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The conservative view: is it necessary to implant a stent into the dopamine transporter?

Schmid D, Koenig X, Bulusu S, Schicker K, Freissmuth M, Sitte HH, Sandtner W - Br. J. Pharmacol. (2015)

View Article: PubMed Central - PubMed

Affiliation: Center of Physiology and Pharmacology, Medical University Vienna, Vienna, Austria.

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The principal argument in favour of the molecular stent hypothesis was the observation of a persistent current through DAT expressed in Xenopus laevis oocytes upon removal of (S+)amphetamine ((S+)AMPH) from the bath solution... However, we showed that such a current was not a property of DAT expressed in HEK-293 cells (supplementary Fig.1 of )... This observation was at odds with the conclusions reached by DeFelice and coworkers and prompted us to develop a more comprehensive model that was able to explain the results from both experimental systems... In their commentary, DeFelice and Cameron assert that we observed a persistent current in HEK-293 cells but failed to acknowledge it. “In summary, Sandtner et al. see the same relative persistent current in oocytes and in HEK cells, contrary to their assertion expressed in (1), that no persistent current exists in HEK cells” The current in HEK-293 cells expressing DAT or SERT decayed fully to baseline and hence, we prefer not to label it “persistent”... Another point raised by DeFelice and Cameron is that the difference in decay kinetics observed between these different experimental systems (HEK-293 cells and oocytes) could be due to the presence of the patch electrode in HEK-293 cells... We had indeed neglected the patch electrode in our model for the sake of simplicity. “Even though the relative persistent current is the same in oocytes and HEK cells, the more rapid decay of the persistent current in HEK cells compared with oocytes still remains a mystery... One possibility is that whereas sharp electrodes penetrate the oocyte, relatively large, whole-cell electrodes penetrate the HEK cell and are likely to perfuse the cell with the electrode solution. ” What DeFelice and Cameron seem to imply is that the currents in HEK-293 cells would have decayed similarly to those in Xenopus laevis oocytes if they had not been measured with a patch electrode... These amplitudes were plotted as a function of the polar surface area (PSA) - a good predictor of membrane permeability... They found no correlation between these values and concluded that the lipophilicity-based model proposed by us is incorrect. “Based on Sandtner et al. hypothesis, we would expect to observe the largest persistent current with SMETH due to its low PSA value... We would also expect SAMPH, RAMPH, S MCAT, and SMDMA to produce similar size persistent current because their PSA values are very similar... Our data does not support these predictions and disprove the main hypothesis of the Sandtner et al. model. ” We brought up differences in PSA to highlight the greater ability of amphetamines, compared to the cognate substrates of SERT and DAT, to cross membranes... We simulated currents for an exposure time of 60 sec (as was used by DeFelice et al.) and measured the current amplitude 60 sec after removal of the respective compound from the bath solution... In summary, our model is capable of accounting for the results from both laboratories... We are not aware of other evidence in support of this idea... Moreover the molecular stent hypothesis does not account for the dependence of the “shelf current” on external Na, which is expected if amphetamines act by being transported into the cell, a Na-dependent process.

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2A shows a kinetic model of DAT’s transport cycle embedded into our model for substrate fluxes. The rates for DA binding and unbinding (in red) were adapted to account for the EC50 for the induction of DA currents in Xenopus laevis oocytes. For modelling the currents by (S+)AMPH, (S+)METH and (S−)MCAT we used the same rates as for DA. However for modelling currents by (R−)AMPH and (S+)MDMA we utilized a different set of rates (shown in blue) to account for the lower affinity of these compounds. 2B shows a table of substrate specific parameters that are critical in the prediction of the currents. 2C shows examples of simulated current traces. The red trace shows the response to 10μM DA for an application period of 60s. The blue trace is the response to 10μM (S+) AMPH respectively. 2D Shown is a comparison of the persistent current observed by DeFelice et al. 60 s after removal and the values predicted by the model. Open circles always indicate observed values whereas predicted values are indicated by open triangles. (S+)METH, (S+)AMPH, (R−) AMPH, (S−) MCAT,(S+)MDMA and DA are shown in the colours green, blue, yellow, magenta, grey and red respectively.
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Figure 2: 2A shows a kinetic model of DAT’s transport cycle embedded into our model for substrate fluxes. The rates for DA binding and unbinding (in red) were adapted to account for the EC50 for the induction of DA currents in Xenopus laevis oocytes. For modelling the currents by (S+)AMPH, (S+)METH and (S−)MCAT we used the same rates as for DA. However for modelling currents by (R−)AMPH and (S+)MDMA we utilized a different set of rates (shown in blue) to account for the lower affinity of these compounds. 2B shows a table of substrate specific parameters that are critical in the prediction of the currents. 2C shows examples of simulated current traces. The red trace shows the response to 10μM DA for an application period of 60s. The blue trace is the response to 10μM (S+) AMPH respectively. 2D Shown is a comparison of the persistent current observed by DeFelice et al. 60 s after removal and the values predicted by the model. Open circles always indicate observed values whereas predicted values are indicated by open triangles. (S+)METH, (S+)AMPH, (R−) AMPH, (S−) MCAT,(S+)MDMA and DA are shown in the colours green, blue, yellow, magenta, grey and red respectively.

Mentions: In figure 2 of their comment DeFelice and Cameron show the amplitudes of persistent currents evoked by a set of different compounds. These amplitudes were plotted as a function of the polar surface area (PSA) - a good predictor of membrane permeability. They found no correlation between these values and concluded that the lipophilicity-based model proposed by us is incorrect.


The conservative view: is it necessary to implant a stent into the dopamine transporter?

Schmid D, Koenig X, Bulusu S, Schicker K, Freissmuth M, Sitte HH, Sandtner W - Br. J. Pharmacol. (2015)

2A shows a kinetic model of DAT’s transport cycle embedded into our model for substrate fluxes. The rates for DA binding and unbinding (in red) were adapted to account for the EC50 for the induction of DA currents in Xenopus laevis oocytes. For modelling the currents by (S+)AMPH, (S+)METH and (S−)MCAT we used the same rates as for DA. However for modelling currents by (R−)AMPH and (S+)MDMA we utilized a different set of rates (shown in blue) to account for the lower affinity of these compounds. 2B shows a table of substrate specific parameters that are critical in the prediction of the currents. 2C shows examples of simulated current traces. The red trace shows the response to 10μM DA for an application period of 60s. The blue trace is the response to 10μM (S+) AMPH respectively. 2D Shown is a comparison of the persistent current observed by DeFelice et al. 60 s after removal and the values predicted by the model. Open circles always indicate observed values whereas predicted values are indicated by open triangles. (S+)METH, (S+)AMPH, (R−) AMPH, (S−) MCAT,(S+)MDMA and DA are shown in the colours green, blue, yellow, magenta, grey and red respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561504&req=5

Figure 2: 2A shows a kinetic model of DAT’s transport cycle embedded into our model for substrate fluxes. The rates for DA binding and unbinding (in red) were adapted to account for the EC50 for the induction of DA currents in Xenopus laevis oocytes. For modelling the currents by (S+)AMPH, (S+)METH and (S−)MCAT we used the same rates as for DA. However for modelling currents by (R−)AMPH and (S+)MDMA we utilized a different set of rates (shown in blue) to account for the lower affinity of these compounds. 2B shows a table of substrate specific parameters that are critical in the prediction of the currents. 2C shows examples of simulated current traces. The red trace shows the response to 10μM DA for an application period of 60s. The blue trace is the response to 10μM (S+) AMPH respectively. 2D Shown is a comparison of the persistent current observed by DeFelice et al. 60 s after removal and the values predicted by the model. Open circles always indicate observed values whereas predicted values are indicated by open triangles. (S+)METH, (S+)AMPH, (R−) AMPH, (S−) MCAT,(S+)MDMA and DA are shown in the colours green, blue, yellow, magenta, grey and red respectively.
Mentions: In figure 2 of their comment DeFelice and Cameron show the amplitudes of persistent currents evoked by a set of different compounds. These amplitudes were plotted as a function of the polar surface area (PSA) - a good predictor of membrane permeability. They found no correlation between these values and concluded that the lipophilicity-based model proposed by us is incorrect.

View Article: PubMed Central - PubMed

Affiliation: Center of Physiology and Pharmacology, Medical University Vienna, Vienna, Austria.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

The principal argument in favour of the molecular stent hypothesis was the observation of a persistent current through DAT expressed in Xenopus laevis oocytes upon removal of (S+)amphetamine ((S+)AMPH) from the bath solution... However, we showed that such a current was not a property of DAT expressed in HEK-293 cells (supplementary Fig.1 of )... This observation was at odds with the conclusions reached by DeFelice and coworkers and prompted us to develop a more comprehensive model that was able to explain the results from both experimental systems... In their commentary, DeFelice and Cameron assert that we observed a persistent current in HEK-293 cells but failed to acknowledge it. “In summary, Sandtner et al. see the same relative persistent current in oocytes and in HEK cells, contrary to their assertion expressed in (1), that no persistent current exists in HEK cells” The current in HEK-293 cells expressing DAT or SERT decayed fully to baseline and hence, we prefer not to label it “persistent”... Another point raised by DeFelice and Cameron is that the difference in decay kinetics observed between these different experimental systems (HEK-293 cells and oocytes) could be due to the presence of the patch electrode in HEK-293 cells... We had indeed neglected the patch electrode in our model for the sake of simplicity. “Even though the relative persistent current is the same in oocytes and HEK cells, the more rapid decay of the persistent current in HEK cells compared with oocytes still remains a mystery... One possibility is that whereas sharp electrodes penetrate the oocyte, relatively large, whole-cell electrodes penetrate the HEK cell and are likely to perfuse the cell with the electrode solution. ” What DeFelice and Cameron seem to imply is that the currents in HEK-293 cells would have decayed similarly to those in Xenopus laevis oocytes if they had not been measured with a patch electrode... These amplitudes were plotted as a function of the polar surface area (PSA) - a good predictor of membrane permeability... They found no correlation between these values and concluded that the lipophilicity-based model proposed by us is incorrect. “Based on Sandtner et al. hypothesis, we would expect to observe the largest persistent current with SMETH due to its low PSA value... We would also expect SAMPH, RAMPH, S MCAT, and SMDMA to produce similar size persistent current because their PSA values are very similar... Our data does not support these predictions and disprove the main hypothesis of the Sandtner et al. model. ” We brought up differences in PSA to highlight the greater ability of amphetamines, compared to the cognate substrates of SERT and DAT, to cross membranes... We simulated currents for an exposure time of 60 sec (as was used by DeFelice et al.) and measured the current amplitude 60 sec after removal of the respective compound from the bath solution... In summary, our model is capable of accounting for the results from both laboratories... We are not aware of other evidence in support of this idea... Moreover the molecular stent hypothesis does not account for the dependence of the “shelf current” on external Na, which is expected if amphetamines act by being transported into the cell, a Na-dependent process.

Show MeSH