SOLO-based task to improve self-evaluation and capacity to integrate concepts in first-year physiology students.

An accurate self-assessment of student work can enhance student learning and subsequently improve academic performance. Instructors can facilitate this process by providing "standards" that students can utilize as feedback when self-evaluating their understanding. Traditional forms of feedback, such as marked assessment tasks, are limited in their ability to serve as standards, as they do not adequately capture variations corresponding to different levels of understanding. To develop a complex understanding in physiology, students have to integrate concepts pertaining to different subcomponents of body systems. The present study attempted to ascertain if exposing students to variations in complexity would refine their ability to self-evaluate their understanding and capacity to integrate concepts. Students were tasked to answer an essay-length, open-ended physiology question to expose their current understanding of the topic. The change in students' self-marking of their answer before and after being exposed to the variations in conceptual understanding of the topic were used to determine whether improvements in self-evaluation accuracy occurred. These variations were presented as instructor-generated answers to the open-ended question, framed using the structure of the observed learning outcome (SOLO) taxonomy. Student scores in the integrative questions of the end-of-semester exam were used as a measure of student ability to integrate concepts. Findings indicated that this intervention led to improvements in student self-evaluation and exam performance, and the positive outcomes were replicated across multiple iterations of the activity.

Effects of long-term opioid analgesics on cognitive performance and plasma cytokine concentrations in patients with chronic low back pain: a cross-sectional pilot study.

INTRODUCTION: Cognitive performance and inflammation are altered in people with chronic low back pain (CLBP). Yet, the magnitude of these changes has been unclear because of the potential influence of opioid analgesics. OBJECTIVES: This cross-sectional pilot study aimed to explore whether patients with CLBP receiving long-term opioid analgesics differed from patients not taking opioids on measures of cognitive performance and plasma cytokine concentrations. METHODS: Patients with CLBP who were either taking (N = 18) or not taking (N = 22) opioids daily for 3 or more months were recruited from a tertiary care private hospital and compared with healthy adults (N = 20). All groups were administered validated questionnaires to assess depression, anxiety, and stress; a cognitive test of memory, attention, and executive function; and a peripheral blood draw to measure proinflammatory (IL-1ß, IL-2, IL-8, IL-12p70, TNF-α, and IFN-γ), anti-inflammatory (IL-4, IL-10, and IL-13), and pleiotropic (IL-6) cytokine concentrations. Patients also completed pain-specific questionnaires. RESULTS: Patients receiving opioid analgesics performed significantly (P < 0.05) worse in attention and had significantly (P < 0.05) lower pain self-efficacy beliefs than those patients not taking opioids. Patient groups did not differ in mean pain severity or pain interference scores, tests of memory and executive function, and mean plasma cytokine concentrations, despite long-term opioid analgesics. CONCLUSION: Patients receiving long-term opioid analgesics for CLBP have minor differences when compared with patients not taking opioids. This has important clinical implications when considering long-term treatment for patients with CLBP.

Changes in Biology Self-Efficacy during a First-Year University Course.

Academic self-efficacy encompasses judgments regarding one's ability to perform academic tasks and is correlated with achievement and persistence. This study describes changes in biology self-efficacy during a first-year course. Students (n = 614) were given the Biology Self-Efficacy Scale at the beginning and end of the semester. The instrument consisted of 21 questions ranking confidence in performing biology-related tasks on a scale from 1 (not at all confident) to 5 (totally confident). The results demonstrated that students increased in self-efficacy during the semester. High school biology and chemistry contributed to self-efficacy at the beginning of the semester; however, this relationship was lost by the end of the semester, when experience within the course became a significant contributing factor. A proportion of high- and low- achieving (24 and 40%, respectively) students had inaccurate self-efficacy judgments of their ability to perform well in the course. In addition, female students were significantly less confident than males overall, and high-achieving female students were more likely than males to underestimate their academic ability. These results suggest that the Biology Self-Efficacy Scale may be a valuable resource for tracking changes in self-efficacy in first-year students and for identifying students with poorly calibrated self-efficacy perceptions.

Extracellular loop 3 of the noradrenaline transporter contributes to substrate and inhibitor selectivity.

The human noradrenaline transporter (NET) and 5-hydroxytryptamine (5-HT) transporter (SERT) are inhibited by antidepressants and psychoactive drugs such as cocaine. Both substrates and inhibitors bind in the transmembrane core of the protein, but molecular divergence at the binding site is not sufficient to account for the NET-selective and SERT-selective inhibition of the antidepressants, desipramine and citalopram, respectively. We considered that the poorly conserved third extracellular loop may contribute to these differences. We substituted single amino acid residues of the third extracellular loop in NET for equivalents from SERT, transiently transfected COS-7 cells with WT NET, 13 mutant NETs and WT SERT, and measured [(3)H]noradrenaline uptake, [(3)H]nisoxetine binding and [(3)H]5-HT uptake. Mutants F299W, Y300Q, R301K and K303L, at the C-terminal end of EL3, all showed significantly decreased [(3)H]nisoxetine binding, indicative of a reduced cell surface expression. Most mutants differed little, if at all, from WT NET regarding [(3)H]noradrenaline uptake; however, the I297P mutant showed no significant uptake activity despite intact cell surface expression, and the A293F mutant showed a significantly slower transporter turnover than WT NET in addition to [(3)H]5-HT uptake that was significantly greater than that of WT NET. The A293F mutation also decreased desipramine potency and increased the inhibition of [(3)H]noradrenaline uptake by citalopram compared to WT NET. These results suggest that the third extracellular loop allosterically regulates the ability of the transmembrane domains to transport substrates and bind inhibitors and thus contributes to the selectivity of substrates and antidepressants for NET and SERT.

A set of vertically integrated inquiry-based practical curricula that develop scientific thinking skills for large cohorts of undergraduate students.

Science graduates require critical thinking skills to deal with the complex problems they will face in their 21st century workplaces. Inquiry-based curricula can provide students with the opportunities to develop such critical thinking skills; however, evidence suggests that an inappropriate level of autonomy provided to underprepared students may not only be daunting to students but also detrimental to their learning. After a major review of the Bachelor of Science, we developed, implemented, and evaluated a series of three vertically integrated courses with inquiry-style laboratory practicals for early-stage undergraduate students in biomedical science. These practical curricula were designed so that students would work with increasing autonomy and ownership of their research projects to develop increasingly advanced scientific thinking and communication skills. Students undertaking the first iteration of these three vertically integrated courses reported learning gains in course content as well as skills in scientific writing, hypothesis construction, experimental design, data analysis, and interpreting results. Students also demonstrated increasing skills in both hypothesis formulation and communication of findings as a result of participating in the inquiry-based curricula and completing the associated practical assessment tasks. Here, we report the specific aspects of the curricula that students reported as having the greatest impact on their learning and the particular elements of hypothesis formulation and communication of findings that were more challenging for students to master. These findings provide important implications for science educators concerned with designing curricula to promote scientific thinking and communication skills alongside content acquisition.

Assessing core manipulative skills in a large, first-year laboratory.

Responding to the concern from our faculty that undergraduate students do not have robust laboratory skills, we designed and implemented a strategy to individually teach and assess the manipulative skills of students in first-year laboratories. Five core laboratory skills were selected for the course entitled Human Biology, a large, first-year class of students, most of whom were enrolled in Bachelor of Pharmacy and Human Movement Studies. Here, we report details for the 365 students enrolled primarily in Pharmacy and Human Movement Studies bachelor degree programs in semester 1 of 2006. We designed a specific strategy to assess five core laboratory skills: 1) accurate and precise use of a micropipette, 2) calculation of dilutions and preparation of diluted samples of saline, 3) accurate representation of data using a graph, 4) use of a light microscope, and 5) acquisition of digital data by measuring the latent period for the Achilles reflex. Graduate tutors were trained to teach and assess each student on each skill. The development of competency was tracked for all students across all five skills. Most students demonstrated proficiency on their first attempt. The development of proficiency across the core skills depended on both the skill and degree program. In semester 2 of 2006, 854 students mostly enrolled in the Bachelor of Science degree program and were similarly taught and assessed on the same five core skills. This approach was an effective teaching and assessment strategy that, when applied beyond first year, should increase the level of laboratory skills across undergraduate programs in physiology.

The personal response: A novel writing assignment to engage first year students in large human biology classes.

The teaching of highly valued scientific writing skills in the first year of university is challenging. This report describes the design, implementation, and evaluation of a novel written assignment, The Personal Response and accompanying Peer Review, in the course, Human Biology (BIOL1015) at The University of Queensland. These assignments were the first assessment tasks of the course and were set early in the first semester of university. BIOL1015 had a diverse cohort of 319 first year students from five bachelor degree programs, primarily from Pharmacy and Human Movement Studies. Audio files in the form of interviews with eminent biomedical scientists were obtained from a leading public radio program. Students used these files as triggers to submit a short but highly structured assignment written from a personal perspective and in an expressive style. Evaluations revealed that overall, students found the task interesting and challenging. Students performed well, regardless of their background knowledge, disciplinary interest, or preference for topics within human biology. This study demonstrated that The Personal Response was an appropriate task for these first year students of human biology. It represents an alternative to traditional essay writing.

Biohorizons: An eConference to assess human biology in large, first-year classes.

We detail the design, implementation and evaluation of an eConference entitled "Biohorizons", using a presage-process-product model to describe the development of an eLearning community. Biohorizons was a summative learning and assessment task aiming to introduce large classes of first-year Human Biology students to the practices of professional scientists. It was implemented in semester 1 for students enrolled in Pharmacy and Human Movement Studies degree programs, and in semester 2, for Science students. Pairs of students selected a topic of interest in Human Biology, registered into on-line clusters, then developed and wrote a short scientific paper and accompanying PowerPoint presentation. They then individually participated in an online Discussion. All three tasks were assessed using standards-referenced assessment rubrics. Learning was supported by eTutors, working in asynchronous mode. Biohorizons was evaluated by analyzing student achievement data, surveys and focus group interviews. Most students were able to achieve high academic standards (global mean scores for semester 1, 85-96%; semester 2, 81-90%). Student evaluations support: 1) the successful integration of eLearning into large classes of Human Biology, 2) the engagement of first-year students through collaborative learning, and 3) the fostering of learning through challenging assessment relevant to the core practices of professional scientists.

Investigation of the functional roles of the MELAL and GQXXRXG motifs of the human noradrenaline transporter using cysteine mutants.

The study examines the roles of the highly conserved MELAL and GQXXRXG motifs, located in the second transmembrane domain and the first intracellular loop of the human noradrenaline transporter (hNET). We have previously shown that this region does not directly participate in the NET substrate translocation pathway [Sucic, S., and Bryan-Lluka, L.J., 2005. Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis. J. Neurochem. 94, 1620-1630.], while the current report focuses on the importance of this region in determining other functional properties of the hNET. Mutation to cysteine of the wild-type residues was carried out by site-directed mutagenesis of hNET cDNA. The wild-type and mutant hNETs were expressed in transiently transfected COS-7 cells and the effects of these mutations were pharmacologically examined. The results indicate that the GQXXRXG motif is important for the binding of cocaine, but not antidepressants. The hN120C mutant caused an 11-fold increase in the binding affinity of cocaine, compared to the wild-type hNET, while hQ118C, hY119C, hR121C and hE122C showed smaller increases. Interestingly, the apparent affinities of cocaine for some of these mutants were either decreased or unchanged, contrasting with the effects observed from the binding studies. The hE113C mutant in the MELAL motif caused very marked (over 400-fold) reductions in the binding affinities of substrates, but had no effects on the binding affinities of cocaine or antidepressants. Overall, the MELAL and GQXXRXG motifs are important determinants of NET cell surface expression and substrate and inhibitor binding. The results further suggest that the binding sites for substrates, cocaine and antidepressants on the NET are distinct but overlapping.

A role for serotonin (5-HT) in hepatic stellate cell function and liver fibrosis.

Hepatic stellate cells (HSCs) are key cellular components of hepatic wound healing and fibrosis. There is emerging evidence that the fibrogenic function of HSCs may be influenced by neurochemical and neurotrophic factors. This study addresses the potential for the serotonin (5-HT) system to influence HSC biology. Rat and human HSCs express the 5-HT1B, 5-HT1F 5-HT2A 5-HT2B, and 5-HT7 receptors, with expression of 5-HT1B 5-HT2A and 5-HT2B being induced on HSC activation. Induction of 5-HT2A and 5-HT2B was 106+/-39- and 52+/-8.5-fold that of quiescent cells, respectively. 5-HT2B was strongly associated with fibrotic tissue in diseased rat liver. Treatment of HSCs with 5-HT2 antagonists suppressed proliferation and elevated their rate of apoptosis; by contrast 5-HT was protective against nerve growth factor-induced apoptosis. 5-HT synergized with platelet-derived growth factor to stimulate increased HSC proliferation. HSCs were shown to express a functional serotonin transporter and to participate in both active uptake and release of 5-HT. We conclude that HSCs express key regulatory components of the 5-HT system enabling them to store and release 5-HT and to respond to the neurotransmitter in a profibrogenic manner. Antagonists that selectively target the 5-HT class of receptors may be exploited as antifibrotic drugs.

Dose determination of haloperidol, risperidone and olanzapine using an in vivo dopamine D2-receptor occupancy method in the rat.

The purpose of the present study was to determine antipsychotic doses that achieve 80% striatal dopamine D2-receptor occupancy for haloperidol, risperidone and olanzapine in rats. Wistar rats were treated with normal saline vehicle (controls), haloperidol (0.25 and 0.5 mg/kg/day), risperidone (3, 5 and 6 mg/kg/day) and olanzapine (5 and 10 mg/kg/day) for 7 days via osmotic minipumps. Striatal and cerebellar tissue were collected and in vivo dopamine D2-receptor occupancies were determined using 3H-raclopride. The doses required to achieve dopamine D2-receptor occupancy of 80% in 11- and 24-week old rats were: haloperidol 0.25 mg/kg/day, risperidone 5 mg/kg/day and olanzapine 10 mg/kg/day.

Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis.

The aim was to investigate the roles of transmembrane domain 2 and the adjacent region of the first intracellular loop in determining human noradrenaline transporter (hNET) function by pharmacological and substituted-cysteine accessibility method (SCAM) analyses. It was first necessary to establish a suitable background NET for SCAM. Alanine mutants of endogenous hNET cysteines, hC86A, hC131A and hC339A, were examined and showed no marked effects on expression or function. hNET and the mutants were also resistant to methanethiosulfonate (MTS), ethylammonium (MTSEA) and MTStrimethylammonium (MTSET). Hence, wild-type hNET is an appropriate background for production of cysteine mutants for SCAM. Pharmacological investigation showed that all mutants except hT99C and hL109C showed reduced cell-surface expression, while all except hM107C showed a reduction in functional activity. The mutations did not markedly affect the apparent affinities of substrates, but apparent affinities of cocaine were decreased 7-fold for hP97C and 10-fold for hF101C and increased 12-fold for hY98C. [3H]Nisoxetine binding affinities were decreased 13-fold for hP97C and 5-fold for hF101C. SCAM analysis revealed that only hL102C was sensitive to 1.25 mm MTSEA, and this sensitivity was protected by noradrenaline, nisoxetine and cocaine. The results suggest that this region of hNET is important for interactions with antidepressants and cocaine, but it is probably not involved in substrate translocation mechanisms.

Quantitative analysis of immunolabeling for serotonin and for glutamate transporters after administration of imipramine and citalopram.

Serotonin (5-hydroxytryptamine, 5-HT) is an amine neurotransmitter derived from tryptophan and is important in brain systems regulating mood, emotional behavior, and sleep. Selective serotonin reuptake inhibitor (SSRI) drugs are used to treat disorders such as depression, stress, eating disorders, autism, and schizophrenia. It is thought that these drugs act to prolong the action of 5-HT by blocking reuptake. This may lead to decreased 5-HT content in the nerve fibers themselves; however, this has not previously been directly demonstrated. We have studied the effects of administration of two drugs, imipramine and citalopram, on levels of 5-HT in nerve fibers in the murine brain. Quantitative analysis of the areal density of 5-HT fibers throughout the brain was performed using ImageJ software. While a high density of fibers was observed in mid- and hind-brain regions and areas such as thalamus and hypothalamus, densities were far lower in areas such as cortex, where SSRIs might be thought to exert their actions. As anticipated, imipramine and citalopram produced a decline in 5-HT levels in nerve fibers, but the result was not uniform. Areas such as inferior colliculus showed significant reduction whereas little, if any, change was observed in the adjacent superior colliculus. The reason for, and significance of, this regionality is unclear. It has been proposed that serotonin effects in the brain might be linked to changes in glutamatergic transmission. Extracellular glutamate levels are regulated primarily by glial glutamate transporters. Qualitative evaluation of glutamate transporter immunolabeling in cortex of control and drug-treated mice revealed no discernable difference in intensity of glutamate transporter immunoreactivity. These data suggest that changes in intracellular and extracellular levels of serotonin do not cause concomitant changes in astroglial glutamate transporter expression, and thus cannot represent a mechanism for the delayed efficacy of antidepressants when administered clinically.

Nitric oxide donors inhibit 5-hydroxytryptamine (5-HT) uptake by the human 5-HT transporter (SERT).

1. The aim was to test the hypothesis that nitric oxide (NO) donor drugs can inhibit the 5-hydroxytryptamine (5-HT) transporter, SERT. 2. The NO donors, MAHMA/NO (a NONOate; (Z)-1-[N-methyl-N-[6-(N-methylammoniohexyl)-amino]]diazen-1-ium-1,2-diolate), SIN-1 (a sydnonimine; 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride), FK409 (an oxime; (+/-)-(4-ethyl-2E-(hydroxyimino)-5-nitro-3E-hexenamide)) and peroxynitrite, but not Angeli's salt (source of nitroxyl anion) or sodium nitrite, caused concentration-dependent inhibition of the specific uptake of [3H]-5-HT in COS-7 cells expressing human SERT. 3. Superoxide dismutase (150 U ml(-1)) plus catalase (1200 U ml(-1)), used to remove superoxide and hence prevent peroxynitrite formation, prevented the inhibitory effect of SIN-1 (which generates superoxide) but not of MAHMA/NO or FK409. 4 The inhibitory effects of the NO donors were not affected by the free radical scavenger, hydroxocobalamin (1 mM) or the guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 3 microM). 5. L-Cysteine (1 mM; source of excess thiol residues) abolished or markedly reduced the inhibitory effects of MAHMA/NO, SIN-1, FK409 and peroxynitrite. 6. It is concluded that inhibition of SERT by the NO donors cannot be attributed exclusively to NO free radical nor to nitroxyl anion. It does not involve guanosine-3',5'-cyclic monophosphate, but may involve nitrosation of cysteine residues on the SERT protein. Peroxynitrite mediates the effect of SIN-1, but not the other drugs. 7. Data in mice with hypoxic pulmonary hypertension suggest that SERT inhibitors may attenuate pulmonary vascular remodelling. Thus, NO donors may be useful in pulmonary hypertension, not only as vasodilators, but also because they inhibit SERT, provided they display this effect in vivo at appropriate doses.

chi-Conopeptide MrIA partially overlaps desipramine and cocaine binding sites on the human norepinephrine transporter.

The interactions of chi-conopeptide MrIA with the human norepinephrine transporter (hNET) were investigated by determining the effects of hNET point mutations on the inhibitory potency of MrIA. The mutants were produced by site-directed mutagenesis and expressed in COS-7 cells. The potency of MrIA was greater for inhibition of uptake by hNET of [3H]norepinephrine (Ki 1.89 microM) than [3H]dopamine (Ki 4.33 microM), and the human dopamine transporter and serotonin transporter were not inhibited by MrIA (to 7 microM). Of 18 mutations where hNET amino acid residues were exchanged with those of the human dopamine transporter, MrIA had increased potency for inhibition of [3H]norepinephrine uptake for three mutations (in predicted extracellular loops 3 and 4 and transmembrane domain (TMD) 8) and decreased potency for one mutation (in TMD6 and intracellular loop (IL) 3). Of the 12 additional mutations in TMDs 2, 4, 5, and 11 and IL1, three mutations (in TMD2 and IL1) had reduced MrIA inhibitory potency. All of the other mutations tested had no influence on MrIA potency. A comparison of the results with previous data for desipramine and cocaine inhibition of norepinephrine uptake by the mutant hNETs reveals that MrIA binding to hNET occurs at a site that is distinct from but overlaps with the binding sites for tricyclic antidepressants and cocaine.

5-Hydroxytryptamine and platelets: uptake and aggregation in hypoxic pulmonary hypertensive rats.

Pulmonary hypertension is associated with various alterations in 5-hydroxytryptamine (5-HT) physiology. In this study in platelets from hypoxic pulmonary hypertensive rats (10% O(2); 1 week) and normoxic rats (room air), (i) initial rates of specific [3H]5-HT uptake were measured and (ii) potentiation of collagen- and ADP-induced aggregation by 5-HT was quantified. The platelet count was almost halved in hypoxic rats. In uptake experiments, there was a decrease in 5-HT uptake in platelets from hypoxic compared with normoxic rats, due to a 36% reduction in the maximal initial rate of uptake. The aggregation experiments showed that 5-HT (1-100 microM) increased the magnitude of responses to collagen and the duration of responses to ADP, but there was no difference between hypoxic and normoxic rats. Abnormalities in platelet function may conceivably lead to increases in plasma 5-HT levels in hypoxic pulmonary hypertension, but are unlikely to aggravate pulmonary thromboembolism.

The role of the conserved GXXXRXG motif in the expression and function of the human norepinephrine transporter.

Highly conserved motifs in the monoamine transporters, e.g. the human norepinephrine transporter (hNET) GXXXRXG motif which was the focus of the present study, are likely to be important structural features in determining function. This motif was investigated by mutating the glycines to glutamate (causing loss of function) and alanine, and the arginine to glycine. The effects of hG117A, hR121G and hG123A mutations on function were examined in COS-7 cells and compared to hNET. Substrate K(m) values were decreased for hG117A and hG123A, and their K(i) values for inhibition of [3H]nisoxetine binding were decreased 3-4-fold and 4-6-fold, respectively. Transporter turnover was reduced to 65% of hNET for hG117A and hR121G and to 28% for hG123A, suggesting that substrate translocation is impaired. K(i) values of nisoxetine and desipramine for inhibition of [3H]norepinephrine uptake were increased by 5-fold for hG117A, with no change for cocaine. The K(i) value of cocaine was increased by 3-fold for hG123A, with no change for nisoxetine and desipramine. However, there were no effects of the mutations on the K(d) of [3H]nisoxetine binding or K(i) values of desipramine or cocaine for inhibition of [3H]nisoxetine binding. Hence, glycine residues of the GXXXRXG motif are important determinants of NET expression and function, while the arginine residue does not have a major role. This study also showed that antidepressants and psychostimulants have different NET binding sites and provided the first evidence that different sites on the NET are involved in the binding of inhibitors and their competitive inhibition of substrate uptake.

Functional significance of a highly conserved glutamate residue of the human noradrenaline transporter.

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+- and Cl--dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site-directed mutagenesis and the mutants were expressed in transfected COS-7 or HEK-293 cells. Cell surface expression of hE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in Vmax values for noradrenaline and dopamine uptake of 2-3-fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82-260-fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.

Pharmacological properties of the naturally occurring Ala(457)Pro variant of the human norepinephrine transporter.

Recently, another research group has reported an almost complete loss of function of the human norepinephrine transporter (hNET) in patients who had orthostatic intolerance and who were heterozygous for a guanine to cytosine exchange, resulting in a hNET Ala(457)Pro variant. To explore the reason for the deficiency in NET function, we compared in detail the pharmacology of the Ala(457)Pro variant with that of the wild-type hNET in COS-7 cells transiently transfected with hNET or Ala(457)Pro cDNA. Compared to the wild-type hNET, the Ala(457)Pro variant exhibited a five-fold higher affinity for cocaine, but a two-fold lower affinity for the NET inhibitor nisoxetine, and an unchanged affinity for the antidepressant desipramine. Plasma membrane expression (measured as Bmax of [3H]nisoxetine binding) of the Ala(457)Pro variant was only 40% of that of the wild-type hNET. The Ala(457)Pro variant showed a six- to 10-fold decrease in affinity for the substrates dopamine and 1-methyl-4-phenylpyridinium (MPP(+)). Compared with the wild-type hNET, the maximum rate (V(max)) of norepinephrine uptake by the Ala(457)Pro variant was slightly reduced, whereas the turnover number (calculated from V(max)/B(max)) was approximately two-fold higher. However, the Ala(457)Pro variant exhibited a 50-fold higher K(m) (i.e. lower apparent affinity) for norepinephrine than the wild-type hNET. Thus, the previously reported loss of function of the Ala(457)Pro variant associated with orthostatic intolerance is only partly due to a reduction in plasma membrane expression of the transporter, and is mainly caused by the pronounced reduction in the apparent affinity of norepinephrine.

Amino acids involved in differences in the pharmacological profiles of the rat and human noradrenaline transporters.

It has been suggested from a previous study in our laboratory that differences in the pharmacology of the species variants of the noradrenaline transporter (NET) are the result of four non-conservative amino acid exchanges from the total of 26 amino acids that are divergent between the rat NET (rNET) and human NET (hNET). The aim of this study was to examine the effects of changing the rNET at each of these four amino acid residues, which markedly alter local charge distribution, to the amino acid found in hNET.Site-directed mutagenesis was used to create mutant cDNAs from rNET cDNA. The mutant NETs (rK7D, rE62K, rK375N and rR612Q), rNET and hNET were expressed in transiently transfected COS-7 cells to determine the effects of the mutations on the differing pharmacological properties of the species variants. The ratios of V(max) for noradrenaline uptake and B(max) for nisoxetine binding (which are a measure of the turnover number of the transporter, i.e. the number of transport cycles per min) were greater for rNET and rR612Q than for hNET, rK7D, rE62K and rK375N. The K(m) of noradrenaline was lower for hNET, rK7D, rE62K and rK375N than for rNET or rR612Q. There were no differences between the K(i) values for inhibition of noradrenaline uptake by nisoxetine for rNET, hNET or the mutants, but the K(i) values of cocaine were lower for hNET, rE62K and rR612Q than rNET or rK375N.Hence, the study showed that: (1) the aspartate 7, lysine 62 and asparagine 375 amino acid residues are important in determining the lower substrate translocation by hNET than rNET; (2) the aspartate 7 and lysine 62 residues in the N-terminus of hNET determine the higher affinities of substrates for the hNET than the rNET; and (3) the lysine 62 and glutamine 612 residues in the N- and C-termini, respectively, of hNET are determinants of the higher cocaine affinity for the hNET than rNET.