The trans-effects of organic cations on MPP efflux (a) and MPP influx (b) in depolarized oocytes. Water-injected control oocytes or oocytes expressinghOCT2 were injected with 0.1 pmol of [³H]MPP (efflux) or with different amounts of nonradioactive cations (preloading for uptake measurements). The indicated intracellular cation concentrations were estimated from the injected amounts of cations by assuming an intracellular aqueous space of 0.5 μl. a, Initial [³H]MPP efflux rates were estimated by measuring the efflux of washed oocytes between 10- and 70-sec incubations in Ori buffer (Na⁺,trans-zero) or in K oocyte buffer without or with the indicated cations (depolarized oocytes: K⁺ trans-zero or K⁺ cations). ▨,trans-zero condition in Fig. 5c in which the oocytes had been stored in the presence of 1 mm choline. b, The initial MPP uptake rates were estimated from the uptake after a 5-min incubation in Ori buffer (Na⁺ without preloading) or in K oocyte buffer (K⁺ without preloading or K⁺cation concentration) containing 0.1 μm[³H]MPP. Mean ± standard error values from 8–10 oocytes are presented.

Time course and potential dependence of hOCT2-mediated uptake of MPP in HEK 293 cells. HEK 293 cells that were constantly transfected with hOCT2 were incubated with 0.1 μm [³H]MPP, and the cellular MPP concentration was determined after different time intervals. The measurements were performed in the absence (•, ▪) or presence (▵) of 200 μm cyanine 863 with either Na⁺ (•, ▵) or K⁺ (▪) in the bath. Mean ± standard error values of three measurements are presented.

Distribution of hOCT2 in brain of the human analyzed by Northern blotting. Top, 2 μg/lane of poly(A)⁺ mRNA from different brain areas was separated by agarose gel electrophoresis and hybridized under high stringency conditions with a DIG-labeled cRNA probe specific forhOCT2. Bottom, intactness of the mRNAs in the different lanes was controlled by hybridization with cRNA of β-actin.

In situ hybridization of cortex and hippocampus from the human with hOCT2-specific cRNA. Cryosections through area 18 of the cerebral cortex (a and b) and through the hippocampus (c, d, and e) were fixed and hybridized with an antisense (a, c, and e) and a sense (b and d) fragment of cRNA as described in Experimental Procedures. Specific hybridizations were observed in pyramidal cells of the cerebral cortex and hippocampus.Arrow in e, hybridization with mRNA in a dendrite.Scale bars: a–d 250 μm; e, 25 μm.

Light microscopic immunohistochemical localization of hOCT2 in pyramidal cells of human hippocampus. a, Cryosections through human hippocampus were stained with a rabbit antiserum against an hOCT2-specific peptide. b, Staining of the pyramidal cells was observed, which was blocked when the antiserum was preincubated with the antigenic peptide. Scale bars, 50 μm.

Transport expression by hOCT2 in X. laevis oocytes of monoamine neurotransmitters and memantine.X. laevis oocytes were injected with 50 nl of water without or with 10 ng of hOCT2 cRNA and incubated for 3 days. Uptake of 90 μm of [³H]norepinephrine, [³H]serotonin, [³H]histamine, or [³H]dopamine and of 60 μm[¹⁴C]memantine was measured in the absence or presence of the hOCT2 inhibitor cyanine 863 (36 μm). The cyanine-inhibited uptake is indicated. Median ± standard error values from 10 parallel measurements are given.

Electrical measurements with X. laevis oocytes that were injected withhOCT2-cRNA or water. For expression, the oocytes were incubated for 3 days in Ori buffer. For the measurements, the oocytes with superfused with Ori buffer or with Ori containing 50 μm amantadine (■), 50 μm memantine (▨), or 5 mm dopamine (▪). a, The membrane potential was measured in the absence and presence of the indicated cations. b, The oocytes were clamped to the indicated membrane potentials, and the currents were determined that were induced by superfusion of hOCT2-expressing oocytes with 50 μm amantadine, 50 μm memantine, or 5 mm dopamine.

Induction of currents inhOCT2-expressing oocytes clamped to different membrane potentials after superfusion with different concentrations of amantadine, memantine, or dopamine. a, hOCT2-cRNA-injected oocytes were clamped at −50 mV and superfused for 30 sec with the indicated cation concentrations. Mean ± standard error values of 8–10 measurements of induced currents in different oocytes are presented. The values from different oocytes were normalized against the currents induced by 1 mm TEA. b, The potential dependence of the currents induced by 5 mm dopamine or 50 μmamantadine in hOCT2-expressing oocytes is presented (mean ± standard error).

Membrane potentials of hOCT2-expressing oocytes without and with preincubation with organic cations that were measured with Na⁺ or K⁺ in the bath. hOCT2-cRNA-injected oocytes were preincubated for 12 hr without organic cations or with the indicated concentrations of choline, MPP, amantadine, and dopamine. The membrane potential was measured with Ori buffer in the bath (▨) or after replacement of Na⁺ in the Ori buffer by K⁺ (■). Mean ± standard deviation values from six oocytes are shown.

Concentration dependence of hOCT2-mediated uptake of [³H] MPP and [³H] dopamine in HEK 293 cells. Initial uptake rates at different concentrations of MPP or dopamine were determined after 1-sec incubation of nontransfected HEK 293 cells or HEK 293 cells that were constantly transfected withhOCT2. The measurements were performed in the presence of Na⁺ without or with 200 μm cyanine 863 in the bath. Cyanine-inhibited uptake rates are presented that were calculated from three to six parallel measurements. Mean ± standard error values are indicated. The curves were obtained by fitting the Michaelis-Menten equation to the data.

Efflux of MPP and choline fromhOCT2-transfected HEK 293 cells measured undertrans-zero conditions. Nontransfected HEK 293 cells (○, ■, ▵) and HEK 293 cells stably transfected withhOCT2 (•, ▴, ▪) were preloaded with 19 μm [³H]MPP (■, ▪, ▵, ▴) or 210 μm [³H]choline (○, •). After washing at 0°, the efflux of the radioactively labeled cations was measured at 37° under trans-zero conditions with Na⁺or K⁺ in the bath. Mean ± standard deviation values of four determinations are presented.

The trans-effects of organic cations on the efflux (a) and influx (b) of MPP in depolarized HEK 293 cells stably transfected with hOCT2. a,hOCT2-transfected HEK 293 cells and nontransfected control cells were preloaded with 0.1 μm[³H]MPP and washed at 0°, and the [³H]MPP efflux was measured after a 30-sec incubation at 37° in the K buffer. b, The transfected and nontransfected cells were preincubated without and with the indicated organic cations and washed at 0° with K buffer. The cells were incubated for 1 sec at 37° in K buffer containing 0.1 μm [³H]MPP, and the cellular [³H]MPP was analyzed. Mean ± standard deviation values from four determinations are presented.