3. METHODS
3.2 Cell biological methods
3.2.1 Expression of hOAT1 in Xenopus laevis oocytes
3.2 Cell biological methods
3.2.1 Expression of hOAT1 in Xenopus laevis oocytes
The Xenopus laevis oocyte expression system was used for functional characterization the mutations produced in hOAT1. Oocytes were injected with cRNA synthesized from the cDNA of interest (wt or mutant hOAT1) and, after a 3 day expression period, uptake assays were carried out as described below. Figure 3.1 illustrates a schematic representation of the oocyte assay system.
3.2.1.1 Preparation of oocytes
Reagents used
Barth’s 88 mM NaCl, 1 mM KCl, 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 15 mM HEPES, 12 μg/ml gentamycin, pH set at 7.6 with NaOH
ORI
(oocyte Ringer’s solution)
90 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH set at 7.6 with 1 mM Tris
Individual stage V-VI oocytes were defolliculated by collagenase treatment. It involved overnight incubation of several ovarian lobes in 20 ml Barth’s solution containing 0.5 mg/ml collagenase (Type CLSII, Biochrom KG) at 18°C. On the following day, oocytes were washed several times with oocyte Ringer’s solution (ORI), incubated for 10 min in Ca2+ free medium and then washed again 2-3 times with ORI solution. Oocytes were sorted and the “healthy” looking oocytes were used for the cRNA injection.
METHODS
OA* OA*
Trans-stimulation Uptake-assay
OA* OA*
Trans-stimulation Uptake-assay
OA* OA*OA*
OA*
Trans-stimulation Uptake-assay
Figure 3.1 Schematic representation of the oocyte assay system. Xenopus laevis oocytes are injected with in vitro synthesized cRNA, with cap and poly-A tail. Then follows the expression period, necessary for translation of the polypeptide encoded by the cRNA, its processing and insertion into membrane. On the 3rd day, the functions of transporter could be studied by: (A) uptake assay, when the uptake of radioactively labeled substrates (OA*) in the absence or presence of inhibitor compounds (I) was measured or (B) trans-stimulation, when oocytes were preloaded by injecting with glutaric acid (GA), and then trans-stimulation of labeled OA* (PAH) uptake was measured.
METHODS
3.2.1.2 Oocyte injection
cRNA were injected into the oocyte cytoplasm using a nanoliter microinjector with glass capillaries (World Precision Instruments). Oocytes were injected with 20-30 ng of cRNA in a 23-46 nl volume, or the equivalent volume of water as a control. Oocytes were arranged on a specially designed plastic support chamber with grooves to facilitate the injection process. Injection was carried out in ORI solution, and upon injection, the oocytes were incubated for three days at 18°C in Barth’s solution. On the third day after injection, surviving oocytes were sorted to remove unhealthy or maturated oocytes and used for transport assays.
3.2.1.3 Transport experiments
The selected oocytes were divided into groups of 9-12 and after equilibration in ORI medium, transferred to a 10 ml vial with 1 ml of ORI uptake medium, containing radioactively labeled substrate. Radio-labeled substances used were: [3H]PAH (5 μCi/ml) (aminohippuric acid, p-[glycyl-2-3H]-, 1-5 Ci/mmol, NEN), [1,5-14C]glutaric acid ([14C]GA) 100 µCi/ml, 55 mCi/mmol, or [3H(G)]-ochratoxin A ([3H]OTA) 1 mCi/ml, 7.2 Ci/mmol. Uptake was assayed at room temperature for time periods of 15 minutes to 1 hour, as required. After completion of the incubation period, the uptake was terminated by aspiration of the incubation medium and 3 x 4 ml washes with ice-cold ORI buffer. Individual oocytes were then transferred to 5 ml scintillation vials and dissolved in 100 μl of 1 N NaOH for 2 hours with shaking and, after neutralization with 100 μl of 1 N HCl and addition of 2.5 ml Lumasafe scintillation fluid (Lumac-LSC), the
3H or 14C content was assayed in a scintillation counter over 5 minutes.
3.2.1.4 Transport under chloride free conditions
Cl free ORI was prepared by substitution with gluconate (90 mM Na+ -gluconate, 3 mM K+-gluconate, 2 mM Ca2+-gluconate, 1 mM Mg2+-gluconate, 5 mM HEPES-Tris, pH 7.6). For transport assays in the absence of Cl, oocytes were initially washed three
METHODS
appropriate timeperiods, in the Cl free ORI containing the radiolabel. Following the incubation, oocytes were washed with ice cold Cl free ORI essentially and the same procedures as described in the previous section were followed.
3.2.1.5 Cis-inhibition experiments
For cis-inhibition experiments 1mM malonic, glutaric or adipic acid solutions were prepared in ORI and pH was adjusted to 7.6. Uptake of [3H]PAH was assayed, in the presence of each dicarboxylate, at room temperature for 1 hour using uptake in [3H]PAH in ORI without any dicarboxylate as control. The same general protocol for transport experiments described in section 3.2.1.2 was followed.
3.2.1.6 Trans-stimulation experiments
Trans-stimulation experiments were performed by injecting oocytes with 46 nl of 5 mM unlabeled glutaric acid solution in water (pH adjusted to 7.6). They were then washed and kept over ice for 15 minutes and then transferred to a 10 ml vial with 1 ml of ORI uptake medium, containing radioactively labeled substrate. Uptake of [3H]PAH was assayed over one hour. Uninjected oocytes and oocytes not preloaded with glutarate served as controls.
3.2.1.7 Kinetics
Apparent Km determinations – An indirect approach was applied for determining changes in substrate affinity between mutant and wild type transporters under normal and chloride free conditions (Malo and Berteloot, 1991). For the determination of the kinetics of PAH transport in wt and mutant hOAT1, oocytes expressing the respective transporter were assayed for 1µM [3H]PAH uptake over 30 min in ORI with or without chloride and in the presence of increasing concentrations of unlabeled PAH (0-500 µM).
METHODS
For both processes, the addition of unlabeled PAH inhibited uptake of [3H]PAH by a process adequately described by the Michaelis-Menten equation for competitive interaction of the labeled and unlabeled substrate:
Vmax[*S]
Kt+ [*S] + [S] + C V = Vmax[*S]
Kt+ [*S] + [S] + C V =
where V is the rate of [3H]PAH transport from a concentrationof labeled substrate equal to [*S]; Vmax is the maximum rate of mediated PAH transport; Kt is the PAH concentration that resulted in half-maximal transport (Michaelis constant); [S] is the concentration of unlabeled PAH in the transport reaction; and C is a constant representing the component of total PAH uptake that was not saturated (over the range of substrate concentrationstested) and presumably reflected the combined influence of diffusiveflux, nonspecific binding, and/or incomplete washing.