An N-terminal threonine mutation produces an efflux-favorable, sodium-primed conformation of the human dopamine transporter

Abstract

The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr62 revealed T62D-human (h)DAT partitions in a predominately efflux-preferring conformation. Compared with wild-type (WT), T62D-hDAT exhibits reduced [(3)H]DA uptake and enhanced baseline DA efflux, whereas T62A-hDAT and WT-hDAT function in an influx-preferring conformation. We now interrogate the basis of the mutants' altered function with respect to membrane conductance and Na(+) sensitivity. The hDAT constructs were expressed in Xenopus oocytes to investigate if heightened membrane potential would explain the efflux characteristics of T62D-hDAT. In the absence of substrate, all constructs displayed identical resting membrane potentials. Substrate-induced inward currents were present in oocytes expressing WT- and T62A-hDAT but not T62D-hDAT, suggesting equal bidirectional ion flow through T62D-hDAT. Utilization of the fluorescent DAT substrate ASP(+) [4-(4-(dimethylamino)styryl)-N-methylpyridinium] revealed that T62D-hDAT accumulates substrate in human embryonic kidney (HEK)-293 cells when the substrate is not subject to efflux. Extracellular sodium (Na(+) e) replacement was used to evaluate sodium gradient requirements for DAT transport functions. The EC50 for Na(+) e stimulation of [(3)H]DA uptake was identical in all constructs expressed in HEK-293 cells. As expected, decreasing [Na(+)]e stimulated [(3)H]DA efflux in WT- and T62A-hDAT cells. Conversely, the elevated [(3)H]DA efflux in T62D-hDAT cells was independent of Na(+) e and commensurate with [(3)H]DA efflux attained in WT-hDAT cells, either by removal of Na(+) e or by application of amphetamine. We conclude that T62D-hDAT represents an efflux-willing, Na(+)-primed orientation-possibly representing an experimental model of the conformational impact of amphetamine exposure to hDAT.

Fig. 1.

Expression of WT-, T62A-, and T62D-mutant hDATs causes similar membrane depolarization in Xenopus oocytes. Xenopus oocytes were injected with 23 ng of cRNA for WT-, T62A-, or T62D-hDAT. Expression of hDAT statistically reduces the recorded RMP of oocytes when compared with control oocytes (Ctl) injected with 23 nl or water (***P < 0.001).

Fig. 2.

Dopamine-induced inward currents in T62D-hDAT oocytes are partially rescued by zinc. (A) Representative continuous traces of currents measured in oocytes clamped at –60 mV are shown. Inward currents are observed in WT- and T62A-hDAT oocytes after application of 10 μM DA. Coapplication of 2 μM ZnCl₂ partially restored a measurable inward current in T62D-hDAT oocytes. (B) Change in accumulated current (Δ current = DA current – baseline current) in WT-, T62A-, and T62D-hDAT oocytes held at –60 mV. The data are expressed as the average ± S.E.M with numbers of oocytes for each condition given. Unpaired t test with Welch’s correction for DA ± zinc application shows a significant increase in the DA-induced currents in WT- (*P < 0.05), T62A- (***P < 0.001), and T62D-hDAT (***P < 0.001)–expressing oocytes. Zinc did not affect the DA current measurement in control oocytes. Ctl, control.

Fig. 3.

Current-voltage relationships in hDAT-expressing Xenopus oocytes. A step–voltage clamp protocol was used from –150 to 30 mV at a holding potential of –60 mV and with 10-mV step increments for a 30-second duration at each step. Oocytes were challenged with 10 μM DA ± 2 μM ZnCl₂. The current is displayed as the difference from baseline measured in the absence of substrate (ΔI). At hyperpolarizing potentials, DA (▪) induces an inward current in (A) WT- and (B) T62A-hDAT oocytes. Coapplication with zinc (□) potentiates DA-induced inward currents. (C) In T62D-hDAT oocytes, DA inward currents are only measurable in the presence of zinc. Application of zinc alone (○) produced a small current in all constructs. Each current-voltage plot was generated from four to six oocytes expressing the indicated hDAT construct.

Fig. 4.

Affinity of the fluorescent substrate ASP⁺ is unaffected by the T62A- or T62D-hDAT mutations. As detailed in Materials and Methods, (A) uptake of 10 nM [³H]DA for 3 minutes or (B) binding of [³H]WIN 35,428 for 30 minutes in WT- (▪), T62A- (▴), and T62D-(◊) hDAT HEK cells was measured as a function of varying ASP⁺ concentrations. Data are expressed as the percent of the control without ASP⁺ and are shown as the mean ± S.E.M; n = 2 experiments performed in triplicate. The IC₅₀ (μM) values with 95% confidence intervals are reported in Results.

Fig. 5.

Accumulation of fluorescent substrate ASP⁺ in T62A- and T62D-hDAT HEK cells. Representative confocal images of time-dependent ASP⁺ accumulation are displayed in columns (A) WT-, (B) T62A-, and (C) T62D-hDAT, and (D) parental HEK cells. Acquired representative images of differential interference contrast and ASP⁺ signals are overlain, at times 0 and 180 seconds after exposure to 2 μM ASP⁺. (E) The optical density of the fluorescent signal was quantified in intracellular regions of WT- (▪), T62A- (▴), T62D- (◊) hDAT, and parental (°) HEK cells using ImageJ software. Data are plotted for the full 3-minute image acquisition of 10-second intervals; WT, n = 140 cells; T62A, n = 126 cells; T62D, n = 148 cells; and HEK, n = 148 cells.

Fig. 6.

The reliance on extracellular Na⁺ to promote DA uptake is not altered by the Thr62-hDAT mutations. Extracellular sodium (Na⁺_e) stimulation of [³H]DA uptake in WT- and T62-mutant hDAT HEK cells. Uptake of 10 nM [³H]DA was conducted for 3 minutes at room temperature. Na⁺ in the uptake buffer was replaced with NMDG-Cl to maintain osmolarity. The IC₅₀ (mM) for Na⁺_e in WT- (▪), T62A- (▴), and T62D- (◊) hDAT was 39.6, 40.5, and 58.7, respectively. Data are from n = 4 experiments done in triplicate and reported as a percentage of the maximum uptake achieved at the normal, highest [Na⁺_e] tested.

Fig. 7.

Initial [³H]DA efflux in T62D-hDAT is Na⁺-independent and resembles inducible DA efflux of WT-hDAT HEK cells. (A) WT-, (B) T62A-, and (C) T62D-hDAT HEK cells were preloaded with 0.5 μM [³H]DA (in normal KRH buffer) for 20 minutes. Basal and AMPH-induced DA efflux were determined while varying [Na⁺_e]. After 15 minutes cells were stimulated with AMPH (10 μM, black arrow) while varying [Na⁺_e] for 5 minutes. Data are averaged from three to six experiments performed in three or four replicates. In WT (A) and T62A (B), **P < 0.01 for 0 versus 125 mM for basal DA efflux at times 5, 10, and 15 minutes; ⁺⁺⁺P < 0.001; ⁺⁺P < 0.001; or ⁺P < 0.05 for AMPH-induced DA efflux at 20 minutes compared with 15 minutes (before AMPH) at each corresponding [Na⁺_e]. (D) Comparisons of basal and AMPH (black arrow) DA efflux in WT (circles) and T62D (squares) at 0 mM (open symbols) and 125 mM (closed symbols) Na⁺_e. To account for variation in DA uptake, the data are represented as a percentage of the total [³H]DA inside of the cell after DA preloading. Analyses of the three-way interaction (time versus [Na⁺_e] versus cell type) is not significantly different between WT and T62D at 0 mM Na⁺ (with the exception of 15 minutes, P = 0.005). DA efflux after AMPH (20 minutes) is significantly greater in WT compared with T62D at 125 mM Na⁺ (P < 0.0001), with no difference between cell types at 0 mM Na⁺ (P = 0.23). The effect of Na⁺_e on DA efflux (time versus cell type versus [Na⁺_e]) was significant in WT- (P ≤ 0.001) but not in T62D-hDAT. Analyses of cell type versus [Na⁺_e] versus time shows a significant change after the addition of AMPH (15 versus 20 minutes) in WT-hDAT at both 0 mM (P = 0.002) and 125 mM Na⁺ (P < 0.0001); and in T62D-hDAT at 0 mM Na⁺ (P = 0.03), but not 125 mM (P = 0.235).