Identification of the Endogenous Key Substrates of the Human Organic Cation Transporter OCT2 and Their Implication in Function of Dopaminergic Neurons

BACKGROUND: The etiology of neurodegenerative disorders, such as the accelerated loss of dopaminergic neurons in Parkinson's disease, is unclear. Current hypotheses suggest an abnormal function of the neuronal sodium-dependent dopamine transporter DAT to contribute to cell death in the dopaminergic system, but it has not been investigated whether sodium-independent amine transporters are implicated in the pathogenesis of Parkinson's disease. METHODOLOGY/PRINCIPAL FINDINGS: By the use of a novel tandem-mass spectrometry-based substrate search technique, we have shown that the dopaminergic neuromodulators histidyl-proline diketopiperazine (cyclo(his-pro)) and salsolinol were the endogenous key substrates of the sodium-independent organic cation transporter OCT2. Quantitative real-time mRNA expression analysis revealed that OCT2 in contrast to its related transporters was preferentially expressed in the dopaminergic regions of the substantia nigra where it colocalized with DAT and tyrosine hydroxylase. By assessing cell viability with the MTT reduction assay, we found that salsolinol exhibited a selective toxicity toward OCT2-expressing cells that was prevented by cyclo(his-pro). A frequent genetic variant of OCT2 with the amino acid substitution R400C reduced the transport efficiency for the cytoprotective cyclo(his-pro) and thereby increased the susceptibility to salsolinol-induced cell death. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that the OCT2-regulated interplay between cyclo(his-pro) and salsolinol is crucial for nigral cell integrity and that a shift in transport efficiency may impact the risk of Parkinson's disease

Substrate discrimination by ergothioneine transporter SLC22A4 and carnitine transporter SLC22A5: Gain-of-function by interchange of selected amino acids

AbstractETT (originally designated as OCTN1; human gene symbol SLC22A4) and CTT (OCTN2; SLC22A5) are highly specific transporters of ergothioneine and carnitine, respectively. Despite a high degree of sequence homology, both carriers discriminate precisely between substrates: ETT does not transport carnitine, and CTT does not transport ergothioneine. Our aim was to turn ETT into a transporter for carnitine and CTT into a transporter for ergothioneine by a limited number of point mutations. From a multiple alignment of several mammalian amino acid sequences, those positions were selected for conversion that were momentously different between ETT and CTT from human but conserved among all orthologues. Mutants were expressed in 293 cells and assayed for transport of ergothioneine and carnitine. Several ETT mutants clearly catalyzed transport of carnitine, up to 35% relative to wild-type CTT. Amazingly, complementary substitutions in CTT did not provoke transport activity for ergothioneine. In similar contrast, carnitine transport by CTT mutants was abolished by very few substitutions, whereas ergothioneine transport by ETT mutants was maintained even with the construct most active in carnitine transport. To explain these results, we propose that ETT and CTT use dissimilar pathways for conformational change, in addition to incongruent substrate binding sites. In other words, carnitine is excluded from ETT by binding, and ergothioneine is excluded from CTT by turnover movement. Our data indicate amino acids critical for substrate discrimination not only in transmembrane segments 5, 7, 8, and 10, but also in segments 9 and 12 which were hitherto considered as unimportant

Simultaneous fitting of real-time PCR data with efficiency of amplification modeled as Gaussian function of target fluorescence

<p>Abstract</p> <p>Background</p> <p>In real-time PCR, it is necessary to consider the efficiency of amplification (EA) of amplicons in order to determine initial target levels properly. EAs can be deduced from standard curves, but these involve extra effort and cost and may yield invalid EAs. Alternatively, EA can be extracted from individual fluorescence curves. Unfortunately, this is not reliable enough.</p> <p>Results</p> <p>Here we introduce simultaneous non-linear fitting to determine – without standard curves – an optimal common EA for all samples of a group. In order to adjust EA as a function of target fluorescence, and still to describe fluorescence as a function of cycle number, we use an iterative algorithm that increases fluorescence cycle by cycle and thus simulates the PCR process. A Gauss peak function is used to model the decrease of EA with increasing amplicon accumulation. Our approach was validated experimentally with hydrolysis probe or SYBR green detection with dilution series of 5 different targets. It performed distinctly better in terms of accuracy than standard curve, DART-PCR, and LinRegPCR approaches. Based on reliable EAs, it was possible to detect that for some amplicons, extraordinary fluorescence (EA > 2.00) was generated with locked nucleic acid hydrolysis probes, but not with SYBR green.</p> <p>Conclusion</p> <p>In comparison to previously reported approaches that are based on the separate analysis of each curve and on modelling EA as a function of cycle number, our approach yields more accurate and precise estimates of relative initial target levels.</p

The garlic ingredient diallyl sulfide inhibits cytochrome P450 2E1 dependent bioactivation of acrylamide to glycidamide. Toxicol Lett

Abstract Genotoxic effects of acrylamide are supposed to result from oxidative biotransformation to glycidamide. After incubation of rat liver slices with acrylamide we detected free glycidamide using a liquid chromatography tandem mass spectrometric method. Glycidamide formation was diminished in the presence of the cytochrome P450 2E1 inhibitor diallyl sulfide (DAS), which is a specific ingredient of garlic. This may be relevant to human health since the suggested carcinogenic risk of dietary acrylamide may be reduced by concomitant intake of garlic