4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP+) is a fluorescent substrate for the human serotonin transporter

Abstract

Monoamine transporters terminate synaptic neurotransmission and are molecular targets for antidepressants and psychostimulants. Fluorescent reporters can monitor real-time transport and are amenable for high-throughput screening. However, until now, their use has mostly been successful to study the catecholamine transporters but not the serotonin (5HT) transporter. Here, we use fluorescence microscopy, electrophysiology, pharmacology, and molecular modeling to compare fluorescent analogs of 1-methyl-4-phenylpyridinium (MPP(+)) as reporters for the human serotonin transporter (hSERT) in single cells. The fluorescent substrate 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP(+)) exhibits superior fluorescence uptake in hSERT-expressing HEK293 cells than other MPP(+) analogs tested. APP(+) uptake is Na(+)- and Cl(-)-dependent, displaced by 5HT, and inhibited by fluoxetine, suggesting APP(+) specifically monitors hSERT activity. ASP(+), which was previously used to study catecholamine transporters, is 10 times less potent than APP(+) at inhibiting 5HT uptake and has minimal hSERT-mediated uptake. Furthermore, in hSERT-expressing oocytes voltage-clamped to -60 mV, APP(+) induced fluoxetine-sensitive hSERT-mediated inward currents, indicating APP(+) is a substrate, whereas ASP(+) induced hSERT-mediated outward currents and counteracted 5HT-induced hSERT currents, indicating ASP(+) possesses activity as an inhibitor. Extra-precise ligand receptor docking of APP(+) and ASP(+) in an hSERT homology model showed both ASP(+) and APP(+) docked favorably within the active region; accordingly, comparable concentrations are required to elicit their opposite electrophysiological responses. We conclude APP(+) is better suited than ASP(+) to study hSERT transport fluorometrically.

FIGURE 1.

Structures of MPP⁺ and its fluorescent analogs APP⁺ and ASP⁺ and screening for a fluorescent substrate of hSERT. A, the addition of an electron donating dimethyl amine group to the phenyl ring of MPP⁺ results in the fluorescent compound APP⁺, which can be compared with ASP⁺, the previously characterized fluorescent substrate for hNET (chemical structures shown in two-dimensional (2D) views, top row). Two three-dimensional (3D) views showing energy minimized MPP⁺, APP⁺, and ASP⁺ (bottom row). Although the aromatic rings in APP⁺ do not attain a co-planar conformation and favor twisted dihedral angles, ASP⁺ adopts a co-planar conformation. B, images of hSERT-HEK cells exposed to fluorescent compounds (from top to bottom: APP⁺ only, differential interference contrast, and APP⁺ and differential interference contrast merged). C, fluorescence intensity was calculated by integrating emission spectra curves for each compound. Fluorescence was normalized to the brightest compound, APP⁺. Structure nomenclature: 332, N,N-dimethyl-4-(pyridin-4-yl)aniline; APP⁺, 4-(4-(dimethylamino)styryl)-N-methylpyridinium; 321, 4-(4-(dimethylamino)phenyl)-1-ethylpyridinium; 326, 4-(4-(dimethylamino)-2-methylphenyl)-1-methylpyridinium; 330, 4-(4-(dimethylamino)-3-fluorophenyl)-1-methylpyridinium; 377, N,N-dimethyl-4-(pyridin-3-yl)aniline; 378, 3-(4-(dimethylamino)phenyl)-1-methylpyridinium.

FIGURE 2.

APP⁺ displays two rates of uptake. A, APP⁺ accumulation depends on concentration. Time-lapses of hSERT-HEK cells exposed to different concentrations of APP⁺ (from 250 nm to 10 μm) were acquired at a rate of one image per second for 10 min. Dotted lines represent the two rates observed, the initial (slow) and subsequent (fast) components. B, to determine the V_max and k_m, straight lines were fitted, and slopes were obtained for both the slow and fast phases acquired at each concentration. For the fast phase, measurements from 20–100 s were used, and for the slow phase, the measurements from 200 s to the end of the acquisition were used. Slopes were plotted against concentration and these values were fit to the Hill equation, y = V_min + (V_max − V_min) × xⁿ/(kⁿ + xⁿ). The V_max, V_min, and k_m for the fast phase were 0.65 ± 0.07 AFU/s, 0.062 ± 0.008 AFU/s, and 2.29 ± 0.65 μm, respectively, and for the slow phase, the V_max,V_min, and k_m were 0.38 ± 0.04 AFU/s, 0.029 ± 0.003 AFU/s, and 2.36 ± 0.55 μm, respectively. The Hill coefficient was 2.91 ± 1.09 for the fast component and 2.53 ± 0.68 for the slow component.

FIGURE 3.

APP⁺ is a fluorescent substrate of hSERT. Time-lapses of hSERT-HEK cells exposed to 2 μm APP⁺. A, temperature regulates APP⁺ accumulation. APP⁺ fluorescence accumulation is greater when measured under physiological (37 °C) temperature, and much lower at 20 °C as compared with the moderate (27 °C) temperature. B, removal of both Na⁺ and Cl⁻ diminished APP⁺ accumulation. C, an immediate decrease in APP⁺ accumulation occurs when 5HT (10 μm) was added 6 min into the time-lapse (indicated by arrow). D, co-treatment with fluoxetine (FLX; from 1 to 10 μm) abolishes APP⁺ fluorescence accumulation.

FIGURE 4.

Comparing the effects of ASP⁺ and APP⁺ on hSERT. A and B, images of hSERT-HEK or parental HEK293 cells before (row labeled with 0) and after exposure to 10 μm ASP⁺ (A) or APP⁺ (B) for 1 or 3 min. ASP⁺ fluoresces red and labels the exterior membrane of cells in both hSERT-HEK and parental cells, whereas APP⁺, which fluoresces green, seems to accumulate only inside of hSERT-HEK cells. C and D, time-lapses comparing fluorescence accumulation rates between hSERT-HEK and HEK293 cells for ASP⁺ (30 μm; C) and APP⁺ (5 μm;D). E, concentration-response curve for APP⁺ and ASP⁺ inhibition of [³H]5HT uptake into hSERT-HEK cells. [³H]5HT accumulation was measured in hSERT-HEK cells in the presence of increasing APP⁺ and ASP⁺ concentrations and normalized to data in the absence of the competing substrate. The data were fit to the Hill equation, y = V_max + (V₀ − V_max) × xⁿ/(kⁿ + xⁿ), and k_i values were determined using the Cheng-Prusoff equation to correct for substrate concentration. The fits yield k_i (APP⁺) = 19.7 ± 2.23 μm and k_i (ASP⁺) = 180.1 ± 20.3 μm. Values are represented as means ± S.E. (n = 3). The Hill coefficients for ASP⁺ and APP⁺ are 0.91 ± 0.15 and 1.23 ± 0.08, respectively. F, electrophysiological effect of substrates on hSERT. Currents in an hSERT-expressing X. laevis oocyte clamped to −60 mV are measured in response to 10 μm 5HT, APP⁺, ASP⁺, and 1 μm fluoxetine (FLX). Bars display perfusion duration of each compound. G, the effect of voltage (from 0 to −100 mV) on hSERT-induced currents. Currents induced by 5HT, APP⁺, ASP⁺ are plotted relative to the baseline set as 0 at each potential (n = 4). Control (uninjected) oocytes show no response to 5HT, ASP⁺, or APP⁺ (not shown).

FIGURE 5.

APP⁺ exhibits substrate-like activity at hSERT. A, APP⁺ concentration-response curve. Representative trace of hSERT currents are measured in response to APP⁺ (0.1–25 μm) applied to hSERT-expressing X. laevis oocyte clamped to −60 mV. B, summary data were normalized to 5 μm 5HT-induced currents and fit to the Hill equation, y = I_max + (I_min − I_max) × xⁿ/(kⁿ + xⁿ) (n = 8). The I_max, I_min, k_m, and Hill coefficient were 23.75 ± 2.51, 3.02 ± 2.08, 1.13 ± 0.28 μm, and 1.23 ± 0.60, respectively. C, application of fluoxetine (FLX; 1 μm) blocks the 5HT-induced hSERT current ([5HT] = 2 μm). D, application of fluoxetine (1 μm) blocks the APP⁺-induced hSERT current ([APP⁺] = 10 μm).

FIGURE 6.

ASP⁺ exhibits inhibitor-like activity at hSERT. A, ASP⁺ concentration-response curve. Representative trace of hSERT currents are measured in response to APP⁺ (0.5–100 μm) applied to hSERT-expressing X. laevis oocyte clamped to −60 mV. B, summary data were normalized to 2 μm 5HT-induced currents and fit to the Hill equation, y = I_max + (I_min − I_max) × xⁿ/(kⁿ + xⁿ) (n = 8). The I_max, I_min, k_m, and Hill coefficient were 28.32 ± 2.17, 3.48 ± 0.44, 12.25 ± 2.71 μm, and 1.34 ± 0.31, respectively. C, ASP⁺ inhibits 5HT-induced hSERT currents. During currents induced by 5HT (2 μm), ASP⁺ is co-applied at indicated concentrations (from 1 to 250 μm, as indicated above upper bars in traces) (n = 8). D, summary data of ASP⁺ inhibition of 5HT-induced hSERT currents (from currents in C).

FIGURE 7.

APP⁺ is not fluorescent at the plasma membrane. A, plasma membrane of hSERT-HEK cells is labeled with DiI (red, left) and APP⁺ (green, middle) is added to cells to determine co-localization (DiI and APP⁺ merged, right). B, line scan (red arrow) through an hSERT-HEK cell with APP⁺/DiI (left) and plotted fluorescence intensity of line scan through cell (right).