Selective transport of monoamine neurotransmitters by human plasma membrane monoamine transporter and organic cation transporter 3

Abstract

The plasma membrane monoamine transporter (PMAT) and organic cation transporter 3 (OCT3) are the two most prominent low-affinity, high-capacity (i.e., uptake(2)) transporters for endogenous biogenic amines. Using the Flp-in system, we expressed human PMAT (hPMAT) and human OCT3 (hOCT3) at similar levels in human embryonic kidney 293 cells. Parallel and detailed kinetics analysis revealed distinct and seemingly complementary patterns for the two transporters in transporting monoamine neurotransmitters. hPMAT is highly selective toward serotonin (5-HT) and dopamine, with the rank order of transport efficiency (V(max)/K(m)) being: dopamine, 5-HT ≫ histamine, norepinephrine, epinephrine. The substrate preference of hPMAT toward these amines is substantially driven by large (up to 15-fold) distinctions in its apparent binding affinities (K(m)). In contrast, hOCT3 is less selective than hPMAT toward the monoamines, and the V(max)/K(m) rank order for hOCT3 is: histamine > norepinephrine, epinephrine > dopamine >5-HT. It is noteworthy that hOCT3 demonstrated comparable (≤2-fold difference) K(m) toward all amines, and distinctions in V(max) played an important role in determining its differential transport efficiency toward the monoamines. Real-time reverse transcription-polymerase chain reaction revealed that hPMAT is expressed at much higher levels than hOCT3 in most human brain areas, whereas hOCT3 is selectively and highly expressed in adrenal gland and skeletal muscle. Our results suggest that hOCT3 represents a major uptake(2) transporter for histamine, epinephrine, and norepinephrine. hPMAT, on the other hand, is a major uptake(2) transporter for 5-HT and dopamine and may play a more important role in transporting these two neurotransmitters in the central nervous system.

Fig. 1.

A, quantification of hOCT3 and hPMAT mRNA levels in stably transfected Flp-in HEK293 cells. Total RNA from Flp-293-pcDNA5, hOCT3, and hPMAT cells were extracted and reverse-transcribed. hOCT3 and hPMAT mRNA copy numbers were determined by Taqman real-time RT-PCR using specific primers and probes. Diluted hOCT3 and hPMAT expression vectors with known copy numbers were used as standards to determine absolute mRNA copy numbers. B, localization of hOCT3 and hPMAT in Flp-in HEK293 cells stably transfected with hPMAT or hOCT3. Cells transfected with pcDNA5 vector were used as control. Confluent cells were immunostained with anti-OCT3 or anti-PMAT primary antibodies and Alexa Fluor-conjugated secondary antibodies. Images were taken under a fluorescent microscope with corresponding filters.

Fig. 2.

Uptake of MPP+, TEA, and monoamines by hOCT3 and hPMAT. Flp-293 pcDNA5, hOCT3, and hPMAT cells were run in parallel with substrate concentrations at 1 μM.

Fig. 3.

Time-dependent uptake of MPP+, TEA, and monoamines by hOCT3 and hPMAT. Uptake experiments were conducted as described under Materials and Methods for up to 30 min. Time points were chosen within the linear uptake phases (which range between 2 and 5 min) to determine kinetics parameters. The concentrations of substrates were: MPP+, 1 μM; dopamine, 10 μM; 5-HT, 10 μM; norepinephrine, 20 μM; epinephrine, 20 μM; histamine, 10 μM; TEA, 20 μM.

Fig. 4.

Concentration-dependent uptake for MPP+, TEA, and monoamines in hOCT3 and hPMAT cells. Uptake for a specific substrate was carried out in parallel in Flp-293 pcDNA5, hOCT3, and hPMAT cells. Uptake in pcDNA5 cells were subtracted from hOCT3 or hPMAT cells to obtain transporter-specific uptake. Uptake time points were 5 min for TEA and 2 min for all other substrates.

Fig. 5.

hOCT3 and hPMAT mRNA levels in various human tissues and brain regions. Total RNA from human tissues were reverse-transcribed to cDNA. hOCT3, hPMAT, and hGUSB transcripts levels were quantified by using Taqman real-time RT-PCR. A, absolute copy numbers of hOCT3 and hPMAT mRNA in 10 ng of total RNA. B, hOCT3 and hPMAT mRNA levels normalized to hGUSB.

Fig. 6.

Effects of inhibitors used in synaptosome uptake studies on MPP+ uptake mediated by hOCT3 and hPMAT. Flp-293 pcDNA5, hOCT3, and hPMAT cells were run in parallel with MPP+ concentrations at 1 μM and in the absence or presence of inhibitors. NT, no treatment; Cit, citalopram (10 μM); Cort, corticosterone (100 μM); D22 (10 μM); GBR, GBR12909 (1 μM); Des, desipramine (1 μM). Data are expressed as percentage of MPP+ uptake obtained from cells with no treatment.

Fig. 7.

Uptake of 0.5 μM 5-HT (A), dopamine (B), and norepinephrine (C) in adult mouse brain synaptosomes in the presence or absence (no treatment) of various inhibitors. Cit, citalopram (10 μM); Cort, corticosterone (100 μM); D22 (10 μM); GBR, GBR12909 (1 μM); Des, desipramine (1 μM). *, p < 0.05 compared with no treatment (NT); **, p < 0.05 compared with citalopram, GBR12909, or desipramine alone.