Abstract
The physiological role of neurotransmitter transporter (NTT) proteins is the reuptake of released neurotransmitter from the synaptic cleft. NTTs accomplish uptake by undergoing a transport cycle, which relies on a return step in the empty state. In addition, NTTs can also run in the reverse direction and transport substrates out of the cells. This can be observed under conditions, where the transmembrane sodium gradient dissipates, e.g., if sodium accumulates within the cell. This reverse transport mode is also induced by amphetamines and the exact mechanism underlying the amphetamine action is still enigmatic and involves complex regulatory processes. In the current chapter, we describe various methods that can be used to assess the efflux of neurotransmitter from cells heterologously expressing the NTTs of interest or from preparations derived from intact brain tissue.
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Key words
- Carrier-mediated efflux
- Transport reversal
- Neurotransmitter transporter
- Superfusion
- Radiolabeled tracer flux
- Heterologous cell expression systems
- Synaptosomes
- Brain slices
1 Introduction
Three different possibilities exist to terminate synaptic transmission: (1) diffusion of neurotransmitter out of the synaptic cleft , (2) enzymatic degradation, or (3) reuptake by neurotransmitter transporters (NTT; [1]). The latter results in reaccumulation of neurotransmitters; the sodium gradient provides the driving force for reuptake from the synaptic cleft. Thus, it is by definition a secondary-active transport [2], which affords an economical and rapid reuse of released neurotransmitter. The monoamine transporters of the solute carrier 6 family (SLC6 ) comprise the transporters for dopamine, DAT (SLC6A3 ), norepinephrine, NET (SLC6A2 ), and serotonin, SERT (SLC6A4) [3], also abbreviated as 5-HTT [4]. The monoamine NTT family is of clinical importance since they serve as target to alleviate or effectively treat a number of psychiatric disorders including depression and attention deficit hyperactivity disorder [5].
Chemical neurotransmission was established by the pioneering experiments of Otto Loewi: electrical stimulation of a nerve released a diffusible neurotransmitter—in Loewi’s case the “Vagusstoff,” i.e., acetylcholine, and this principle was shown to be universally true in both the peripheral and the central nervous system . It took some 40 years until Axelrod and Hertting documented that neurotransmission by monoamines (and most other released neurotransmitters) was terminated by a transport process rather than enzymatic degradation. [6]. Several models were proposed to account for the ability of a protein to translocate a solute over the lipid bilayer : Jardetzky condensed these ideas into a concept, which posited an alternating access mechanism [7]. This model was vindicated by the X-ray crystal structures of many transporters, including those of the bacterial transporter LeuTAa , which revealed several conformational states consistent with the sequence of events postulated by the alternating access model [8–10]. The fact that LeuTAa is highly homologous to NTTs allows for educated guesses on the mechanistic details of the transport process [11–14] and provides a reference framework for dynamic studies at the single-molecule level [15, 16].
Importantly, reuptake from the extracellular space to the cytosol is the major but not the only possible transport direction: changes in the intracellular milieu, in particular the sodium concentration, can result in reverse transport and thus lead to NTT-mediated efflux . Reverse transport can also be observed after the administration of sympathomimetic amines such as tyramine or amphetamine-like drugs [6, 17–21]. In vivo, reverse transport can be detected by microdialysis in the awake animals [22–24] and by high-speed chronoamperometry [25]. As an alternative approach, reverse transport by psychoactive amines has also been extensively studied in brain slices or synaptosomes [26–30]. However, the interpretation of mechanistic studies is confounded by two inherent limitations: (1) both slices and synaptosomes often contain several different NTTs (e.g., all monoamine transporters are included in a striatal slice preparation ); (2) in addition, slices—and to a lesser extent synaptosomes—also contain the complete machinery for synaptic vesicle exocytosis [31, 32]. Accordingly, isolation of a single transporter requires the others to be blocked by specific NTT-blockers (to detect only effects mediated by the transporter of interest) and/or receptor inhibitors (because auto-receptors may be stimulated by the psychoactive compounds under study and regulate both vesicular and carrier-mediated release ).
The cloning of the monoamine NTT cDNA’s made it possible to heterologously express a specific monoamine transporter in appropriate cell lines such as human embryonic kidney 293 cells (HEK293) . Thereby, the problems are eliminated, which arise from interferences with vesicular storage and the stimulation of monoaminergic receptors: these paradigms have been used to study reverse transport induced by any substrates of NSS members (e.g., dopamine, tyramine , amphetamine and its derivatives or others [33–37]).
In this chapter, we outline the techniques, which have been successfully used to assess reverse transport mediated by monoamine transporters. Initially, we provide the methodologies to prepare brain slices and synaptosomes and subsequently describe the procedures to work with heterologous expression systems in both static and dynamic systems , i.e., using batch release and a superfusion system , respectively. After a description of the superfusion system, we then elaborate on the experimental procedures of the release experiment. The next part focuses on the evaluation and interpretation of the data. Finally, we will discuss troubleshooting issues and point out limitations and drawbacks of the methods.
2 Materials
2.1 Synaptosome Preparation from Animal Brain Tissue
Krebs-HEPES buffer or Krebs–Henseleit buffer :
Krebs-HEPES buffer (KHB): 25 mM HEPES , 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, and 1.2 mM MgSO4 supplemented with 5 mM d-glucose, adjusted with NaOH to pH = 7.4.
Krebs–Henseleit buffer (KHensB) : 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.25 mM CaCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, 11 mM glucose.
Prepare KHensB freshly every day and oxygenate with 95 % O2/5 % CO2 for 1 h before use to adjust pH to 7.4.
Animal brain removed from either mouse or rat.
Tissue douncer with appropriately sized teflon-coated pestle for the preparation of synaptosomes.
Phosphate-buffered saline (PBS) containing protease inhibitors (Roche Complete™).
Protein determination kit (e.g., BCA kit , Pierce/Thermo Scientific ).
24-well plates.
Whatman GF/B filters (1 mm diameter).
2.2 Slice Preparation from Animal Brain Tissue
Krebs-HEPES buffer or KHensB buffer (see above for composition).
Animal brain removed from either mouse or rat.
McIlwain tissue chopper (Fig. 1) for the preparation of brain slices.
2.3 HEK293 Cell Culture and Reagents
DMEM medium (any source is good provided that they adhere to the original recipe, e.g., Gibco, Invitrogen Life Science, Bethesda, MD).
Fetal bovine serum (FBS) .
0.05 % Trypsin/EDTA (Sigma-Aldrich, St. Louis, MO).
l-Glutamine (Gibco, Invitrogen Life Science, Bethesda, MD).
10 cm or 15 cm dishes; 24-well or 96-well culture plates (Greiner, Sarstedt, BD Biosciences, Falcon).
Penicillin (10,000 U/mL) and streptomycin (10 mg/mL) solutions are frozen at −20 °C (Gibco, Invitrogen Life Science); 5 mL is added to 0.5 L of DMEM complete culture medium.
Cell line: HEK293 cells transiently or stably expressing NTT of interest.
0.1 mg/mL poly-d-lysine solution in sterile H2O.
Round coverslip (5 mm diameter).
2.4 Equipment, Software, and Accessories
Cell culture incubator , 37 °C, 5 % CO2.
Sterile hood for the handling of the cell lines.
Vacuum pump for cell washes.
A personal computer with appropriate data calculation and graphics software.
For the identification of the brain structures, use pertinent brain atlases (e.g., Franklin and Paxinos for mouse brain or the rat brain atlas by König and Klippel). Note: for the preparation of synaptosomes and brain slices, brains need preferentially to be dissected freshly and on a cold plate (not frozen).
3 Methods
In the methods section, we will describe in the first paragraphs how the three preparations covered in this chapter are produced (Sects. 3.1–3.3) before we describe the superfusion assay (Sect. 3.4) and the evaluation of the data (Sect. 3.5).
3.1 Preparation of Rodent Brain Synaptosomes
Kill mouse or rat by cervical dislocation or decapitation .
Remove the brain carefully (Note: make sure that the meninges are removed thoroughly otherwise you risk serious damage to the delicate brain tissue during dissection) and keep on ice.
Dissect both left and right region(s) of interest.
Homogenize in ice-cold 0.32 M sucrose in phosphate-buffered saline (PBS) containing protease inhibitors (Roche Complete™).
Centrifuge the suspension for 10 min at 1000 × g. Keep the supernatant , discard the pellet.
Centrifuge the supernatant for 15 min at 12,600 × g. Discard the supernatant and resuspend the pellet (referred to as P2) in KHB.
Measure the wet weight of P2 or determine the protein concentration of P2 after resuspension in buffer to estimate the amount of synaptosomes.
Use the resulting synaptosomes directly or freeze at −80 °C. Synaptosomes can be stored at −80 °C for at least 2 years with only minor loss in functional activity .
3.2 Preparation of Mouse Brain Slices
Retrieve a rodent brain as described above and place it into ice-cold buffer (KHB or KHensB ).
Dissect the regions of interest (e.g., striatum , hippocampus , or cortical tissue) and store in ice-cold buffer in a watch glass on ice.
Take dissected brain region and place onto a Whatman filter paper saturated with ice-cold buffer; place filter with brain onto the cutting stage of the McIlwain tissue chopper and operate the knife slowly to cut the first tissue slice (usually, 0.3 mm thickness is recommended). Discard the first section and start collecting subsequent sections using a fine brush by gently swiping them and placing them in ice-cold buffer for storage (Fig. 2).
Repeat until all slices of your region of interest are retrieved.
Handle the slices cautiously with the brush avoiding damage to the tissue.
Tissue slices can be kept in ice-cold buffer for a couple of hours but tissue quality and integrity decrease with time.
3.3 Heterologously Expressing Cell Lines
The expression of the NTT of interest in appropriate cell lines can be bothersome from time to time and expression levels may vary significantly. This may cause differences in results for NTT-mediated efflux from lab to lab but even among different cell lines within one lab. The reason for the differences might simply be that the properties of the cell lines differ considerably with regard to the expression of kinases and other important regulatory factors , e.g., protein kinase C , [38], or αCamKII, [39–43]. Hence, it would be good to start the approach by screening the literature and selecting the right cell line based on the experience of other groups. For instance, DAT has been expressed in a number of different cell lines, including HEK293 cells [32], PC-12 cells, [44], SK-NMC cells [45] and LLC-PKC1 cells [46, 47]. All cell lines have their unique advantages: HEK293 cells for instance can be most easily transfected and afford high expression levels. In contrast, LLC-PKC1 cells support much lower expression levels but these cells are endowed with a large complement of protein kinases . Clearly, it depends on the goal, which is being pursued: the high expression in HEK293 cells is best suited to study transporter-ligand interactions while the low expression profile of LLC-PKC1 cells can be utilized to assess the influence of kinases on transporter regulation [47].
For transfection of HEK293 cells we usually resort to the CaPO4 transfection method since it is cheap, easy and reliable; only if this method does not lead to the desired cell expression result, we use a lipofection method. In the following, we outline how we perform CaPO4 transfections, followed by one lipofection method which is given as an example for a large variety of different commercially available products. Still, the number of products is increasing and it provides a simple and efficient way of transfection. However, lipofection interferes with the lipidome of the cells and may therefore interfere with lipid-transporter interactions [48, 49]. At the very least, these aspects must be considered, when selecting the transfection method.
Ca 2+ Transfections
HeBS buffer (2×): | |
HEPES | 50 mM |
NaCl | 280 mM |
Na2HPO4·2H2O | 1.5 mM |
Adjust the pH to 7.08 with 10 N NaOH; accurate pH is critical for efficient transfection. Sterilize by filtration through a 0.45-μm nitrocellulose filter . Store at −20 °C. Thaw only once!
Phosphate buffered saline (PBS): | |
KCl | 2.7 mM |
KH2PO4 | 1.5 mM |
NaCl | 137 mM |
Na2HPO4·2H2O | 4.3 mM |
Dissolve in sterile water (Milli-Q), adjust to pH 7.3–7.4 (by adding 10 N NaOH).
Seed cells to achieve approximately 40 % confluence in a 10 cm dish on the day of transfection . Mix reagents as follows (CaCl2 + H2O = 1:8.6):
H2O | 430 μL | |
CaCl2 | (2.3 M) | 50 μL |
DNA (1 μg/μL) | 20 μL | |
HeBS (2×) | 500 μL |
Incubate the mixture for 6 min at room temperature to allow the reaction to take place: here, DNA-Ca2+ should form a fine precipitate. The precipitate should not be too coarse. Drop the suspension onto the medium covering the cells.
Apply the “glycerol shock ” after 4–6 h of incubation time at 37 °C to increase transfection efficiency: remove the transfection media, add 1 mL of glycerol shock solution (thoroughly mix 13.8 mL glycerol and 86.2 mL PBS , sterile filtered afterwards) and remove it immediately after and rapidly by aspiration.
Wash the cells with 10 mL PBS and supply 10 mL of fresh, warm media.
Wait for at least 24 h before performing experiments on transiently expressing cells. To establish a stably expressing cell line, start the selection process with the appropriate antibiotic (e.g., geneticin ) 48–72 h after the glycerol shock.
Turbofection
Seed cells into 10 cm dishes and use them for turbofection when they are approximately 80–90 % confluent.
Combine 1 μg of DNA of interest with “empty” vectors (e.g., pcDNA3.1) to reach a total amount of 5 μg of DNA (Note: dilution of transporter DNA is usually recommended as lipofection can induce massive overexpression of the protein of interest; in order to achieve more “physiological” expression, dilution series of transporter DNA should be performed and tested).
Add 500 μL of DMEM to the DNA mix followed by the addition of 5 μL Turbofect (Fermentas; vortex Turbofect well before adding).
Vortex the mixture and incubate for 15–20 min at room temperature.
In the meantime, remove the media from the cells and replace with 4.5 mL of fresh media including FCS and antibiotics.
After the incubation of Turbofect–DNA mixture is ready, add the solution to the cells. Incubate the cells for another 24–48 h at 37 °C.
It is advisable to first verify that the monoamine NTT of interest is expressed in the given cell line at adequate levels prior to carrying out a release assay . Expression can be confirmed by performing an uptake assay or by labeling the NTT of interest with a fluorescent protein (e.g., GFP or mCherry ). Note: the addition of a fluorescent protein can compromise NTT function ; for instance, only SERT tolerates the addition of a fluorescent protein to the carboxyl terminus [38], whereas in all other monoamine NTT surface expression is substantially reduced because their C-terminal PDZ-binding motif cannot be masked [50, 51].
Intuitively, stable transfection is more appealing, but it may not be necessary to have a cell line with a homogeneous expression level. In addition, it may not be possible to express a transporter of interest in a stable manner. This is for instance true for mutants, which generate a large leak current . A point in case is the mutant DAT-Y335A [52]; this mutation converts the transporter into a ion-channel-like pore [53]. Here, stable expression has not been possible, because the large leak conductance of DAT-Y335A apparently precluded long term cell survival (unpublished results).
3.4 Release Assays
While the preparations (synaptosomes , brain slices or cells on coverslips or in plates) differ, the superfusion is done in a very similar manner; the variations are modest. The static batch release assay will be described in Sect. 3.4.2.
3.4.1 Superfusion Assay
We will shortly describe the superfusion apparatus and how to set it up before each experiment before the actual experiments involving the three different preparations will be explained.
The system is designed to allow for rapid removal of the released neurotransmitter by continuous superfusion of the preparation of interest. The goal is to preclude reuptake of the released neurotransmitter (regardless of whether released spontaneously or after a stimulus) by the cognate NTT or other transporters. An initial superfusion is employed to define the baseline , i.e., to estimate the spontaneous release or leak in the respective preparation.
Our superfusion apparatus was designed to comprise 12 individual channels (Fig. 3). Hence, 12 brain slices, 12 synaptosomal fractions or 12 cell coverslips can be used per assay in parallel. This allows for an adequate number of replicates; we rely on triplicate determinations for each experimental condition. A schematic representation of a superfusion chamber is given in Fig. 4b. Of importance to control the temperature in the chambers, the tubing ought to be immersed in a water bath, which should be kept at a temperature higher than the desired value in the chamber to yield a temperature of 25 °C in the chambers proper.
The preparations are continuously superfused with buffer at a flow rate of 0.7 mL/min. It is essential that no air bubbles are trapped in the superfusion tubings . Air bubbles can be easily removed by rinsing the superfusion system with 30 % isopropanol in ddH2O before the experiment. After the isopropanol, the system is washed extensively with ddH2O (10 min) to remove any residual isopropanol before equilibrating the system buffer (10 min).
The superfusate must obviously have a constant temperature to eliminate a source of variability. The control of the temperature is achieved by immersing the tubing (total diameter = 1 mm, a luminal diameter = 0.35 mm) over a length of 60 cm in a water bath set at the pertinent temperature (usually 25 °C). Note that the flow rate of the buffer results in mechanical stress for the tested preparations. The flow-related shear forces are limited by the size of the chambers, which harbor the preparations: their volume is 200 μL with a diameter of 8 mm.
For superfusion of synaptosomes , synaptosomal P2 pellets are resuspended in the respective buffer to achieve a concentration of 1 mg wet weight/15 μL or 100 μg of protein.
For superfusion of brain slices, individual sections are used per channel.
Synaptosomes are incubated with a [3H]-labeled substrate of interest: for instance when DAT is the NTT in focus, [3H]MPP+ or [3H]dopamine can be used to load the tissue. When SERT is the NTT of interest, [3H]5-HT should be used.
CAVEAT: biogenic amines are substrates for monoamine oxidases and prone to spontaneous oxidation. Hence, they can get rapidly metabolized in native tissue such as synaptosomes or tend to spontaneously oxidize. It is therefore advisable to block MAOs throughout the assay by adding for instance 100 nM pargyline (a MAO blocker) and to prevent spontaneous oxidation by including 100 nM ascorbate (as antioxidant) in the buffer .
CAVEAT: radiolabeled substrates may be taken up by multiple transporters, e.g., [3H]dopamine is not only a substrate for DAT but also for NET and SERT. Hence, when DAT is the transporter of interest, NET and SERT should be blocked. We therefore add 100 nM desipramine (NET blocker) and 100 nM paroxetine to the buffer. In contrast, when SERT is the transporter of interest, we add 100 nM nomifensine to the buffer, which blocks both DAT and NET at similar potencies . If NET is to be investigated, the addition of a 100 nM GBR12909 or GBR12935 (specific DAT inhibitors) and 100 nM paroxetine is recommended.
Preincubation time for synaptosomes and slices :
Preincubate synaptosomes and slices for 30 min at 37 °C with [3H]-labeled substrate (e.g., 100 nM [3H]dopamine or 100 nM [3H]5-HT) in working buffer ( + 100 nM pargyline + 100 nM ascorbate + respective blockers for other NTT , e.g., 100 nM desipramine (to block NET) and 100 nM paroxetine (to block SERT) or 100 nM nomifensine to block DAT and NET, respectively).
Preincubation time for cells:
Preincubate cells for 20 min at 37 °C with [3H]-labeled substrate in buffer (depending on the cell line using, addition of pargyline to block MAO or ascorbate as an antioxidant is recommended. Also, if neuronal-like cell lines are used, it might be advisable to add the respective inhibitors for NTT that are not the primary focus of investigation, e.g., both SHY5Y cells and PC12 cells endogenously express NET)
Insert preparations into superfusion apparatus and perform washout :
After preincubation , preparations are transferred to the superfusion apparatus.
To insert preparations, stop perfusion flow and open all the channels.
Synaptosomes : prepare a plastic dish with GF/B filters (8 mm diameter) and pipet 15 μL of preincubation solution onto Whatman GF/B filters . Immediately put one loaded filter into one channel, close the channels and perfuse with buffer .
Slices: insert one slice per channel .
Cells: insert one coverslip per channel.
After the preparations have been placed into the chambers, a washout phase is started to equilibrate the system and to establish a stable baseline efflux of radioactivity. Usually a washout time of 45 min is sufficient (keep in mind that MAO blocker, ascorbate and inhibitors of the other NTTs should also be present in the buffer during this washout phase).
During washout, prepare the required amount of scintillation vials in a rack and fill with 2 mL scintillation cocktail (we usually use 50 mL scintillation vials filled with 2 mL of scintillation liquid).
Also during the washout period , prepare the working solutions needed for the efflux experiment, i.e., prepare the releaser solution :
For example “releaser” solution (for DAT, we usually use 3–10 μM of d-amphetamine , for SERT 3–10 μM of para-chloroamphetamine [pCA] in working buffer since this allows for maximal substrate release at monoamine transporters ; keep in mind that amphetamines usually have a bell-shaped dose-response curve , i.e., further increases in d-amphetamine or pCA concentrations do not increase efflux but rather have inhibitory effects on release; see Seidel et al. [38] for details).
Note: depending on the transporter under investigation, one should choose the respective amphetamine: amphetamines usually are promiscuous molecules but they still show preferences for the one or other transporter. For instance, d-amphetamine has higher affinities for DAT and NET , whereas pCA shows an increased affinity for SERT. Hence, it is advisable to select the most potent amphetamine for a given transporter under investigation, because this minimizes off-target effects.
“Releaser + inhibitor” (control) : another way to test, whether release through the NTT of interest is specific it is recommended to include a condition where release is blocked by the pertinent inhibitor for this transporter, e.g., 3–10 μM d-amphetamine + 1 μM GBR12909 for DAT.
After washout , the actual experiment is started by collecting at least 3 × 2-min fractions where the preparation is superfused with buffer only. After the first three fractions, the tubings can be switched to the releaser solution . When also looking at the effect of an inhibitor, we usually collect three 2-min with inhibitor only (e.g., 1 μM GBR12909) or buffer only before collecting five 2-min fractions with releaser and releaser + inhibitor, respectively.
At the end of the experiment, synaptosomes or slices are collected in tubes filled with 2 mL of 1 % SDS to recover the remaining amount of [3H] substrate present in the tissue.
Superfusion experiments with cells grown on coverslips are terminated by collecting three 2-min fractions where the superfusion system is washed with 1 % SDS to lyse the cells.
At the end of the experiment, put the lid onto the scintillation vials and shake tubes well before measuring in the liquid scintillation counter.
3.5 Data Analysis
Calculations are based as fractional release : hence the first step is to calculate the radioactivity initially present in the preparation, which is sum of the radioactivity present in all collected 2-min fractions and radioactivity present at the end, i.e., the radioactivity released after solubilization of the tissue with 1 % SDS. The released radioactive [3H]substrate in any given 2-min fraction is expressed as percentage of the radioactivity present in the slice at the start of this very collection period , i.e., fractional release = cpm2-min fraction/(total radioactivity − sum of previously release) × 100.
Usually the baseline efflux of radioactivity is stable and amounts to about 1–1.5 % fractional release per 2-min buffer superfusion. Efflux rises to 5–10 % after the addition of amphetamine (Fig. 5).
3.5.1 Static Batch Release Assay
The problem of the batch release assay format is its static nature: Here, the diffusion of substrate and of the compounds under investigation cannot be controlled like in the superfusion apparatus . Superfusion provides a robust assay format to assess transporter-mediated efflux, because confounding effects arising form back diffusion are eliminated [54]. The disadvantage of superfusion, however, is the large volume of superfusate and hence the need of a larger amount of the compound under study. Therefore, static batch release is the preferred method when the amount of compound under scrutiny is small [55], but its limitations must be kept in mind. In the following, we will outline how the static release assay has worked out well in our hands.
Cells are grown in poly-d-lysine-coated 96-well plates (4 × 104 cells per well). The cells are preloaded with 0.05 μM [3H]substrate for 20 min at 37 °C in a final volume of 0.1 mL/well. The residual extracellular radioactivity is removed by three gentle wash steps with Krebs-Ringer-HEPES buffer . The cells are then incubated with the test compounds at room temperature and compared to a reference compound (for instance, d-amphetamine can be used for all three monoamine transporters ). All compounds are used at the concentration, which results in a 50 % inhibition of substrate uptake. The specificity of drug-induced release needs to be assessed by the addition of a specific inhibitor at appropriate concentration (for instance, 10 μM of mazindole for DAT and NET or 10 μM paroxetine for SERT) to the test compound. After 10 min, the incubation buffer is removed and transferred into a counting vial; the cells remaining in the well are overlaid with a solution containing 1 % SDS to extract the radioactivity. The resulting solution is transferred into a counting vial. All samples are subjected to standard liquid scintillation counting. It is preferable to perform all determinations in triplicates. The sum of the radioactivity in the incubation buffer and the cell lysate represents the total [3H]substrate included in the assay. This sum is the reference value, to which the released radioactivity is related: the data are expressed as released [3H]substrate as percent of total available radioactivity .
4 Notes
The application of the above mentioned superfusion apparatus provides a robust technique to measure NTT-mediated reverse transport . However, various circumstances must be taken into consideration, which might result in flawed data interpretations.
-
1.
Liquid scintillation counting is our method of choice to determine the amount of tritium within the superfusates . Liquid scintillation cocktails (e.g., Rotiscint®) convert the energy of the beta-decay of tritium into light signals. Some drugs may absorb photons emitted by scintillators and quench the cognate signal. Therefore, one has to ensure that the applied substances do not influence the readout at the tested concentrations. For example, a substance-related quench would seemingly reduce the amount of released tritiated substrate and could be misinterpreted as transporter-related effect.
-
2.
Uptake inhibition assays do not provide the possibility to differentiate between non-transported competitive NTT inhibitors or amphetamine-like releasers [56]. In general, amphetamines are accepted as NTT substrates and compete with the endogenous substrate for the binding site . As a result, amphetamines dose-dependently inhibit uptake of tritiated substrates. The use of the superfusion technique allows bypassing this limitation of inwardly directed radiotracer flux assays. The presence of amphetamines enhances the efflux of tritiated substrates whereas non-transported inhibitors, e.g., cocaine , do not result in NTT-mediated reverse transport [57]. In addition, NTTs utilize the preexisting sodium gradient as driving force for their concentrative transport. Disrupting the sodium gradient by addition of the Na+/H+ ionophore monensin [58] will selectively enhance efflux triggered by true substrates/amphetamines [56, 59]. However, the use of monensin alters the intracellular pH, as monensin serves as a leak for intracellular protons. Serotonin (5-HT) is weak base with a pKa value of 10.4 [60]. A reduction in intracellular H+ inevitably increases the deprotonated fraction of 5-HT, which can passively diffuse across the plasma membrane. This results in a transporter-independent “pseudo efflux ” [59]. It is worth mentioning that inhibition of NTTs by various inhibitors can unmask a basal loss of substrates by diffusion. Biogenic amines do have a limited—albeit measurable—potential to cross the plasma membrane by passive diffusion [61]. Therefore, if results obtained from superfusion experiments with [3H]-5HT or [3H]-DA are ambiguous, [3H]-MPP+ should be used as NTT substrate. MPP+ carries a permanent charge and passive diffusion is thus negligible [54]. The cells or the synaptosomal preparation under investigation may also express additional gradient-driven transporters : Members of the SLC22 family, epitomized by organic cation transporters 1–3 (OCTs, SLC22A1–3) or the plasmalemmal monoamine transporter (PMAT , SLC29A4), translocate DA, NE , 5-HT, and MPP+ across cellular membranes [62, 63]. Their coexistence with SLC6 family members may confound the interpretation of outwardly directed transport of tritiated biogenic amines or MPP+. OCTs and PMAT are very broadly expressed; they can be found in both peripheral tissue and in the central nervous system , where they are expressed in neuronal and non-neuronal cell types [63–67]. As a consequence, gradient-driven transporters may contribute to efflux measured during superfusion experiments. OCTs and PMAT are not blocked by most NTT-inhibitors. Therefore, in the presence of OCTs and/or PMAT, inhibition of NTTs can enhance a basal loss of substrate, because the compensatory re-uptake by NTTs is eliminated by the NTT-inhibitor. This effect is pronounced in systems containing vesicular storage pools and/or upon application of amphetamine-like releasers. Efflux triggered by substances targeting NTTs, in particular their blockers, should be interpreted cautiously, if there is evidence for the presence of other gradient-driven transporters. As a safeguard, we recommend the use of the OCT and PMAT blocker decynium-22 (D22) . D22 exhibits rather low affinities for NTTs. Therefore, as control experiment, the experimenter might test, if the efflux in presence of NTT blockers is sensitive to increasing concentrations of D22.
References
Iversen LL (1971) Role of transmitter uptake mechanisms in synaptic neurotransmission. Br J Pharmacol 41:571–591
Rudnick G, Clark J (1993) From synapse to vesicle: the reuptake and storage of biogenic amine neurotransmitters. Biochim Biophys Acta 1144:249–263
Kristensen AS, Andersen J, Jorgensen TN et al (2011) SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol Rev 63:585–640
Nelson N (1998) The family of Na+/Cl− neurotransmitter transporters. J Neurochem 71:1785–1803
Iversen L (2000) Neurotransmitter transporters: fruitful targets for CNS drug discovery. Mol Psychiatry 5:357–362
Axelrod J, Whitby LG, Hertting G (1961) Effect of psychotropic drugs on the uptake of 3 H-Norepinephrine by tissues. Science 133:383–384
Jardetzky O (1966) Simple allosteric model for membrane pumps. Nature 211:969–970
Singh SK, Piscitelli CL, Yamashita A et al (2008) A competitive inhibitor traps LeuT in an open-to-out conformation. Science 322:1655–1661
Singh SK, Yamashita A, Gouaux E (2007) Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 448:952–956
Yamashita A, Singh SK, Kawate T et al (2005) Crystal structure of a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters. Nature 437:215–223
Forrest LR, Rudnick G (2009) The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters. Physiology (Bethesda) 24:377–386
Forrest LR, Zhang YW, Jacobs MT et al (2008) Mechanism for alternating access in neurotransmitter transporters. Proc Natl Acad Sci U S A 105:10338–10343
Penmatsa A, Gouaux E (2014) How LeuT shapes our understanding of the mechanisms of sodium-coupled neurotransmitter transporters. J Physiol 592:863–869
Shi L, Quick M, Zhao Y et al (2008) The mechanism of a neurotransmitter:sodium symporter—inward release of Na+ and substrate is triggered by substrate in a second binding site. Mol Cell 30:667–677
Zhao Y, Terry D, Shi L et al (2010) Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature 465:188–193
Zhao Y, Terry DS, Shi L et al (2011) Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature 474:109–113
Barger G, Dale HH (1910) Chemical structure and sympathomimetic action of amines. J Physiol 41:19–59
Tainter ML, Chang DK (1927) The antagonism of sympathetic and adrenaline content of the spleen, kidney, and salivary glands in the sheep. J Pharmacol Exp Ther 30:193–207
Furchgott RF, Kirpekar SM, Rieker M et al (1963) Actions and interactions of norepinephrine, tyramine and cocaine on aortic strips of rabbit and left atria of guinea pig and cat. J Pharmacol Exp Ther 142:39–58
Ross SB, Kelder D (1977) Efflux of 5-hydroxytryptamine from synaptosomes of rat cerebral cortex. Acta Physiol Scand 99:27–36
Glowinski J, Axelrod J (1965) Effect of drugs on the uptake, release, and metabolism of H3-norepinephrine in the rat brain. J Pharmacol Exp Ther 149:43–49
Agneter E, Sitte HH, Stockl-Hiesleitner S et al (1995) Sustained dopamine release induced by secretoneurin in the striatum of the rat: a microdialysis study. J Neurochem 65:622–625
Gainetdinov RR, Fumagalli F, Jones SR et al (1997) Dopamine transporter is required for in vivo MPTP neurotoxicity: evidence from mice lacking the transporter. J Neurochem 69:1322–1325
Gainetdinov RR, Jones SR, Fumagalli F et al (1998) Re-evaluation of the role of the dopamine transporter in dopamine system homeostasis. Brain Res Brain Res Rev 26:148–153
Daws LC, Toney GM, Davis DJ et al (1997) In vivo chronoamperometric measurements of the clearance of exogenously applied serotonin in the rat dentate gyrus. J Neurosci Methods 78:139–150
Gobbi M, Frittoli E, Mennini T et al (1992) Releasing activities of d-fenfluramine and fluoxetine on rat hippocampal synaptosomes preloaded with [3H]serotonin. Naunyn Schmiedebergs Arch Pharmacol 345:1–6
Gobbi M, Funicello M, Gerstbrein K et al (2008) N,N-Dimethyl-thioamphetamine and methyl-thioamphetamine, two non-neurotoxic substrates of 5-HT transporters, have scant in vitro efficacy for the induction of transporter-mediated 5-HT release and currents. J Neurochem 105:1770–1780
Gobbi M, Mennini T, Garattini S (1997) Mechanism of neurotransmitter release induced by amphetamine derivatives: pharmacological and toxicological aspects. Curr Top Pharmacol 3:217–227
Rothman RB, Baumann MH (2002) Serotonin releasing agents. Neurochemical, therapeutic and adverse effects. Pharmacol Biochem Behav 71:825–836
Rothman RB, Baumann MH (2003) Monoamine transporters and psychostimulant drugs. Eur J Pharmacol 479:23–40
Whittaker VP, Michaelson IA, Kirkland RJ (1964) The separation of synaptic vesicles from nerve-ending particles (‘synaptosomes’). Biochem J 90:293–303
Scholze P, Norregaard L, Singer E et al (2002) The role of zinc ions in reverse transport mediated by monoamine transporters. J Biol Chem 277:21505–21513
Eshleman AJ, Henningsen RA, Neve KA et al (1994) Release of dopamine via the human transporter. Mol Pharmacol 45:312–316
Wall SC, Gu H, Rudnick G (1995) Biogenic amine flux mediated by cloned transporters stably expressed in cultured cell lines: amphetamine specificity for inhibition and efflux. Mol Pharmacol 47:544–550
Pifl C, Agneter E, Drobny H et al (1999) Amphetamine reverses or blocks the operation of the human noradrenaline transporter depending on its concentration: superfusion studies on transfected cells. Neuropharmacology 38:157–165
Pifl C, Drobny H, Reither H et al (1995) Mechanism of the dopamine-releasing actions of amphetamine and cocaine: plasmalemmal dopamine transporter versus vesicular monoamine transporter. Mol Pharmacol 47:368–373
Pifl C, Singer EA (1999) Ion dependence of carrier-mediated release in dopamine or norepinephrine transporter-transfected cells questions the hypothesis of facilitated exchange diffusion. Mol Pharmacol 56:1047–1054
Seidel S, Singer E, Just H et al (2005) Amphetamines take two to tango: an oligomer-based counter-transport model of neurotransmitter transport explores the amphetamine action. Mol Pharmacol 67:140–151
Fog JU, Khoshbouei H, Holy M et al (2006) Calmodulin kinase ii interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport. Neuron 51:417–429
Steinkellner T, Montgomery TR, Hofmaier T et al (2015) Amphetamine action at the cocaine- and antidepressant-sensitive serotonin transporter is modulated by alphaCaMKII. J Neurosci 35:8258–8271
Steinkellner T, Mus L, Eisenrauch B et al (2014) In vivo amphetamine action is contingent on alphaCaMKII. Neuropsychopharmacology 39:2681–2693
Steinkellner T, Yang JW, Montgomery TR et al (2012) Ca(2+)/calmodulin-dependent protein kinase IIalpha (alphaCaMKII) controls the activity of the dopamine transporter: implications for Angelman syndrome. J Biol Chem 287:29627–29635
Rickhag M, Owens WA, Winkler M-T et al (2013) Membrane-permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release. J Biol Chem 288:27534–27544
Melikian HE, Buckley KM (1999) Membrane trafficking regulates the activity of the human dopamine transporter. J Neurosci 19:7699–7710
Pifl C, Wolf A, Rebernik P et al (2009) Zinc regulates the dopamine transporter in a membrane potential and chloride dependent manner. Neuropharmacology 56:531–540
Foster JD, Yang J-W, Moritz AE et al (2012) Dopamine transporter phosphorylation site threonine 53 regulates substrate reuptake and amphetamine-stimulated efflux. J Biol Chem 287:29702–29712
Moritz AE, Foster JD, Gorentla BK et al (2013) Phosphorylation of dopamine transporter serine 7 modulates cocaine analog binding. J Biol Chem 288:20–32
Buchmayer F, Schicker K, Steinkellner T et al (2013) Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate. Proc Natl Acad Sci U S A 110:11642–11647
Hamilton PJ, Belovich AN, Khelashvili G et al (2014) PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein. Nat Chem Biol 10:582–589
Scholze P, Freissmuth M, Sitte H (2002) Mutations within an intramembrane leucine heptad repeat disrupt oligomer formation of the rat GABA transporter 1. J Biol Chem 277:43682–43690
Chiu CS, Jensen K, Sokolova I et al (2002) Number, density, and surface/cytoplasmic distribution of GABA transporters at presynaptic structures of knock-in mice carrying GABA transporter subtype 1-green fluorescent protein fusions. J Neurosci 22:10251–10266
Loland CJ, Norregaard L, Litman T et al (2002) Generation of an activating Zn(2+) switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle. Proc Natl Acad Sci U S A 99:1683–1688
Meinild A, Sitte H, Gether U (2004) Zinc potentiates an uncoupled anion conductance associated with the dopamine transporter. J Biol Chem 279:49671–49679
Scholze P, Sitte H, Singer E (2001) Substantial loss of substrate by diffusion during uptake in HEK-293 cells expressing neurotransmitter transporters. Neurosci Lett 309:173–176
Rosenauer R, Luf A, Holy M et al (2013) A combined approach using transporter-flux assays and mass spectrometry to examine psychostimulant street drugs of unknown content. ACS Chem Neurosci 4:182–190
Baumann MH, Partilla JS, Lehner KR et al (2013) Powerful cocaine-like actions of 3,4-methylenedioxypyrovalerone (MDPV), a principal constituent of psychoactive ‘bath salts’ products. Neuropsychopharmacology 38:552–562
Sitte HH, Freissmuth M (2010) The reverse operation of Na(+)/Cl(−)-coupled neurotransmitter transporters—why amphetamines take two to tango. J Neurochem 112:340–355
Mollenhauer HH, Morre DJ, Rowe LD (1990) Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity. Biochim Biophys Acta 1031:225–246
Sitte HH, Scholze P, Schloss P et al (2000) Characterization of carrier-mediated efflux in human embryonic kidney 293 cells stably expressing the rat serotonin transporter: a superfusion study. J Neurochem 74:1317–1324
Chattopadhyay A, Rukmini R, Mukherjee S (1996) Photophysics of a neurotransmitter: ionization and spectroscopic properties of serotonin. Biophys J 71:1952–1960
Scholze P, Zwach J, Kattinger A et al (2000) Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter. J Pharmacol Exp Ther 293:870–878
Koepsell H, Lips K, Volk C (2007) Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications. Pharm Res 24:1227–1251
Courousse T, Gautron S (2015) Role of organic cation transporters (OCTs) in the brain. Pharmacol Ther 146:94–103
Cui M, Aras R, Christian WV et al (2009) The organic cation transporter-3 is a pivotal modulator of neurodegeneration in the nigrostriatal dopaminergic pathway. Proc Natl Acad Sci U S A 106:8043–8048
Iversen LL (1997) The uptake of catechol amines at high perfusion concentrations in the rat isolated heart: a novel catechol amine uptake process. 1964. Br J Pharmacol 120:267–282, discussion 264–266
Vialou V, Balasse L, Callebert J et al (2008) Altered aminergic neurotransmission in the brain of organic cation transporter 3-deficient mice. J Neurochem 106:1471–1482
Kristufek D, Rudorfer W, Pifl C et al (2002) Organic cation transporter mRNA and function in the rat superior cervical ganglion. J Physiol 543:117–134
Singer EA (1988) Transmitter release from brain slices elicited by single pulses: a powerful method to study presynaptic mechanisms. Trends Pharmacol Sci 9:274–276
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The authors wish to thank the Austrian Science Fund for continuous support (grant F35).
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Steinkellner, T. et al. (2016). Tracer Flux Measurements to Study Outward Transport by Monoamine Neurotransmitter Transporters. In: Bönisch, H., Sitte, H. (eds) Neurotransmitter Transporters. Neuromethods, vol 118. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3765-3_2
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