubMed2001

Autocrine regulation of norepinephrine transporter expression.

The norepinephrine transporter (NET) is a neurotransmitter scavenger and site of drug action in noradrenergic neurons. The aim of this study was to identify mechanisms that regulate NET expression during the development of quail (q) sympathetic neuroblasts, which develop from neural crest stem cells. Neurotrophin-3 (NT-3) and transforming growth factor beta1 (TGF-beta1) cause an increase of qNET mRNA levels in neural crest cells. When combined, the growth factors are additive in increasing qNET mRNA levels. Both NT-3 and TGF-beta1 are synthesized by neural crest cells. Onset of NET expression precedes the onset of neural crest stem cell emigration from the neural tube. In older embryos, qNET is expressed by several crest-derived and noncrest tissues. The data show that qNET expression in presumptive sympathetic neurons is initiated early in embryonic development by growth factors that are produced by neural crest cells themselves. Moreover, the results support our previous observations that norepinephrine transport contributes to the regulation of the differentiation of neural crest stem cells into sympathetic neurons.

Animals↗
PubMed2001

Molecular cloning and characterization of a murine hemicholinium-3-sensitive choline transporter.

In cholinergic neurons, a specific requirement for precursor choline in the biosynthesis of acetylcholine (ACh) is thought to be sustained by a presynaptic, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). This transporter exhibits micromolar affinity for choline and transport activity is Na(+)- and Cl(-)-dependent. Based on the sequence information available with the recent cloning of rat and human CHTs [Okuda, Haga, Kanai, Endou, Ishihara and Katsura (2000) Nat. Neurosci. 3, 120-125; Apparsundaram, Ferguson, George Jr and Blakely (2000) Biochem. Biophys. Res. Commun. 276, 862-867; Okuda and Haga (2000) FEBS Lett. 484, 92-97], we have identified a murine CHT orthologue (mCHT) by reverse transcriptase-PCR of spinal cord mRNA and confirmed this sequence using assembled mouse genomic DNA. Inferred splice junctions for mCHT exons are conserved with those of hCHT. The mCHT cDNA encodes a protein of 580 amino acids with 93% and 98% amino acid identity to human CHT and rat CHT1, respectively. Hydropathy analysis of the predicted amino acid sequence of mCHT indicates a protein containing 13 transmembrane domains (TMDs), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analysis of mouse tissues reveals the expression of mCHT as a single transcript of approximately 5 kb with highest expression in regions that are rich in cholinergic cell bodies, e.g. the spinal cord, brainstem, mid-brain and striatum, whereas hybridization signals are absent in regions lacking cholinergic soma, e.g. the cerebellum and kidney. Expression of mCHT in COS-7 cells results in high-affinity [(3)H]HC-3-binding sites (K(d)=5 nM), and Na(+)- and Cl(-)-dependent HC-3-sensitive choline uptake (K(m)=2 microM), assessed in resealed membrane vesicles. The availability of cloned, functional mCHT and its cognate genomic DNA should prove useful for studies of mCHT regulation and should open possibilities for evaluation of CHT dysfunction in murine models.

Animals↗
PubMed2001

Familial orthostatic tachycardia due to norepinephrine transporter deficiency.

UNLABELLED: Orthostatic intolerance (OI) or postural tachycardia syndrome (POTS) is a syndrome primarily affecting young females, and is characterized by lightheadedness, palpitations, fatigue, altered mentation, and syncope primarily occurring with upright posture and being relieved by lying down. There is typically tachycardia and raised plasma norepinephrine levels on upright posture, but little or no orthostatic hypotension. The pathophysiology of OI is believed to be very heterogeneous. Most studies of the syndrome have focused on abnormalities in norepinephrine release. Here the hypothesis that abnormal norepinephrine transporter (NET) function might contribute to the pathophysiology in some patients with OI was tested. In a proband with significant orthostatic symptoms and tachycardia, disproportionately elevated plasma norepinephrine with standing, impaired systemic, and local clearance of infused tritiated norepinephrine, impaired tyramine responsiveness, and a dissociation between stimulated plasma norepinephrine and DHPG elevation were found. Studies of NET gene structure in the proband revealed a coding mutation that converts a highly conserved transmembrane domain Ala residue to Pro. Analysis of the protein produced by the mutant cDNA in transfected cells demonstrated greater than 98% reduction in activity relative to normal. NE, DHPG/NE, and heart rate correlated with the mutant allele in this family. CONCLUSION: These results represent the first identification of a specific genetic defect in OI and the first disease linked to a coding alteration in a Na+/Cl(-)-dependent neurotransmitter transporter. Identification of this mechanism may facilitate our understanding of genetic causes of OI and lead to the development of more effective therapeutic modalities.

Amino Acid Sequence↗
PubMed2001

Interactions of tryptamine derivatives with serotonin transporter species variants implicate transmembrane domain I in substrate recognition.

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is responsible for the inactivation of synaptic 5-HT and is also a target for multiple psychostimulants. Despite the critical role of SERT in 5-HT inactivation and psychostimulant response, many aspects of the transporter's recognition of ligands are poorly defined. We took advantage of sequence divergence of SERT species variants to identify structural determinants of substrate recognition. Tryptamine derivatives with substitutions at the 4 and 7 positions on the phenyl ring, the indole nitrogen, and the beta position show up to 40-fold potency differences for inhibiting [(3)H]5-HT transport in cells transfected with either human or Drosophila melanogaster SERT cDNAs. Species selectivities of these derivatives were largely recapitulated in antagonist binding. Human/D. melanogaster SERT chimera studies implicated the first two SERT transmembrane domains (TMDs) in the potency of the indole nitrogen-substituted compounds N-isopropyltryptamine (NIT), 5-methoxy-N-isopropyltryptamine (5-MNIT), and the 7-substituted compound 7-benzyloxytryptamine (7BT). Potency differences of analogs with substitutions at the 4 and beta positions are influenced by sequences distal to this region. Within TMD I-II, species-scanning mutagenesis implicated a single residue (Y95 in human SERT, F90 in D. melanogaster SERT) in the recognition of NIT, 5-MNIT, and 7BT. Remarkably, this is the same site we established previously in species-specific recognition of the antagonists citalopram and mazindol. These findings support a critical role for TMD I residues in defining shared aspects of SERT substrate and antagonist recognition.

Animals↗
PubMed2001

Trafficking-dependent and -independent pathways of neurotransmitter transporter regulation differentially involving p38 mitogen-activated protein kinase revealed in studies of insulin modulation of norepinephrine transport in SK-N-SH cells.

Presynaptic, cocaine- and antidepressant-sensitive norepinephrine (NE) transporters (NETs) dictate levels of extracellular NE after vesicular release. Recent studies suggest that G protein-coupled receptors linked to protein kinase C (PKC) down-regulate cell surface NET protein levels and diminish NE uptake capacity. We identified distinct phosphatidylinositol 3-OH kinase (PI3K)-linked pathways supporting basal and insulin-triggered NE transport in the human noradrenergic neuroblastoma, SK-N-SH. Acute (0-60 min) insulin treatments produced a time- and concentration-dependent stimulation of NE transport, resolved in kinetic studies as an enhancement of NE transport capacity (Vmax) without an alteration in NE Km. Basal and insulin-modulated NET activities were reduced by the tyrosine kinase inhibitor genistein and the PI3K inhibitors wortmannin and LY-294002, but not by the PKC inhibitor staurosporine. PI3K activation was found to support phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). However, basal and insulin-stimulated NET activities were differentiated by their reliance on p38 MAPK activation. Thus, the p38 MAPK inhibitor SB203580 and SB202190 abolished insulin activation of NE transport yet failed to impact basal NET activity. Moreover, p38 MAPK activation and insulin activation of NETs were found to be sensitive to external Ca2+ depletion, blockade of voltage-sensitive Ca2+ channels, and inhibition of protein phosphatase 2A. Effects of tyrosine kinase and PI3K inhibitors on basal NET uptake appear to arise from a loss of cell surface NET protein, whereas the p38 MAPK-dependent enhancement of NE transport occurs without a detectable enhancement of surface NET. Our findings establish two distinct pathways for regulation of NE uptake involving PI3K, one linked to transporter trafficking and a second linked to Ca2+-dependent, p38 MAPK phosphorylation that promotes activation of cell surface NETs.

Blotting, Western↗
PubMed2000

Ultrastructural localization of the serotonin transporter in superficial and deep layers of the rat prelimbic prefrontal cortex and its spatial relationship to dopamine terminals.

Dopamine levels within the prefrontal cortex (PFC) can be manipulated by selective inhibitors of the serotonin transporter (SERT). However, the cellular mechanisms underlying these effects are not clear. The present study sought to examine the distribution of immunogold-silver labeling for SERT (SERT-ir) in the rat prelimbic PFC and to describe its ultrastructural spatial relationship to dopamine axons labeled by immunoperoxidase staining for tyrosine hydroxylase (TH-ir). SERT was localized to axonal profiles that ranged in size from fine caliber fibers containing dense SERT-ir, primarily along the membrane, and rarely forming synapses to large, spherical varicosities exhibiting less dense staining, mainly within the cytoplasm, and more commonly forming synapses. Synaptic contacts of SERT profiles were typically asymmetric, axospinous, and more frequent in superficial (38%) than deep (19%) layers. For TH-ir profiles, only 24% were within the same 13.8 microm(2) microenvironment as SERT-ir profiles. Furthermore, TH-ir and SERT-ir profiles were rarely directly apposed to each other or convergent onto common dendritic structures. Instead, these two profiles were typically separated by an average distance of 1.30 microm in the coronal plane, a value that did not vary with the size of SERT-ir axons, the amount of SERT labeling, or the cortical layer examined. These results are consistent with two populations of SERT profiles within the rat prelimbic PFC that may arise from different raphe nuclei or that represent varicose and intervaricose portions of the same axons. Moreover, the functional interactions between cortical serotonin and dopamine systems that may contribute to the therapeutic efficacy of antidepressant drugs are likely to occur over distances greater than 1 microm.

Animals↗
PubMed2000

Cocaine and antidepressant-sensitive biogenic amine transporters exist in regulated complexes with protein phosphatase 2A.

Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration.

Animals↗
PubMed2000

Molecular cloning of a human, hemicholinium-3-sensitive choline transporter.

Under many physiological circumstances, Na(+)- and Cl(-)-dependent, hemicholinium-3 (HC-3)-sensitive, high-affinity choline uptake (HACU) in cholinergic neurons is thought to be rate-limiting in the biosynthesis of acetylcholine (ACh). Based on sequence information provided by the Human Genome Project and the recently reported rat CHT1 (rCHT1), we cloned a human CHT cDNA from spinal cord. The hCHT cDNA encodes a protein of 580 amino acids having 93% identity to rCHT1 and 51% identity to the Caenorhabditis elegans homolog CHO-1, and is distantly related to members of the Na(+)-coupled glucose transporter (SGLT) gene family of Na(+)-coupled glucose (SGLT), nucleoside and iodide transporters. Northern blot analysis reveals the expression of a approximately 5 kb transcript in human brain regions rich in cholinergic neurons including the putamen, spinal cord, and medulla. Expression of hCHT cDNA in COS-7 cells results in saturable, Na(+)/Cl(-)-dependent choline uptake (K(m) = 1.2 microM) in membrane vesicles and [(3)H] HC-3 binding (K(d) = 4 nM) in membrane fractions, consistent with characteristics reported in mammalian cholinergic neurons. Using radiation hybrid mapping techniques, we localized the hCHT gene to human chromosome 2q12. These studies elucidate the primary structure and chromosomal localization of hCHT and provide a basis for mechanistic analysis of HACU regulation and an investigation of the role of hCHT in disease states.

Amino Acid Sequence↗