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

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          Abstract

          The question of how amphetamines affect the transporters for the neurotransmitters serotonin (5-HT), dopamine (DA) and noradrenalin (NA) has been subject of numerous studies. Over three decades ago Fischer and Cho (Fischer and Cho, 1979) proposed that amphetamine- induced DA release occurs as a consequence of facilitated exchange diffusion. The exchange diffusion hypothesis posits that an amphetamine will release monoamines if (i) the transporter operates according to an alternate access mechanism and (ii) amphetamines are substrates of the transporter. When both of these assumptions are fulfilled, monoamine release is predicted to occur by exchange. An alternative view, proposed by Lester (Su et al., 1996) and DeFelice (Adams et. al., 2003, Petersen and DeFelice, 1999) is the idea that monoamine transporters work more like channels rather than by alternating access. The molecular stent hypothesis of amphetamine action proposed by DeFelice fits better with this model, because it can work without reference to an alternate access mechanism. 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 (Rodriguez-Menchaca et al., 2012). We also observed persistent currents in Xenopus laevis oocytes. However, we showed that such a current was not a property of DAT expressed in HEK-293 cells (supplementary Fig.1 of Sandtner et al., 2013). 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. For this we had to take into account a very important attribute of amphetamines: as a class they are more membrane permeant than the natural substrates of the transporters. This allows them to leak out of the cell after uptake and maintain an extracellular concentration that leads to continued influx of amphetamine and Na+; both of which facilitate substrate efflux. Moreover, their permeability is the reason they readily cross the blood brain barrier to release monoamines from CNS neurons. By incorporating this property into our previously described model of the SERT transport cycle (Schicker et al., 2011), we were able to account for the disparate findings in HEK-293 cells and oocytes and relate them to differences in cell size. Furthermore we were able to link the different ways that cognate substrate and amphetamines affect SERT associated currents to their different physicochemical properties. 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”. However, their statement raises important questions regarding the nature of the slow decay of this current. Both our analyses presume that amphetamine builds up in the cytoplasm during prolonged incubation. It is this cytoplasmic amphetamine that would bind to an intracellular site on DAT or SERT in the molecular stent hypothesis. In our model, the cytoplasm is a reservoir of amphetamine that leaks out and acts on DAT or SERT from the extracellular side. If DeFelice et al. assign the decay to slow dissociation of amphetamine from the transporter, then the absolute value of this rate is critical, because the dissociation of a ligand from its binding site should not be dependent on the cellular expression system. The interaction of amphetamine at this binding site could also be rapid and of low affinity, and then the extent of binding would be in equilibrium with the intracellular concentration. If this is what DeFelice and Cameron believe, we can agree that the internal volume of a cell matters and that the persistent current in Xenopus laevis oocytes is a consequence of the large internal reservoir of amphetamine. 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. Obviously this is difficult to test experimentally, so we decided to model the time course of amphetamine diffusion out of a HEK-293 cell with and without the patch electrode. We built a diffusion model of a cell attached to the patch electrode to emulate the whole cell patch clamp configuration (see the supplement for a description of the model) and set up the following simulation (see figure 1A): We assumed that at the beginning of the simulation the cell was filled with 100μM (S+)AMPH, whereas the bath solution and the patch electrode contained no (S+)AMPH. Figure 1B shows the drop in intracellular concentration over time as (S+)AMPH exited the cell. This simulation was conducted in the presence and in the absence of the patch-electrode. As can be seen from these simulations (figure 1B) the respective time courses were affected by the presence of the patch electrode. However, the difference in the time-constant (0.6 s versus 1.8 s) was not large enough to account for currents that persist for minutes (as they do in Xenopus laevis oocytes) even in the absence of the patch electrode. 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. “Based on Sandtner et al. hypothesis, we would expect to observe the largest persistent current with S(+)METH due to its low PSA value. We would also expect S(+)AMPH, R(−)AMPH, S(−) MCAT, and S(+)MDMA 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. Another important determinant is the affinity of each amphetamine to the transporter, which was not considered in DeFelice and Cameron’s Figure 2. For example, in that figure, R(−)AMPH and S(+) MDMA gave lower currents than expected based on PSA alone, but these two derivatives are known to bind to DAT with low affinity (Baumann et al., 2007, Harris and Baldessarini, 1973). Taking both affinity and PSA into account, we modeled the ability of each of the compounds in DeFelice and Cameron’s Figure 2 to elicit a persistent current (Figure 2). For this we utilized a model for DAT’s reaction cycle as previously proposed (Erreger et al., 2008) and as can be seen the simulations agreed quite well with the reported measurements (for a model description see supplement). The model predicts a “shelf current”, shown in Figure 2C, similar to that observed by DeFelice et al. (Rodriguez-Menchaca et al,. 2012). Shown are simulated current traces of currents induced by 10μM DA and 10μM (S+)AMPH respectively. While the DA induced current quickly decayed to baseline values the (S+)AMPH induced current persisted. 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. Moreover, this model does not invoke any special properties of amphetamines other than their ability to act as substrates and to cross membranes easily, and without invoking an intracellular binding site with the ability to open a conductance. The molecular stent hypothesis invokes actions of amphetamines on monoamine transporters, such as their ability to bind to a binding site at the internal vestibule, that cannot be occupied by the cognate substrate (Rodriguez-Menchaca et al., 2012). 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+ (Rodriguez-Menchaca et al., 2012), which is expected if amphetamines act by being transported into the cell, a Na+-dependent process. The requirement for internal K+ concentrations is also expected because an inward-facing K+-bound intermediate is required for substrate-induced currents (Adams and DeFelice, 2003, Sandtner et al., 2013). However, these observations are difficult to explain within the framework of the molecular stent hypothesis. In addition the observed inactivation of substrate-induced currents (Sandtner et al., 2013) is predicted by our model as intracellular amphetamine builds up and competes with K+ but is not predicted by the model proposed by DeFelice and coworkers. Supplementary Material 1 2 3

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          Unifying Concept of Serotonin Transporter-associated Currents*

          Introduction Neurotransmitter transporters such as serotonin transporter (SERT), 2 dopamine transporter (DAT), and norepinephrine transporter (NET) are essential for synaptic transmission. SERT is responsible for serotonin (5-HT) reuptake from the synaptic cleft and therefore shapes synaptic transmission by both terminating receptor-mediated signaling events and by replenishing vesicular neurotransmitter pools (1). SERT is an important drug target for therapeutic compounds such as antidepressant 5-HT reuptake inhibitors and for illicit substances, such as cocaine and amphetamines including methylene-dioxymethamphetamine (ecstasy) (2). A model of the SERT transport cycle was first derived from measuring the flux of radiolabled 5-HT and the influence of intracellular and extracellular ions. The salient features of this model posited that one Na+ and one Cl− ion were co-transported with the substrate and one K+ ion was counter transported with each positively charged 5-HT molecule (3, 4). This stoichiometry predicts that no net charge movement occurs during the transport cycle. In contrast to this prediction, several studies showed that SERT mediates a constant current when challenged with the natural substrate 5-HT or with amphetamines (5–8). Single-channel recordings (9) and experiments measuring uptake and current simultaneously (5, 7), suggested that most if not all of the current was accounted for by uncoupled ion flux. In addition, the presence of a substrate-independent leak current further supports the notion that in SERT, ion permeation is not strictly coupled to transport. Although SERT is the only neurotransmitter transporter for which transport stoichiometry is believed to be electroneutral, the presence of an uncoupled current component was also shown in DAT and NET (10–13). For example, in the case of DAT it was proposed that the contribution of the uncoupled current is responsible for approximately 50% of the current amplitude measured upon dopamine addition (14). With respect to these leak currents, which are uncoupled by definition, both DAT and NET are phenomenologically very similar to SERT. Because the uncoupled currents of SERT, DAT, and NET are not direct indicators of substrate transport, it has been difficult to use measurement of these currents to understand structural and mechanistic aspects of transport. The work presented here attempts to assign a mechanistic basis for these currents. Additionally it is unclear why SERT, NET, or DAT would dissipate part of the energy stored in transmembrane ion gradients without obvious benefits for transport. However, there are indications that substrate-induced currents and reverse transport are correlated (15). In DAT a mechanism has been proposed in which substrate release is carried by a channel mode of the transporter (16). Also for SERT, it was suggested that 5-HT is driven through a channel in the transporter by rheogenic Na+ flux (17). However, this proposal is inconsistent with observations that imposing a membrane potential did not affect the rate or steady-state level of 5-HT uptake (3). Moreover, co-expression of syntaxin 1A eliminated 5-HT-induced and endogenous SERT currents but not the transport of 5-HT (7). There are reports that SERT and DAT currents are regulated in vivo by association with syntaxin (7, 18). In the present study, we characterized the nature of the uncoupled currents. By combining electrophysiological, biochemical, and fluorescence microscopy techniques we show that the uncoupled current in SERT is carried by a K+-dependent conducting state that is in equilibrium with an inward facing conformation of the transporter. EXPERIMENTAL PROCEDURES hSERT Expression in Xenopus Oocytes Stage V and VI Xenopus oocytes were isolated from female frogs (NASCO, Ft. Atkinson, WI), washed with a solution containing 96 mm NaCl, 2 mm KCl, 20 mm MgCl2, and 5 mm HEPES (titrated to pH 7.4 with NaOH), treated with 1 mg/ml collagenase for 0.5 to 1 h, and had their follicular cell layers manually removed. As judged from photometric measurements, ∼5 ng of cRNA was injected into each oocyte with a Drummond microinjector (Broomall, PA). cRNA was synthesized using a T7 promoter cRNA synthesis kit (Ambion). Xenopus laevis oocytes were injected with 50-nl cRNAs of hSERT (5 ng/oocyte). Oocytes were allowed 3–5 days to express the hSERT before attempting recordings. Two-electrode Voltage Clamp Recordings were made in the two-electrode voltage clamp configuration using a TEC 10CD clamp (npi electronic, Tamm, Germany). Oocytes were placed in recording chambers in which the bath flow rate was about 100 ml/h, and the bath level was adjusted so that the total bath volume was 60% (n = 7, Fig. 1, c and d). When the cells were clamped to holding potentials more positive than 0 mV the background noise increased due to activation of endogenous K+ channels, and noise precluded reliable analysis of current amplitudes. These data demonstrate the presence of two different components in 5-HT-induced currents of hSERT. One component is transient and saturates only at high 5-HT concentration whereas the other saturates at low 5-HT and has a slow rise time and no decay. Conducting State Is Linked to K+ Intracellular K+ plays an important role in the function of hSERT (3, 4). The replacement of intracellular K+ with other cations led to a dramatic decrease in 5-HT uptake. Similarly, omission of intracellular K+ reduced the amplitude of hSERT currents recorded from X. laevis oocytes (6). Due to the large size and the slow exchange rate of solution around the cells, currents recorded in oocytes most likely represent only the steady-state component. In the cellular system used here, the steady-state current in response to 10 μm 5-HT was also abolished when K+ was replaced by NMDG. In contrast, the peak current remained unaltered (Fig. 2). These data show that the steady-state component requires internal K+, whereas the peak component does not, suggesting that they represent different processes. FIGURE 2. Low intracellular K+ abolishes the steady-state component of the 5-HT-induced current in hSERT. Single HEK293 cells stably expressing hSERT were voltage clamped to a holding potential of −70 mV, and 5-HT (10 μm) was applied. a, sample currents from two cells recorded either with 163 mm K+ (upper) or 0 mm (substituted with NMDG; lower) in the pipette solution. b, statistical evaluation of peak and steady-state component at the indicated conditions (163 K+ versus 0 K+). Peak, −7.0 ± 1.1 pA versus −8.1 ± 0.7 pA; steady state, −4.9 ± 0.6 pA versus −1–1 ± 0.8 pA. ***, p 3-fold. Agents known to increase the current amplitude such as 5-HT and Li+ also increased the reaction rate, indicating a more open cytoplasmic pathway. 10 μm 5-HT (Fig. 3 a, light gray circles) increased the rate of labeling >2.5-fold, and exchanging Na+ with Li+ (Fig. 3 a, dark gray circles) increased the rate >4-fold. These data show that both 5-HT and Li+ increase the rate of labeling to an extent comparable with ibogaine, implying that they favor accumulation of SERT in an inward facing conformation. FIGURE 3. Conformational probes indicate that ibogaine, 5-HT, and Li+ favor the inward facing conformation of SERT. a, membranes from HeLa cells transfected with SERT S277C in the X5C background were treated for 15 min with the indicated concentrations of MTSEA either in the presence of 150 mm Na+ alone or with 40 μm ibogaine, 10 μm 5-HT, or after substitution of Na+ with Li+. The log IC50 values for MTSEA for all of the above treatments were shifted to the left compared with Na+ (Na+, 17.2 μm (12.8–23.0 μm); 5-HT, 8.2 μm (6.7–10.2 μm); ibogaine (9.6 μm (7.3–12.6 μm)), Li+, 5.8 μm (4.4–7.6 μm)). b, example images taken of HEK293 cells transiently transfected with a hSERT construct tagged with ECFP at the N terminus and EYFP at the C terminus recorded at the indicated filter settings. c, calculated NFRET values. Addition of 5-HT or ibogaine to a Na+-containing bath solution as well as exchange of Na+ for Li+ significantly reduce NFRET. The latter effect is rescued by the addition of 10 μm paroxetine to the bath solution (Na+, 47.9 ± 1.0; 5-HT, 42.5 ± 1.1; ibogaine, 41.3 ± 1.5; Li+, 39.5 ± 1.2; Li+ + paroxetine, 47.2 ± 1.0). *, p 0.05, n = 40 each, Kruskal-Wallis test). e, single hSERT expressing X. laevis oocytes were voltage clamped to −40 mV using the two-electrode voltage clamp technique and continuously superfused with bath solution. Absolute current values are plotted. Addition of 10 μm 5-HT to a Na+-containing bath solution as well as the exchange of Na+ for Li+ significantly increase the current amplitude (Na+, 3.6 ± 0.7 nA; 5-HT, 20.5 ± 2.4 nA; Li+, 24.7 ± 2.4 nA. **, p 5-fold increase in current amplitude at a holding potential of −40 mV (Fig. 3 d, light gray bar). When Na+ was replaced with 100 mm Li+ (Fig. 3 d, dark gray bar), the holding current was also enhanced almost 7-fold. The Na+ leak was defined by its sensitivity to blockade by 10 μm paroxetine. Although ibogaine was effective in changing the conformation of hSERT (Fig. 3, a and c), it did not induce a current in X. laevis oocytes (supplemental Fig. 1). Kinetic Model Describing Function of hSERT Taken together, the data summarized above are consistent with the hypothesis that the uncoupled conductance of SERT requires an inward facing conformation of the transporter bound to K+. We developed a mathematical model for SERT-mediated 5-HT transport and ionic currents, which is represented in Fig. 4 a. In this model, the peak current is due to a synchronized conformational change that results from 5-HT addition to outward facing SERT molecules (ToNaCl in Fig. 4 a). The steady-state current is modeled to occur with formation of TiKCond, a conductive species transiently formed from the inward facing conformation with K+ bound (TiK, Fig. 4 a). Membrane currents were modeled as the linear sum of the coupled transport (peak) component and the uncoupled current. We tested the ability of the model to recapitulate our experimental findings. As demonstrated in Fig. 4, b and c, the model is capable of reproducing the hallmarks of the response of hSERT currents measured in HEK293 cells to 5-HT concentration. FIGURE 4. Simulated substrate-induced currents are in good agreement with the data obtained in voltage clamp experiments. a, alternating access model of hSERT. b, sample currents at different concentrations of 5-HT simulated with the kinetic model using a holding potential of −70 mV. c, concentration response relation of the coupled (open circles) and steady-state component (filled circles) of simulated hSERT currents. Solid lines depict the nonlinear least square fit to the data. The best fit values obtained were: steady state, EC50 = 105.9 nm (104.1–107.8 nm); coupled current, EC50 = 66.1 μm (47.0–92.8 μm). d, sample currents simulated at a holding potential of −100 mV (left) or 0 mV (right). e, simulated current-voltage relation of the coupled current (open circles) and steady-state component (filled circles). f, effect of internal K+ on the steady-state component of simulated hSERT currents. The simulated traces in Fig. 4 b show only a slowly activating current at lower 5-HT concentrations and a transient peak only at higher concentrations. Our model (Fig. 4 a) provides an explanation for the different concentration dependences for the steady-state and peak components. External 5-HT controls the rate of formation of ToNaSCl. At low 5-HT, this species is converted to TiNaSCl by conformational change as fast as it is formed. The slow step in the overall cycle is the reverse conformational change TiK to ToK. Thus, at low 5-HT, most of the transporter will build up in the TiK form, which is in equilibrium with the conducting species, TiKCond. At high 5-HT, ToNaSCl forms faster than it can be converted to TiNaSCl. Because the peak current results from this conformational transition, synchronized by addition of 5-HT to transporters essentially all waiting in the ToNaCl state, the peak current is maximal only at 5-HT concentrations well above 1 μm, high enough to convert all the ToNaCl to ToNaSCl before a significant amount of ToNaSCl is converted to TiNaSCl. These concentrations are much higher than required to saturate the rate of the overall transport reaction or the uncoupled current, both of which are dependent on the concentration of TiK. The analysis of the concentration response relationship yields a EC50 of 106 nm for the steady-state current and 66 μm for the peak current, affinities very similar to those measured in HEK293 cells (Fig. 4 c). Because the model predicts the peak and steady-state currents independently, the simulated 5-HT dependence of the peak current is not subject to a contribution from the uncoupled current. This analysis contrasts with Fig. 1 b where the currents measured shortly after 5-HT addition are a mixture of peak and steady state. Therefore, the right shift in EC50 between the steady-state (uncoupled) and peak (coupled) currents is more clearly visible in Fig. 4 c. The model also emulates the voltage and K+ dependence of each current. As depicted in Fig. 4, d and e, the model predicts a much stronger voltage dependence for the steady-state component relative to the peak component. This is reflected by a reduction in amplitude of almost 80% when stepping from −100 to 0 mV. In contrast, the peak component was reduced by only 35%. The predicted effect of removing internal K+ was also similar to the observation in HEK293 cells (compare Figs. 2 a and 4 f). Omission of internal K+ in the model completely suppresses the steady-state component but does not affect the peak current. Finally, we note that for simplicity we chose to model Na+ and 5-HT binding to occur in a sequential order. However, there is strong evidence that 5-HT can bind in the absence of Na+ (38). A second simplification was not to indicate H+ antiport, which is assumed to occur in the absence of K+ (4, 27) and is represented in Fig. 4 a by the Ti to To transition. Both simplifications are justified by the reason that they do not affect the ability of the model to account for the SERT currents. DISCUSSION Secondary transporters such as hSERT couple the energy stored in ion gradients to the translocation of substrate and thus drive substrate energetically uphill against a concentration gradient. The conformational rearrangements required for transport are thought to occur as originally posited by the alternating access model: substrate binding to an outward facing conformation of the transporter initiates the transition to an inward facing conformation (39, 40). Following the release of substrate, the empty transporter returns to the outward facing conformation and thereby completes the kinetic cycle. Numerous electrophysiological studies demonstrated currents through SERT (5–8), including a 5-HT-induced inward current, a substrate-independent leak current in Na+ and Li+, and a resistive transient current elicited by voltage jumps from positive (∼ +40 mV) to negative membrane potentials (∼−140 mV). Originally, Lester and co-workers speculated on the mechanistic basis of the currents by proposing a model in which the transporter occasionally adopted a channel-like conformation at conformational transition points of the cycle (5). The model envisaged that both outer and inner gates may be simultaneously open when the transporter converts between the outward and inward facing conformations (steps ToNaSCl to TiNaSCl and TiK to ToK in Fig. 4 a). Our analysis allows us to refine this view and to propose that the uncoupled current is carried by a species that is transiently formed from, and in equilibrium with, an inward facing SERT conformation with K+ bound (TiK in Fig. 4 a). This conclusion is based on the following observations: (i) Two independent measurements of conformational equilibrium, FRET between N and C termini and accessibility of a cysteine residue in the cytoplasmic permeation pathway, indicate that increased uncoupled current follows an increase in the abundance of SERT in inward facing conformations. In contrast, conditions that favor an outward facing conformation, i.e. Na+ and the presence of a competitive inhibitor such as paroxetine, support little or no current. (ii) In agreement with Adams and DeFelice (6) we also found that the steady current required internal K+. Together, these observations suggest that the conducting state requires formation of the K+-bound, inward facing conformation. (iii) A plausible kinetic model consistent with previous observations recapitulates the properties of peak and steady-state currents, including differences in their voltage and concentration dependence and kinetics. Ibogaine increased accessibility of the cytoplasmic pathway and decreased FRET between the N and C termini, but did not activate the uncoupled current. Thus, although the inward facing conformation may be required for this current, it is not itself sufficient. Ibogaine is likely to bind to the substrate site of SERT; indeed, the structure of 5-HT is contained within the ibogaine molecule (30). Thus, the ibogaine-bound SERT complex is likely to resemble TINaSCl in Fig. 4 a, which cannot bind K+ and therefore does not mediate the uncoupled current. Other explanations for the absence of ibogaine-induced currents are that ibogaine may directly block the conduction pathway or it may stabilize a different inward facing conformation of the transporter that is not in equilibrium with the conducting state. The conducting state was used as a reference point to construct a comprehensive kinetic model describing transport and current. Kinetic information was extracted from published transport rates and binding studies. We found it necessary to include 10 individual states to account for the stoichiometry of the transporter. We rigorously tested this model with our experimental data, including the kinetics of the recorded currents, their voltage dependence, and their response to 5-HT concentration. The model summarized in Fig. 4 a not only recapitulates our measurements and published findings but also provides a framework for exploring the nature of the 5-HT-induced current and of the substrate-independent leak currents (e.g. the Li+ leak and the Na+ leak). Previously, these currents were ascribed to independent activities of SERT (5) and of other transporters (14, 42). However, our analysis suggests that all currents may be carried by the same state which is in equilibrium with the inward facing, K+-bound conformation. This state may represent the conductance observed by Lester and co-workers as single-channel openings (9). Thus, our comprehensive model provides for a unifying concept of transporter-associated currents. Support for this view comes from observations that treatments that eliminate the 5-HT-induced current (removal of internal K+ (6) and co-expression of syntaxin 1a (7)) also eliminate Li+-induced currents. Furthermore, internal K+ was required for uncoupled currents initiated by both 5-HT and Li+ (see Ref. 6 and Fig. 2 a), suggesting that the transition to an inward facing state was not sufficient unless it resulted in K+ binding. Additionally, identification of the conducting species of SERT and the conformational transitions leading to stoichiometric (peak) currents allows the use of these currents to investigate the mechanism of SERT-mediated transport. Finally, our model provides an explanation for the inwardly directed current peak observed upon fast application of 5-HT onto cells expressing SERT. We surmise that the current peak is capacitive in nature and a consequence of the electrogenic transition that carries substrate and ions through the cell membrane. A similar observation was reported for DAT challenged by fast perfusion with d-amphetamine (25). However, because the stoichiometry in DAT was assumed to be electrogenic, Erreger et al. modeled the steady-state current as strictly coupled. In contrast to DAT, the established stoichiometry in SERT and a large body of experimental evidence indicate that the steady-state current is uncoupled. Structural models are available that provide snapshots of conformations that occur during the kinetic cycle of transporters: the pertinent structural model for SERT is LeuTAa, which exists in at least two different conformations (43). Our results provide limited guidance in searching for the structural equivalent of the conducting state. A recent study by Zhao and Noskov (44) suggests the presence of a water wire from the cytoplasm to the Na2 site in the occluded state of LeuTAa. Limited conformational changes could conceivably open a potential pathway like this enough to conduct cations, possibly initiated by K+ binding to the Na2 site. Our observations and others (6) link formation of the conducting state to binding of K+. However a K+ binding site does not exist in LeuTAa, and thus its location is still unknown. Additionally, there are apparent differences in the action of Li+ on LeuTAa and SERT. In LeuTAa, unlike SERT, Li+ leads to the closure of the inner gate, favoring an outward facing conformation (45). Our observations do not exclude the possibility that the conducting state can also be entered from the outward facing conformation with K+ bound. With physiological ion gradients and in the presence of external 5-HT, the inward facing K+-bound state is highly populated, whereas the K+-bound outward facing conformation is rarely occupied. Future experiments will address whether the conducting state can also be entered from the K+-bound outward facing conformation; this might be possible following an approach shown for EAAC1, where substrate gradient and all ion gradients are reversed for current measurements (46). Such a condition would populate the K+-bound outward facing conformation, and this could therefore be a suitable way to address this question. In conclusion, our approach highlights the usefulness of electrophysiological recordings to refine kinetic models. The time resolution of electrophysiology is high accordingly, currents are rich in detailed kinetic information, which is inaccessible to substrate flux measurements. Electrophysiology can be complemented with simultaneously recorded intramolecular measurements of distance changes with FRET (41). This provides structural reference points to constrain further studies. The feasibility of this strategy is currently being explored. Supplementary Material Supplemental Data
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            3,4-Methylenedioxymethamphetamine (MDMA) neurotoxicity in rats: a reappraisal of past and present findings

            Rationale 3,4-Methylenedioxymethamphetamine (MDMA) is a widely abused illicit drug. In animals, high-dose administration of MDMA produces deficits in serotonin (5-HT) neurons (e.g., depletion of forebrain 5-HT) that have been interpreted as neurotoxicity. Whether such 5-HT deficits reflect neuronal damage is a matter of ongoing debate. Objective The present paper reviews four specific issues related to the hypothesis of MDMA neurotoxicity in rats: (1) the effects of MDMA on monoamine neurons, (2) the use of “interspecies scaling” to adjust MDMA doses across species, (3) the effects of MDMA on established markers of neuronal damage, and (4) functional impairments associated with MDMA-induced 5-HT depletions. Results MDMA is a substrate for monoamine transporters, and stimulated release of 5-HT, NE, and DA mediates effects of the drug. MDMA produces neurochemical, endocrine, and behavioral actions in rats and humans at equivalent doses (e.g., 1–2 mg/kg), suggesting that there is no reason to adjust doses between these species. Typical doses of MDMA causing long-term 5-HT depletions in rats (e.g., 10–20 mg/kg) do not reliably increase markers of neurotoxic damage such as cell death, silver staining, or reactive gliosis. MDMA-induced 5-HT depletions are accompanied by a number of functional consequences including reductions in evoked 5-HT release and changes in hormone secretion. Perhaps more importantly, administration of MDMA to rats induces persistent anxiety-like behaviors in the absence of measurable 5-HT deficits. Conclusions MDMA-induced 5-HT depletions are not necessarily synonymous with neurotoxic damage. However, doses of MDMA which do not cause long-term 5-HT depletions can have protracted effects on behavior, suggesting even moderate doses of the drug may pose risks.
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              A multi-substrate single-file model for ion-coupled transporters.

              Ion-coupled transporters are simulated by a model that differs from contemporary alternating-access schemes. Beginning with concepts derived from multi-ion pores, the model assumes that substrates (both inorganic ions and small organic molecules) hop a) between the solutions and binding sites and b) between binding sites within a single-file pore. No two substrates can simultaneously occupy the same site. Rate constants for hopping can be increased both a) when substrates in two sites attract each other into a vacant site between them and b) when substrates in adjacent sites repel each other. Hopping rate constants for charged substrates are also modified by the membrane field. For a three-site model, simulated annealing yields parameters to fit steady-state measurements of flux coupling, transport-associated currents, and charge movements for the GABA transporter GAT1. The model then accounts for some GAT1 kinetic data as well. The model also yields parameters that describe the available data for the rat 5-HT transporter and for the rabbit Na(+)-glucose transporter. The simulations show that coupled fluxes and other aspects of ion transport can be explained by a model that includes local substrate-substrate interactions but no explicit global conformational changes.
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                Author and article information

                Journal
                7502536
                1895
                Br J Pharmacol
                Br. J. Pharmacol.
                British journal of pharmacology
                0007-1188
                1476-5381
                8 July 2015
                October 2015
                01 October 2015
                : 172
                : 19
                : 4775-4778
                Affiliations
                Center of Physiology and Pharmacology, Medical University Vienna, Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria
                Author notes
                []To whom correspondence should be addressed: Walter Sandtner, Medical University Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Waehringerstr. 13a, A-1090 Vienna, Austria; Tel: +43-1-40160-31328, Fax: +43-1-40160-931300, walter.sandtner@ 123456meduniwien.ac.at
                Article
                EMS64135
                10.1111/bph.12766
                4561504
                24824446
                cb91cfca-d26e-4699-8f91-f32f59fbef69

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                Pharmacology & Pharmaceutical medicine
                Pharmacology & Pharmaceutical medicine

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