Search

The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney‐derived COS‐7, COS‐1 and HEK‐293 but not in other mammalian cell lines (CHO, Y1, Ltk−), we have characterized a putative functional dopamine transporter displaying a high affinity (Km∼250 nM) and a low capacity (∼0.1 pmol/105cells/min) for [3H]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter. Estimated Kivalues of numerous substrates and inhibitors for the COS‐dopamine transporter and the cloned human neuronal transporter (human dopamine transporter) correlate well with the exception of a few notable compounds, including the endogenous neurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine. As with native neuronal and cloned dopamine transporters, the uptake velocity was sodium‐sensitive and reduced by phorbol ester pre‐treatment. Two mRNA species of 3.8 and 4.0 kb in COS‐7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue. A reverse‐transcribed PCR analysis confirmed the existence of a processed dopamine transporter. However, no immunoreactive proteins of expected dopamine transporter molecular size or [3H]WIN 35,428 binding activity were detected. A partial cDNA of ∼1.3 kb, isolated from a COS‐1 cDNA library and encoding transmembrane domains 1–6, displayed a deduced amino acid sequence homology of ∼96% to the human dopamine transporter. Taken together, the data suggest the existence of a non‐neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter.

This Final Report of the European Federation of Professional Psychologists' Associations (EFPPA) Task Force on Health Psychology defines the nature and scope of health psychology and its possible future development to the year 2000 and beyond. Training needs and objectives are specified for professional health psychologists working in Europe. Practical and policy implications of medical progress and societal changes are discussed. The future development of health psychology as a profession depends on putting theory and policy into practice through the implementation of high quality training. Currently there are relatively few European countries where this has yet happened. Training programmes need to be introduced in all European countries within the framework of each member- country's national laws, regulations and practices.

Modification of the transport velocity of both the native neuronal and cloned presynaptic dopamine transporter (DAT) has been reported following activation/inhibition of second messenger system pathways. In order to identify the mechanism by which the functional activity of human DAT (hDAT) is regulated, we assessed the [³H]dopamine uptake kinetics, [³H]CFT binding characteristics, and, via immunofluorescent confocal microscopy, the cellular localization profiles of the hDAT expressed in both Sf9 and COS-7 cells following modulation of protein kinase C (PKC)- and protein kinase A (PKA)-dependent pathways. As with both native neuronal and cloned DATs, acute exposure of hDAT expressing Sf9 cells to the PKC activator PMA (1 μM), but not αPDD, reduced the Vmax (~1 pmol/min/10⁵ cells) for [³H]DA uptake by ~40%, an effect which was blocked by the protein kinase inhibitor staurosporine. Pretreatment of cells with staurosporine (500 nM) alone, however, increased [³H]DA uptake velocity by ~30%, an effect mimicked by the potent PKA inhibitor Rp-cAMPS. Activation of PKA-dependent pathways with Sp-cAMPS did not significantly modify DA uptake. Neither the Km of [³H]DA uptake (~200 nM) nor the affinity of various substrates and transport inhibitors was altered by either PMA or staurosporine treatment. Despite changes in functional dopamine uptake velocity by PKC/PKA-dependent mechanisms, the estimated density of hDAT as indexed by whole-cell [³H]CFT binding was unchanged. Immunofluorescent confocal microscopy demonstrated that the observed functional consequence of PKC activation on [³H]DA uptake is associated with the rapid sequestration/internalization of hDAT protein from the cell surface, while the increase in DA uptake following PKC/PKA inhibition is the result of the recruitment of internalized or intracellular transporters to the plasma membrane. Identical rapid translocation patterns were observed in similarly treated COS-7 cells transiently expressing hDAT. These data suggest that the differential regulation of DAT transport capacity by both PKC- and PKA-dependent pathways are not a result of modifications in DAT catalytic activity. Moreover, the rapid shuttling of DATs between the plasma membrane and intracellular compartments provides an efficient means by which native DAT function may be regulated by second messenger systems, possibly following activation of presynaptic dopaminergic receptors, and suggests a role for cytoskeletal components in the dynamic regulation of DAT function. Synapse 30:79–87, 1998. © 1998 Wiley-Liss, Inc.

Modification of the transport velocity of both the native neuronal and cloned presynaptic dopamine transporter (DAT) has been reported following activation/inhibition of second messenger system pathways. In order to identify the mechanism by which the functional activity of human DAT (hDAT) is regulated, we assessed the [3H]dopamine uptake kinetics, [3H]CFT binding characteristics, and, via immunofluorescent confocal microscopy, the cellular localization profiles of the hDAT expressed in both Sf9 and COS‐7 cells following modulation of protein kinase C (PKC)‐ and protein kinase A (PKA)‐dependent pathways. As with both native neuronal and cloned DATs, acute exposure of hDAT expressing Sf9 cells to the PKC activator PMA (1 μM), but not αPDD, reduced the Vmax(∼1 pmol/min/105cells) for [3H]DA uptake by ∼40%, an effect which was blocked by the protein kinase inhibitor staurosporine. Pretreatment of cells with staurosporine (500 nM) alone, however, increased [3H]DA uptake velocity by ∼30%, an effect mimicked by the potent PKA inhibitor Rp‐cAMPS. Activation of PKA‐dependent pathways with Sp‐cAMPS did not significantly modify DA uptake. Neither the Kmof [3H]DA uptake (∼200 nM) nor the affinity of various substrates and transport inhibitors was altered by either PMA or staurosporine treatment. Despite changes in functional dopamine uptake velocity by PKC/PKA‐dependent mechanisms, the estimated density of hDAT as indexed by whole‐cell [3H]CFT binding was unchanged. Immunofluorescent confocal microscopy demonstrated that the observed functional consequence of PKC activation on [3H]DA uptake is associated with the rapid sequestration/internalization of hDAT protein from the cell surface, while the increase in DA uptake following PKC/PKA inhibition is the result of the recruitment of internalized or intracellular transporters to the plasma membrane. Identical rapid translocation patterns were observed in similarly treated COS‐7 cells transiently expressing hDAT. These data suggest that the differential regulation of DAT transport capacity by both PKC‐ and PKA‐dependent pathways are not a result of modifications in DAT catalytic activity. Moreover, the rapid shuttling of DATs between the plasma membrane and intracellular compartments provides an efficient means by which native DAT function may be regulated by second messenger systems, possibly following activation of presynaptic dopaminergic receptors, and suggests a role for cytoskeletal components in the dynamic regulation of DAT function. Synapse 30:79–87, 1998.© 1998 Wiley‐Liss, Inc.

The cardiac Na+-Ca2+exchanger differs from most other polytopic membrane proteins in that the amino terminus is cleaved during integration into the endoplasmic reticulum membrane. In this study, the cleaved N-terminal signal sequence of the exchanger was deleted (DelSS) or rendered uncleavable by mutation of the cleavage site (MutSS). Functional analysis of the mutants expressed in Xenopus laevisoocytes and sf9 insect cells demonstrates that DelSS exchanger catalyzes Na+-dependent Ca2+transport at wild-type levels, while activity of MutSS exchanger is reduced to approximately 60% of wild-type in oocytes and 20% in sf9 cells. These results indicate that neither the presence nor the cleavage of the signal peptide is required for functional assembly of the exchanger protein in the membrane. Furthermore, these observations support the concept that internal topogenic signals play the major role in membrane insertion of the Na+-Ca2+exchanger.

In order to delineate structural motifs regulating substrate affinity and recognition for the human dopamine transporter (DAT), we assessed [3H]dopamine uptake kinetics and [3H]CFT binding characteristics of COS-7 cells transiently expressing mutant DATs in which the COOH terminus was truncated or substituted. Complete truncation of the carboxyl tail from Ser582allowed for the expression of biphasic [3H]dopamine uptake kinetics displaying both a low capacity (Vmax∼0.4 pmol/105cells/min) high affinity (Km∼300 nM) component and one exhibiting low affinity (Km∼15 µM] and high capacity (Vmax∼5 pmol/105cells/min) with a concomitant 40% decrease in overall apparent Vmaxrelative to wild type (WT) DAT. Truncation of the last 22 amino acids or substitution of the DAT-COOH tail with sequences encoding the intracellular carboxyl-terminal of either dopamine D1 or D5 receptors produced results that were identical to those with the fully truncated DAT, suggesting that the induction of biphasic dopamine uptake kinetics is likely conferred by removal of DAT-specific sequence motifs distal to Pro597. The attenuation of WT transport activity, either by lowering levels of DAT expression or by pretreatment of cells with phorbol 12-myristate 13-acetate (1 µM), did not affect the kinetics of [3H]dopamine transport. The estimated affinity of dopamine (Ki∼180 nM) for all truncated/substituted DAT mutants was 10-fold lower than that of WT DAT (∼2000 nM) and appears selective for the endogenous substrate, since the estimated inhibitory constants for numerous putative substrates or uptake inhibitors were virtually identical to those obtained for WT DATs. In marked contrast, DAT truncation/substitution mutants displayed significantly reduced high affinity [3H]CFT binding interactions with estimated Kivalues for dopamine and numerous other substrates and inhibitors tested from 10-100-fold lower than that observed for WT DAT. Moreover, co-expression of truncated and/or substituted DATs with WT transporter failed to reconstitute functional or pharmacological activities associated with both transporters. Instead, complete restoration of uniphasic low affinity [3H]dopamine uptake kinetics (Km∼2000 nM) and high affinity substrate and inhibitor [3H]CFT binding interactions attributable to WT DATs were evident. These data clearly suggest the functional independence and differential regulation of the dopamine translocation process from the characteristics exhibited by its ligand binding domain. The lack of functional phenotypic expression of mutant DAT activities in cells co-expressing WT transporter is consistent with the contention that native DATs may exist as multisubunit complexes, the formation and maintenance of which is dependent upon sequences encoded within the carboxyl tail.