Human SLC6A3 Stable Cell Line - HEK293

The ability of dopamine (DAT), norepinephrine (NET), and serotonin (SERT) transporters to concentrate substrates is generally considered to be driven by the transmembrane sodium ion (Na+) gradient, though it may also utilize other energy sources, such as membrane potential and/or the transmembrane potassium ion (K+) gradient. To investigate this, researchers used voltage-controlled recordings to monitor the uptake of endogenous substrates or the fluorescent substrate APP+ (4-(4-dimethylaminophenyl)-1-methylpyridinium) in cells expressing DAT, NET, or SERT. The results indicate that DAT and NET differ from SERT in their handling of intracellular potassium ions (K+). For DAT and NET, substrate uptake is voltage-dependent because the transient nature of intracellular K+ binding hinders K+ antiport; in contrast, SERT is capable of K+ antiport, thereby enabling voltage-independent transport. Consequently, there is a trade-off between maintaining a constant uptake rate and utilizing membrane potential to concentrate substrates; the researchers suggest that this difference stems from subtle variations in the co-transported ion binding kinetics among these closely related transporters.

Here, researchers combined the advantages of transporter-targeted radiotracer assays and electrophysiological techniques to establish an experimental system for measuring APP+ (Figure 1A) uptake in a single DAT-, NET-, or SERT-expressing HEK293 cell under voltage-controlled conditions (Figure 1B, left). The schematic in Figure 1C illustrates the transport cycle of Na+/Cl−-coupled secondary active transporters. Figure 1D displays representative dual-channel recording traces obtained from control (untransfected) HEK293 cells or from DAT-, NET-, or SERT-expressing HEK293 cells that were voltage-clamped at −60 mV and exposed to 100 µM APP+. In control HEK293 cells, the perfusion (wash-in) and washout of APP+ caused a rapid, transient rise and fall in the fluorescence signal, respectively, without accompanying changes in current. In DAT-expressing cells, APP+ induced an inward current comprising both peak and steady-state components, indicating that APP+ is a substrate for DAT. The transport of APP+ into DAT-expressing cells also resulted in a slow rise in the fluorescence signal; this increase was linear over time and ceased upon APP+ removal. The fluorescence signal subsequently returned to a new baseline level, indicating intracellular trapping of APP+. In contrast, APP+ induced only an inward peak current-without a steady-state current-in cells expressing NET or SERT. Although the absence of APP+-induced steady-state currents in SERT- and NET-expressing cells might suggest that APP+ is not transported by these carriers, the intracellular accumulation of APP+ in these cells increased linearly over time. This indicates that APP+ transport does occur, albeit with lower efficiency. The rapid rise and fall of the fluorescence signal associated with APP+ wash-in and washout, observed in control cells, were also evident in DAT-, NET-, or SERT-expressing cells.

Figure 1. Simultaneous measurements of fluorescence and currents. (Bhat S, et al., 2021)