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Am J Physiol Renal Physiol 293: F767-F779, 2007. First published June 13, 2007; doi:10.1152/ajprenal.00106.2007
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Identification of cysteines in rat organic cation transporters rOCT1 (C322, C451) and rOCT2 (C451) critical for transport activity and substrate affinity

Alexander Sturm,1,* Valentin Gorboulev,1,* Dmitry Gorbunov,1 Thorsten Keller,1 Christopher Volk,1 Bernhard M. Schmitt,1 Peter Schlachtbauer,1 Giuliano Ciarimboli,2 and Hermann Koepsell1

1Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, and 2Experimental Nephrology, Department of Internal Medicine D, University of Münster, Münster, Germany

Submitted 1 March 2007 ; accepted in final form 9 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effects of the sulfhydryl reagent methylmethanethiosulfonate (MMTS) on functions of organic cation transporters (OCTs) were investigated. Currents induced by 10 mM choline [Imax(choline)] in Xenopus laevis oocytes expressing rat OCT1 (rOCT1) were increased four- to ninefold after 30-s incubation with 5 mM MMTS whereas Imax(choline) by rat OCT2 was 70% decreased. MMTS activated the rOCT1 transporter within the plasma membrane without changing stoichiometry between translocated charge and cation. After modification of oocytes expressing rOCT1 or rOCT2 with MMTS, I0.5(choline) values for choline-induced currents were increased. For rOCT1 it was shown that MMTS increased I0.5 values for different cations by different degrees. Mutagenesis of individual cysteine residues in rOCT1 revealed that modification of cysteine 322 in the large intracellular loop, and of cysteine 451 at the transition of the transmembrane {alpha}-helix (TMH) 10 to the short intracellular loop between the TMH 10 and 11 is responsible for the observed effects of MMTS. After replacement of cysteine 451 by methionine, the IC50(choline) for choline to inhibit MPP uptake by rOCT1 was increased whereas the I0.5(choline) value for choline-induced current remained unchanged. At variance, in double mutant Cys322Ser, Cys451Met, I0.5(choline) was increased compared with rOCT1 wild-type whereas in the single mutant Cys322Ser I0.5(choline) was not changed. The data suggest that modification of rOCT1 at cysteines 322 and 451 leads to an increase in turnover. They indicate that cysteine 451 in rOCT1 interacts with the large intracellular loop and that cysteine 451 in both rOCT1 and rOCT2 is critical for the affinity of choline.

sulfhydryl group modification; mutagenesis; substrate affinity; turnover

POLYSPECIFIC ORGANIC CATION transporters of the SLC22 transporter family play a pivotal role in the elimination and distribution of cationic drugs, toxins, and metabolic compounds (18, 21). The SLC22 family is a member of the major facilitator superfamily (MFS) and contains transporters with 12 transmembrane {alpha}-helices (TMHs), a large extracellular loop with glycosylation sites, and a large intracellular loop with phosphorylation sites (19). Three subtypes of organic cation transporters named OCT1-3 have been identified that mediate facilitative diffusion of structurally diverse organic cations including monoamine neurotransmitters in the liver (OCT1, OCT3), kidney (OCT1-3), and many other organs including the brain and lung. In the human organic cation transporter subtypes, polymorphisms with decreased activity and changed substrate selectivity have been identified that may lead to increased drug susceptibility (16, 22, 31). The tertiary structure of organic cation transporters has not been elucidated. However, functional data, site-directed mutagenesis, and modeling using recently resolved crystal structures of two transporters of the MFS suggest that the organic cation transporters contain large substrate binding regions with partially overlapping binding domains for structurally different substrates that are formed by several TMHs, including TMH 4, 10, and 11 (7, 9, 27, 35, 38).

The expression and function of the polyspecific organic cation transporters are regulated in a subtype- and tissue-specific manner (21). For example, short-term regulation of rat OCT1 (rOCT1) expressed in human embryonic kidney (HEK) 293 cells by PKC was demonstrated (23). Interestingly, after stimulation of rOCT1 by PKC, the cation selectivity of rOCT1 was changed. Upregulation of transport activity by PKC was prevented when any of the four PKC consensus sequences in the large intracellular loop of rOCT1 was inactivated (6). In addition, the IC50 values for the inhibition of rOCT1-mediated uptake of 4-[4-(dimethylamino)styryl]-N-methylpyridinium by tetraethylammonium (TEA) and tetrapentylammonium (TPeA) were increased. At variance during short-term regulation of human OCT2 (hOCT2), Vmax of cation transport was decreased (4). These data suggested that the large intracellular loop modulates substrate binding and/or transport velocity.

In the present study, we characterized cysteine residues in rOCT1 and rOCT2. We observed that the membrane-permeant sulfhydryl reagent methylmethanethiosulfonate (MMTS) increased maximal cation transport activity in oocytes expressing rOCT1 within seconds without changing the amount of transporter molecules in the plasma membrane or the stoichiometry between transported TEA and the charge. At variance, MMTS decreased maximal choline transport by rOCT2. After treatment with MMTS, the selectivity of rOCT1 for organic cations was changed and the affinity of rOCT2 for choline was decreased. Mutagenesis analysis in rOCT1 revealed that modification of cysteine 322 in the large intracellular loop and of cysteine 451 at the cytosolic end of TMH 10 is responsible for the observed effects.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Biochemicals. MMTS and (2-sulfonatoethyl)methanethiosulfonate (MTSES) were purchased from Toronto Research Chemical (Ontario, Canada). [3H]MPP (3.1 TBq/mmol) and [14C]TEA (1.9 TBq/mmol) were obtained from Biotrend (Köln, Germany). The other chemicals were obtained as described earlier (3, 15, 27).

Cloning and site-directed mutagenesis. Mouse OCT1 (mOCT1) was amplified by PCR from mouse liver total RNA using primers designed from the published mOCT1 sequence (30): forward 5'-GCAAGCTTCCCAGCCATGCCCACCGTG-3' and reverse 5'-CGCTCGAGCTCTCTCCACTCAGCTCC-3' (the HindIII and XhoI sites are underlined). The PCR product was digested with HindIII and XhoI and cloned into the oocyte vector pRSSP (5). Sequencing confirmed the absence of PCR errors. Rat OCT3 (rOCT3) in the vector pSPORT1 (14) was supplied by Dr. V. Ganapathy. For immunodetection, an rOCT1 variant with the FLAG epitope (DYKDDDDK) at the COOH terminus [rOCT1(FLAG)] was generated and cloned into the pRSSP vector as described (27). Point mutations were introduced into rOCT1 and rOCT2 by PCR, applying the overlap extension method as described (7). rOCT1, rOCT2, and their mutants were cloned into the vector pRSSP. All PCR-derived parts of the mutant constructs were sequenced to rule out PCR errors.

Expression of organic cation transporters and mutants in oocytes of Xenopus laevis. For injection into oocytes, m7G(5')ppp(5')G-capped cRNAs were prepared using an mMESSAGE mMACHINE kit (Ambion, Huntingdon, UK). To prepare sense cRNA from rOCT1 and mutants of rOCT1 (10), from mOCT1 (30), from rOCT2 and mutants of rOCT2 (3), from hOCT1 (8), hOCT2 (8) and rOCT3 (14), the purified plasmids were linearized with MluI (rOCT1, rOCT2, and their mutants, mOCT1) or with NotI (hOCT1, hOCT2, rOCT3). The cRNA was synthesized using SP6 RNA polymerase (rOCT1, rOCT2, their mutants, mOCT1) or T7 polymerase (hOCT1, hOCT2, rOCT3). cRNA concentrations were estimated from ethidium bromide-stained agarose gels using polynucleotide markers as standards (11).

Stage V-VI oocytes were obtained by partial ovariectomy, defolliculated with collagenase A (34) and stored for several hours in Ori buffer [5 mM 3-(N-morpholino)propanesulfonic acid-NaOH, pH 7.4, 100 mM NaCl, 3 mM KCl, 2 mM CaCl2, and 1 mM MgCl2] containing 50 mg/l gentamycin. The oocytes were injected with 50 nl H2O/oocyte containing 10 ng of cRNA encoding the respective transporter or mutant. For transporter expression, the oocytes were incubated for 3 days at 16°C in Ori buffer containing 50 mg/l gentamycin. Animals were handled in compliance with institutional guidelines and German laws.

Measurements of current and capacitance without and with preincubation with SH-group reagent. Current measurements were performed using the two-electrode voltage-clamp method (3). The oocytes were superfused at room temperature with Ori buffer and clamped to –50 mV. To measure cation-induced inward currents or cation-induced capacitance changes, the oocytes were superfused for 30–120 s with Ori buffer containing the indicated concentrations of choline, TEA, or guanidine. Choline-induced outward currents were measured with oocytes that were incubated overnight with 10 mM choline. The oocytes were superfused with Ori buffer containing 10 mM choline and clamped to –50 mV. Outward currents were measured during superfusion with choline-free Ori buffer. Real-time monitoring of cation-induced capacitance changes and of capacitance changes that were induced by MMTS was performed as described earlier (28, 29). First, choline-induced current and capacitance changes were measured in untreated oocytes. Then, the oocytes were incubated with Ori buffer containing MMTS, washed with Ori buffer, and choline-induced current and capacitance changes were measured again.

Parallel measurements of charge translocation and transport of [14C]TEA and measurements of [3H]MPP uptake. To determine the stoichiometry between translocation of charge and transport of TEA, oocytes expressing rOCT1 or rOCT2 were superfused with Ori buffer and clamped to –50 mV until the baseline of injected current was constant. The superfusion was stopped, the bath solution was exchanged with Ori buffer containing 500 µM [14C]TEA, and the injected current was recorded. After 2 min, the oocytes were superfused with Ori buffer containing 100 µM of the nontransported inhibitor tetrabutylammonium. Under these conditions, the efflux of TEA was inhibited and the injected current reached baseline. The oocytes were washed six times with ice-cold Ori buffer containing 100 µM quinine, solubilized with 5% (wt/vol) sodium dodecylsulfate (SDS), and analyzed for radioactivity. The translocated charge was calculated from the integrated current induced by TEA. Uptake measurements with [3H]MPP were performed as described (3). The oocytes expressing rOCT1 or mutants were incubated for 30 min with 0.1 µM [3H]MPP in the presence of various concentrations of choline. MPP uptake was corrected for uptake that was measured in parallel in noninjected control oocytes.

Isolation of plasma membranes from oocytes using silica beads. Isolation of plasma membranes from oocytes was performed as described (13, 27). Eight defolliculated oocytes were rotated for 30 min at 4°C in Mes-buffered saline (20 mM Mes·HCl, pH 6.0, 80 mM NaCl) containing 1% (wt/vol) colloidal silica (Ludox Cl from Sigma-Aldrich, Taufkirchen, Germany). The oocytes were washed two times with Mes-buffered saline, rotated 30 min at 4°C in Mes-buffered saline containing 0.1% polyacrylic acid (Sigma-Aldrich), and washed two times with Ori buffer. Oocytes were homogenized in 1.5 ml 20 mM Tris·HCl, pH 7.4, 5 mM MgCl2, 5 mM NaH2PO4, 1 mM EDTA, 80 mM sucrose (HbA buffer), and centrifuged for 1 min (4°C) at 10 g. One milliliter of the top of the sample was removed, 1 ml of HbA buffer was added, and the sample was mixed. Centrifugation, removal of 1 ml from the top, addition of 1 ml HbA buffer, and mixing was repeated four times, but the centrifugation was performed twice at 10 g, once at 20 g, and once at 40 g. After the last step, plasma membranes were spun down by 30-min centrifugation at 16,000 g (4°C).

SDS-PAGE and Western blotting. Protein concentrations were determined, and SDS-PAGE and Western blotting were performed as described (15). Samples for SDS-PAGE derived from oocytes expressing an rOCT1 mutant containing a FLAG epitope at the COOH terminus [rOCT1(FLAG)] were pretreated 30 min at 37°C in 60 mM Tris·HCl, pH 6.8, 100 mM dithiothreitol, 2% (wt/vol) SDS, and 7% (vol/vol) glycerol. Proteins separated by SDS-PAGE were transferred by electroblotting to polyvinylidene difluoride membrane. The membrane was blocked and incubated for 2 h at room temperature with a mouse antibody against the FLAG epitope (F1804, Sigma) diluted 1:20,000. After a washing, the blots were incubated for 2 h at room temperature with a protein G-horseradish peroxidase conjugate from Bio-Rad (München, Germany) diluted 1:5,000. The protein G-horseradish peroxidase conjugate was visualized by enhanced chemiluminescence (ECL system; Amersham Bioscience Systems Europe, Freiburg, Germany). Prestained molecular weight markers (Bench Mark, Life Technologies, Karlsruhe, Germany) were used to determine apparent molecular masses.

Purification and reconstitution of rOCT1 into proteoliposomes and measurement of [3H]MPP uptake. rOCT1 containing six histidine residues at the COOH terminus was expressed in Sf9 insect cells, lysed with 1% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and affinity purified using lentil-lectin Sepharose and nickel(II)-charged nitrilotriacetic acid-agarose as described (15). The purified protein was reconstituted into proteoliposomes containing phosphatidylcholine, phosphatidylserine, and cholesterol by a freeze-thaw procedure (15). The proteoliposomes contained 100 mM potassium cyclamate, 20 mM imidazole, pH 7.4, and 0.1 mM magnesium cyclamate (KC buffer). Uptake of [3H]MPP into proteoliposomes was measured in the presence of the potassium ionophore valinomycin (20 µM). Incubation with [3H]MPP was performed for 1 s in the presence of 90 mM sodium cyclamate, 10 mM potassium cyclamate, 20 mM imidazole, pH 7.4, and 0.1 mM magnesium cyclamate. Nonspecific uptake measured in the presence of the OCT1 inhibitor quinine (100 µM) was subtracted. After 1-s incubation, uptake was stopped with ice-cold KC buffer containing 100 µM quinine (stop solution). The proteoliposomes were applied to 0.22-µm cellulose acetate filters (Millipore) and washed with 10 ml ice-cold stop solution. The filters were dissolved in LUMASAFE PLUS cocktail (Lumac, Groningen, The Netherlands) and analyzed for radioactivity (15).

Calculation and statistics. In Figs. 1–5, typical individual experiments from at least three experiments are presented. Apparent I0.5 values for cation-induced currents and IC50 values for inhibition of MPP uptake were determined from measurements that were performed with six to nine different concentrations of substrates or inhibitors. For current and capacitance measurements, I0.5 and IC50 values were determined by fitting the Michaelis-Menten equation (I0.5) or the Hill equation (IC50) to individual experiments performed with oocytes that were derived from at least two different animals. Mean values are presented that were calculated from Imax values, activation factors, I0.5 values, and IC50 values of individual oocytes or of individual tracer uptake experiments in which 7–10 oocytes were measured per inhibitor concentration. Two-sided Student's t-tests were used to prove statistical significance of differences between two groups. A paired t-test was employed to evaluate significance of activation by MMTS. ANOVA with a post hoc Tukey comparison was used when more than two different groups were compared. Means ± SD are presented with the exception of Fig. 8, where means ± SE are shown.


Figure 1
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  		Fig. 1. Effects of methylmethanethiosulfonate (MMTS) on choline-induced inward and outward currents expressed by rat organic cation transporter (rOCT1) or rOCT2. Effects of MMTS were measured in noninjected oocytes (control), rOCT1-expressing oocytes (rOCT1), and in rOCT2-expressing oocytes (rOCT2) clamped to –50 mV. A: for influx measurements, oocytes were subsequently superfused with Ori buffer, with Ori buffer containing 10 mM choline, and with Ori buffer. Superfusion was stopped and oocytes were incubated with 500 µM MMTS. Oocytes were superfused with Ori buffer to remove MMTS, and inward currents were measured that were induced by superfusion with 10 mM choline. B: for efflux measurements, oocytes were incubated overnight with 10 mM choline. Measurements were started by superfusing the oocytes with Ori buffer containing 10 mM choline and clamping them to –50 mV. Holding currents of 0 (control), 39 (rOCT1), and 85 nA (rOCT2) were required, suggesting transporter-mediated inward currents. Choline-induced outward currents were measured by superfusing the oocytes with choline-free Ori buffer followed by Ori buffer containing 10 mM choline. The superfusion was stopped, and oocytes were incubated with Ori buffer containing 10 mM choline and 500 µM MMTS. In the case of rOCT1, this led to an increase in holding current. MMTS was removed from the bath by superfusing the oocytes with Ori buffer containing 10 mM choline. Finally, the choline-induced outward current was measured by switching the superfusate to choline-free Ori buffer. The data indicate that choline-induced currents by rOCT1 were increased whereas choline-induced currents by rOCT2 were decreased after modification of the transporters with MMTS.

 

Figure 5
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  		Fig. 5. Effects of MMTS on capacitance in oocytes expressing rOCT1 or rOCT2 in the absence of organic cations in the bath. Oocytes were injected with cRNA of rOCT1 or cRNA of rOCT2, incubated for 3 days, superfused with Ori buffer, and clamped to –50 mV. The capacitance was measured before, during, and after incubation of the oocytes with 200 µM MMTS to the bath. Typical measurements from 5–9 experiments are shown. The data indicate that MMTS induces transporter-dependent capacitance changes in the absence of extracellular organic cations.

 

Figure 8
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  		Fig. 8. Activation of choline-induced currents by MMTS in mutants of rOCT1 in which cysteine residues were exchanged by alanine or serine. Wild-type (WT) rOCT1 and the indicated rOCT1 mutants were expressed in oocytes. A: currents that were induced after superfusion of oocytes expressing single-cysteine replacement mutants with 10 mM choline. Oocytes were clamped to –50 mV. B: activation of single-cysteine replacement mutants by MMTS. Currents were measured that were induced by 10 mM choline at –50 mV before [Imax(choline) before MMTS] and after 30-s incubation with 5 mM MMTS [Imax(choline) after MMTS], and the activation ratios of the individual oocytes were measured. C: Imax(choline) values and activation ratios observed after treatment with MMTS in oocytes expressing rOCT1 mutants in which two cysteine cyteine residues were replaced. Mean values ± SE are presented. The numbers of analyzed oocytes are given in parenthesis. The data suggest that the increase of Imax(choline) in rOCT1 WT after treatment with MMTS is mainly due to modification of cysteines 322 and 451. *P < 0.05, **P < 0.01, ***P < 0.001 difference from rOCT1 WT. {circ}P < 0.05, {circ}{circ}{circ}P < 0.001 effect of MMTS. bulletbulletP < 0.01, bulletbulletbulletP < 0.001 difference from C451M.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of MMTS on substrate induced currents of organic cation transporters. When oocytes expressing rOCT1 were incubated for 30 s with 5 mM MMTS, inward currents induced by superfusion with 10 mM choline at –50 mV were increased from –18 ± 13 to –107 ± 82 nA (n = 46, P < 0.001, activation factor 7.6 ± 3.7). A similar activation was obtained after incubation with 500 µM MMTS (Fig. 1A). MMTS also activated outward currents that were induced by choline efflux (Fig. 1B). For efflux measurements, rOCT1-expressing oocytes were preloaded with 10 mM choline, superfused with 10 mM choline, clamped to –50 mV, and outward currents after superfusion without choline were measured. Choline-induced outward currents before and after incubation with MMTS were 75 ± 32 and 197 ± 143 nA, respectively (n = 5, P < 0.05, activation factor of 2.5 ± 1.2). The data show that choline-induced inward and outward currents were increased by MMTS; however, the inward current was increased more strongly (P < 0.001).

We also investigated effects of 5 mM MMTS on choline (10 mM)-induced inward currents expressed by OCT transporter orthologs and paralogs (Table 1). MMTS increased the choline-induced inward currents [Imax(choline)] of mouse OCT1 (mOCT1) and human OCT1 (hOCT1) but decreased the choline-induced inward and outward currents of rat OCT2 (rOCT2). At variance, no significant effect of MMTS on the choline-induced currents expressed by rat OCT3 (rOCT3) and human OCT2 (hOCT2) was observed. Choline-induced outward currents in rOCT2-expressing oocytes (preloaded with 10 mM choline, superfused with 10 mM choline, clamped to –50 mV, and superfused without choline) were 160 ± 56 nA without MMTS and 56 ± 12 nA after 30-s superfusion with 5 mM MMTS (n = 5, P < 0.01, activation factor 0.35 ± 0.10). Figure 1 shows current traces before and after incubation of oocytes expressing rOCT2 with 500 µM MMTS.


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  		Table 1. Subtype and species dependence of MMTS effects on choline-induced inward currents expressed by organic cation transporters

 
In Fig. 2, we compared time courses for MMTS activation of rOCT1-mediated currents induced by choline and for MMTS inhibition of rOCT2-mediated currents induced by choline. The half-times for activation of rOCT1 and inactivation of rOCT2 by 50 µM MMTS were ~5 min. They were concentration dependent, being smaller after incubation with higher concentrations of MMTS (data not shown). The effects of MMTS appeared to be irreversible because they persisted after the oocytes were washed for 1 h with Ori buffer (data not shown).


Figure 2
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  		Fig. 2. Time courses of rOCT1 activation and rOCT2 inactivation by MMTS. Oocytes expressing rOCT1 or rOCT2 were superfused with Ori buffer and clamped to –50 mV. For periods of 10–20 s, 10 mM choline was added and the induced current was measured. After addition of 50 µM MMTS to the superfusion buffers, changes in inward currents induced by rOCT1 and rOCT2 were measured. The data indicate similar time courses for activation of rOCT1 and inactivation of rOCT2.

 
Effects of MMTS on organic cation affinity of rOCT1 and rOCT2. Oocytes expressing rOCT1 or rOCT2 were clamped to –50 mV, and choline activation was measured before and after 30-s incubation with 5 mM MMTS (Fig. 3). Half-maximal activation concentrations [I0.5(choline) values] increased 5.4-fold from 0.29 ± 0.18 to 1.57 ± 0.38 mM for rOCT1 (Table 2) and 4.2-fold from 0.38 ± 0.14 to 1.59 ± 0.57 mM for rOCT2 (n = 7, P < 0.001).


Figure 3
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  		Fig. 3. Effects of MMTS on substrate activation of choline-induced currents expressed by rOCT1 or rOCT2. Choline-induced inward currents were measured with oocytes expressing rOCT1 or rOCT2 that were clamped to –50 mV. In each oocyte, the choline-induced currents were first measured before MMTS treatment (open symbols). Then, the oocytes were incubated for 30 s with 5 mM MMTS and superfused for several minutes with Ori buffer. Then, choline-induced currents were measured again (filled symbols). For the measurements, oocytes were superfused for 30-s time periods with increasing concentrations of choline. After each choline pulse, the oocytes were superfused for 1–2 min with Ori buffer to allow the release of choline that had entered the oocytes. Representative experiments of 6 are shown. The data show that MMTS increases the choline-induced inward current (Imax) value of rOCT1 and decreases the Imax value of rOCT2 whereas it decreases the affinities of both transporters.

 

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  		Table 2. Effects of MMTS on activation of rOCT1-expressed currents by different substrates

 
In Table 2, we measured inward currents that were induced by various concentrations of TEA and guanidine before and after 30-s incubation of rOCT1-expressing oocytes with 5 mM MMTS. MMTS increased the currents at saturating concentrations of choline, TEA, and guanidine (Imax values) by similar factors. At variance, MMTS increased the I0.5 values by different degrees. The ratios between Imax and the I0.5 representing the efficacies of expressed transport were not changed significantly for choline but increased 4.7-fold for TEA and 3.5-fold for guanidine. The data show that MMTS changes the selectivity for cation transport of rOCT1-expressing oocytes.

Effect of MMTS on the charge-to-substrate ratio during TEA transport by rOCT1 and rOCT2. To determine whether MMTS-induced modifications change the stoichiometry between transport of cations and translocation of positive charges, we performed parallel measurements of [14C]TEA-induced electrical currents and [14C]TEA uptake. Untreated oocytes or MMTS-incubated oocytes (30 s, 5 mM MMTS) were superfused with Ori buffer, clamped to –50 mV, and superfused for 2 min with Ori buffer containing 500 µM [14C]TEA. The uptake was stopped by superfusion with Ori buffer containing 100 µM nontransported inhibitor tetrabutylammonium (35), preventing efflux of TEA. For technical reasons, we performed these experiments with three batches of rOCT1-expressing oocytes that exhibited a three to four times higher TEA uptake rate as observed routinely. These oocytes exhibited a less pronounced stimulation of cation transport by MMTS than normally. Uptake of 500 µM [14C]TEA in rOCT1-expressing oocytes (in pmol·oocyte–1·min–1 corrected for TEA uptake in control oocytes) was 8.5 ± 1.6 (without MMTS, n = 9) and 24.7 ± 6.9 (after incubation with MMTS, n = 8). In oocytes expressing rOCT2, uptake of 500 µM [14C]TEA was 19.7 ± 2.6 (n = 7) without MMTS treatment and 7.9 ± 2.0 (n = 10) after incubation with MMTS. Without MMTS incubation, the stoichiometries between translocated positive charge and TEA were 1.02 ± 0.18 for rOCT1 and 1.02 ± 0.09 for rOCT2. After MMTS incubation, the stoichiometries were 1.26 ± 0.15 (rOCT1) and 1.15 ± 0.21 (rOCT2), indicating no pronounced effect of MMTS on stoichiometry.

Effects of MMTS on choline-induced capacitance changes by rOCT1 and rOCT2. In rOCT2-expressing oocytes, we previously observed that transported cations, the nontransported cationic inhibitor tetrabutylammonium, and the nontransported uncharged inhibitor corticosterone induced a decrease in membrane capacitance (28, 29). These capacitance changes are supposed to reflect block by ligands of voltage-dependent charge movements within rOCT2. Figure 4 shows typical experiments in which choline-induced capacitance changes at –50 mV were measured in oocytes expressing rOCT1 or rOCT2 before and after treatment with MMTS. Choline concentrations inducing half-maximal capacitance changes [C0.5(choline)] were 0.091 ± 0.025 mM (rOCT1) and 0.195 ± 0.043 mM (rOCT2, n = 3, each). These C0.5(choline) values are not significantly different from the I0.5(choline) values estimated from the concentration dependence of the choline-induced currents. After treatment of rOCT1 expressing oocytes with MMTS, the C0.5(choline) value was increased to 0.76 ± 0.21 mM (n = 3; P < 0.01 for the difference). After MMTS treatment of oocytes expressing rOCT2, for choline-induced capacitance changes a similar C0.5(choline) value (0.35 ± 0.06 mM, n = 3) was measured as in untreated oocytes. This is at variance to choline-induced currents by rOCT2, where MMTS increased the I0.5(choline) for choline activation. The data suggest that choline-induced currents and choline-induced capacitance changes monitor different steps of the transport cycle after substrate binding. Choline-induced currents represent the entire transport cycle, whereas choline-induced capacitance changes may be due to substrate binding and/or a subsequent conformational change that includes movement of electrical charge.


Figure 4
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  		Fig. 4. Effects of MMTS on concentration activation curves of choline-induced capacitance changes. Oocytes expressing rOCT1 or rOCT2 clamped to –50 mV were superfused with different concentrations of choline and the decrease in capacitance by choline [Cmax(choline)] was measured (open symbols). Thereafter, the oocytes were incubated for 30 s with 5 mM MMTS, washed with Ori buffer, and the choline-induced capacitance decrease was measured again (filled symbols). The superfusion with choline was performed as in Fig. 3. Representative experiments of 3 are shown. The data show that MMTS increases the Cmax(choline) value of rOCT1, decreases the Cmax(choline) value of rOCT2 and increases the half-maximal concentrations of choline-induced capacitance changes in oocytes expressing rOCT1.

 
We also compared the effects of MMTS treatment (30 s, 5 mM MMTS) on changes in choline (10 mM choline, –50 mV)-induced currents [Imax(choline)] and capacitance [Cmax(choline)]. Imax(choline) reflects the number of functional active transporter molecules within the plasma membrane and the turnover number of individual transporter molecules. At variance, Cmax(choline) reflects the number of transporter molecules with mobile charges within the electrical field of the plasma membrane as well as the mobility of charges within the transporter. In rOCT1-expressing oocytes, Imax(choline) was 18 ± 13 nA (n = 46) and Cmax(choline) was 1.1 ± 0.9 nF (n = 14) (Table 3). With MMTS treatment, Imax(choline) increased by a factor of 7.6 ± 3.7 whereas Cmax(choline) increased by a factor of 2.9 ± 1.6.


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  		Table 3. Effects of MMTS on choline induced currents and choline induced capacitance changes in oocytes expressing rOCT1 wild-type and mutants of rOCT1

 
In rOCT2-expressing oocytes, Imax(choline) was 208 ± 82 nA and Cmax(choline) was 4.3 ± 1.6 nF (current and capacitance measurements in same oocytes, n = 13). In rOCT2, MMTS decreased choline-induced current and choline-induced capacitance by the same degree (current, factor of 0.42 ± 0.21; capacitance, factor of 0.42 ± 0.19, n = 13). The ratios between Imax(choline) and Cmax(choline) (in nA/nF) were 36 ± 25 (n = 14, rOCT1 without MMTS), 94 ± 49 (n = 19, rOCT1 after MMTS), 59 ± 6 (n = 13, rOCT2 without MMTS), and 60 ± 11 (n = 13, rOCT2 after MMTS). In rOCT1-expressing oocytes, MMTS treatment increased this ratio significantly (P < 0.001). This indicates that MMTS exhibits differential effects on individual steps of the transport cycle by rOCT1 vs. rOCT2.

Effects of MMTS on membrane capacitance in oocytes expressing rOCT1 and rOCT2 in the absence of substrates. When 200 µM MMTS was added for 50 s to the bath of H2O-injected oocytes, no significant change in membrane capacitance was observed (0.26 ± 0.51 nF, n = 7) (Fig. 5). In rOCT1-expressing oocytes, MMTS increased the capacitance by 1.2 ± 0.9 nF (n = 9, P < 0.05) whereas in rOCT2-expressing oocytes MMTS decreased the capacitance by 1.0 ± 0.3 nF (n = 5, P < 0.001) (Fig. 5). The data indicate transporter-specific effects of MMTS on capacitance. This can be explained in two ways; either MMTS exhibits a transporter-specific effect on the equilibrium between transporter molecules within the plasma membrane and vesicles below the plasma membrane or MMTS induces transporter-specific structural changes during which the number of mobile charges within the electrical field of the plasma membrane is changed differently in rOCT1 vs. rOCT2.

MMTS does not change the amount of rOCT1 protein in the plasma membrane. We determined the amount of rOCT1 within the plasma membrane before and after MMTS treatment. For immunochemical quantification, an rOCT1 mutant was used that contained the FLAG-tag sequence at the COOH terminus. Uptake rates and currents induced by superfusion with choline in oocytes expressing the FLAG-tagged rOCT1 mutant were similar to rOCT1 wild-type (data not shown). In three independent experiments, the FLAG-tagged rOCT1 mutant was expressed in oocytes, inward currents induced by superfusion with 10 mM choline (–50 mV) were measured, and plasma membranes of some oocytes were purified by adsorption to colloidal silica (13). The remaining oocytes were treated with MMTS (30 s, 5 mM), washed, choline-induced inward were measured again, and the plasma membranes were purified. In these experiments, MMTS increased Imax(choline) between four- and fivefold. The Western blots in Fig. 6 were developed with an antibody against the FLAG-tag. The relationship between staining intensity for rOCT1 before and after MMTS was 0.99 ± 0.10, indicating that MMTS did not change the amount of rOCT1 in the plasma membrane.


Figure 6
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  		Fig. 6. Quantification of rOCT1 within the plasma membrane before and after treatment with MMTS. In 3 independent experiments, plasma membranes were isolated from 8 oocytes expressing rOCT1 before treatment with MMTS and from 8 oocytes after 30-s incubation with 5 mM MMTS. By MMTS treatment, inward currents measured after superfusion of the oocytes with 10 mM choline at –50 mV were increased 4- to 5-fold. The amount of isolated plasma membrane protein was not significantly altered after MMTS treatment. Proteins of the individual plasma membrane preparations were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and stained with an antibody against the FLAG-tag. Two micrograms of protein were applied per lane. The data indicate that MMTS does not alter the amount of rOCT1 protein in the plasma membrane.

 
MMTS does not alter exocytosis of rOCT1-containing vesicles. In rOCT1-expressing oocytes, we investigated whether block of exocytosis by botulinum toxin B (BTB) or pretreatment of the oocytes with brefeldin A (BFA) changed the increase in choline-induced inward currents by MMTS. BTB inhibits the fusion of intracellular vesicles with the plasma membrane (32) whereas BFA blocks the release of vesicles from the trans-Golgi network (TGN) (17). When 2 ng of BTB/oocyte were injected into oocytes expressing rOCT1 and the oocytes were incubated for 30 min, the inward current induced by 10 mM choline at –50 mV [Imax(choline)] was inhibited by 34 ± 11% (Fig. 7A, n = 5, each, P < 0.05). This indicates that BTB was effective. Importantly, the effect of MMTS on Imax(choline) was not changed. By incubation with MMTS, Imax(choline) was increased 9.2 ± 1.3- and 8.9 ± 2.3-fold before and after injection of BTB, respectively (Fig. 7A, n = 5, each). To test the effect of BFA on the activation of rOCT1 by MMTS, rOCT1-expressing oocytes were incubated for 3 h with 5 µg x ml–1 BFA (17, 33) or with solvent (controls), and Imax(choline) was measured. The oocytes were incubated with MMTS, washed, and Imax(choline) values were measured again (Fig. 7B). Incubation of rOCT1-expressing oocytes with BFA led to a reduction of choline-induced currents by 56 ± 1.0% (n = 6, each, P < 0.01). This indicates that BFA was effective. The activation of Imax(choline) values by MMTS was not changed by BFA. MMTS increased choline-induced currents 8.9 ± 1.0-fold in controls and 9.6 ± 1.9-fold in BFA-treated oocytes (Fig. 7B, n = 6, each). The data indicate that MMTS does not accelerate exocytosis of rOCT1-containing vesicles.


Figure 7
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  		Fig. 7. Effects of MMTS on rOCT1 activity after inhibition of exocytosis. Oocytes expressing rOCT1 were injected either with 2 ng of botulinum toxin B (BTB) per oocyte and incubated for 30 min (A) or incubated for 3 h with 5 µg x ml–1 BFA (B). Thereafter, oocytes were clamped to –50 mV, and currents induced by 10 mM choline were measured before and after 30-s incubation with 5 mM MMTS. Mean values ± SD from measurements of 5 or 6 oocytes from 2 different oocyte batches are shown. The data indicate that the effect of MMTS on Imax(choline) is independent of exocytosis. *P < 0.05, **P < 0.01, ***P < 0.001.

 
Identification of cysteine residues that are responsible for effects of MMTS on rOCT1. We exchanged each of the 16 cysteine residues in rOCT1 individually. The cysteine residues in loops were replaced by serine residues and those in TMHs by alanine residues. Some cysteines were replaced by each of the two amino acids. The cysteine replacement mutants were expressed in oocytes, and uptake of 10 µM [14C]TEA was measured. TEA uptake rates of at least 50% of rates observed with rOCT1 wild-type were obtained after mutation of cysteines 26, 155, 179, 322, 358, 418, 437, 470, or 474. After mutation of cysteine 451 to alanine or serine, TEA uptake was only ~20% compared with wild-type. No significant TEA uptake could be expressed when individual cysteine residues in the large extracellular loop were replaced. This observation is consistent with a recent report showing that mutation of consensus sequences for glycosylation in the large extracellular loop of rabbit OCT2 led to a failure of membrane insertion (26). We speculate that the six conserved SH-groups in the large loop of rOCT1 form disulfide bridges that stabilize the tertiary structure of the loop.

Figure 8 shows the effect of MMTS on the activity [Imax(choline)] of the cysteine mutants that could be expressed functionally [Fig. 8A: currents induced by 10 mM choline at –50 mV before incubation with MMTS; Fig. 8B: factors for activation of Imax(choliine) by MMTS]. Activation factors higher than 4 were observed for the mutants C26A, C155A, C179A, C358A, C418A, C437A, C437S, C470A, and 474A. Mutant C322S was activated 2.9-fold and mutants C451A and C451S not at all. Since <20% of wild-type activity was expressed when cysteine 451 was replaced with alanine or serine, the partial inactivation of rOCT1 by these mutations may prevent activation due to MMTS modification of another cysteine residue.

Trying to obtain a more active C451 mutant, we replaced cysteine 451 with methionine. Methionine was selected because rOCT1 was highly active after modification with MMTS and the rest of methionine (-CH-CH2-S-CH3) has similarity to the rest of a MMTS-modified cysteine (-CH2-S-S-CH3). In fact, about five times higher activity could be expressed with the C451M mutant compared with the C451S mutant (data not shown). The I0.5(choline) value for mutant rOCT1(C451M) i.e., 0.21 ± 0.04 mM, n = 5, was similar to rOCT1 wild-type (Table 3).

After incubation of rOCT1(C451M) with MMTS, the inward current induced by 10 mM choline at –50 mV [Imax(choline)] was increased by a factor of 2.1 ± 0.6 (Table 3). This activation was significantly less than in rOCT1 wild-type. At variance with rOCT1 wild-type, MMTS did not increase the I0.5(choline) in rOCT1(C451M) (Table 3). The data indicate that replacement of cysteine 451 in rOCT1 only partially mimics the functional effects observed after modification of rOCT1 wild-type by MMTS.

We tested whether a second cysteine residue in rOCT1 contributes to the observed effects of MMTS on Imax(choline) and I0.5(choline) of rOCT1 wild-type. We prepared double mutants containing C451M plus cysteine replacement mutations in which the activation of Imax(choline) by MMTS was smaller than in the wild-type (Fig. 8C). After expression in oocytes, Imax(choline) values were obtained that were similar to rOCT1 (C451M) with the exception of double mutant C451M,C322S, which showed a significantly higher Imax(choline) value. Significant activation of Imax(choline) by MMTS was observed with double mutants C451M plus C179A, C451M plus C358A, and C451M plus C418A but not with double mutant C451M plus C322S (Fig. 8C).

For activation of choline-induced inward currents in oocytes expressing rOCT1(C322S), a I0.5(choline) value of 0.40 ± 0.14 mM (n = 4) was determined. After treatment of rOCT1(C322S) with MMTS, the I0.5(choline) value was increased twofold to 0.78 ± 0.10 mM (Table 3). After expression of rOCT1(C322S, C451M) in oocytes, a I0.5(choline) value of 1.31 ± 0.40 mM (n = 8) was obtained, which was significantly higher than in wild-type, rOCT1(C451M), and rOCT1(C322S) (P < 0.001). The finding that the combined mutation of C322S and C451M in rOCT1 resulted in a three- to fourfold decrease in affinity for choline compared with wild-type whereas the affinities for choline to rOCT1(C322S) and rOCT1(C451M) were similar to wild-type (Table 3) suggests neighboring localizations or allosteric interaction between cysteines in positions 322 and 451. After modification of rOCT1(C322S, C451M) with MMTS, no significant increases in Imax(choline) or I0.5(choline) were observed (Fig. 8, Table 3). The I0.5(choline) measured for MMTS-modified rOCT1(C322S, C451M) was 1.71 ± 0.48 mM (n = 8). The data suggest that the functional changes in rOCT1 wild-type observed after modification with MMTS are due to modifications of cysteines 322 and 451.

Role of cysteine residues 451 and 322 in MMTS effects on cysteine-depleted rOCT1 mutants. We exchanged 1) all cysteine residues with the exception of those in the extracellular loop [rOCT1(10{Delta}C)], 2) all cysteine residues with the exception of those in the extracellular loop and of cysteine residues 322 and 451 [rOCT1(8{Delta}C)], or 3) all cysteine residues with the exception of those in the extracellular loop and cysteine 451 [rOCT1(9{Delta}C)]. In oocytes expressing rOCT1(10{Delta}C), the Imax(choline) values were about eight times higher compared with oocytes expressing rOCT1 wild-type (Table 3). For I0.5(choline) of rOCT1(10{Delta}C), a value of 1.41 ± 0.24 mM was obtained that was similar to rOCT1(C322S, C451M) (Table 3). MMTS had no significant effect on Imax(choline) and I0.5(choline) of rOCT1(10{Delta}C) (Table 3). In summary, the functional properties of rOCT1(10{Delta}C) were similar to those of rOCT1(C322S, C451M) except that Imax(choline) was higher.

In oocytes expressing rOCT1(8{Delta}C), a mutant containing cysteine residues 322 and 451, Imax(choline) and I0.5(choline) were similar to rOCT1 wild-type (Table 3). MMTS increased the Imax(choline) 2.9-fold and I0.5(choline) 1.8-fold (Table 3). In oocytes expressing rOCT1(9{Delta}C), a mutant containing cysteine 451, I0.5(choline) was similar to rOCT1 wild-type and rOCT1(8{Delta}C). MMTS increased the Imax(choline) 1.8-fold but had no significant effect on the I0.5(choline) (Table 3). The MMTS-induced increase in Imax was significantly smaller in rOCT1(9{Delta}C) than in rOCT1(8{Delta}C). The data indicate that cysteine 451 is critical for the affinity of choline [I0.5(choline) of rOCT1(9{Delta}C) < I0.5(choline) of rOCT1(10{Delta}C)] and support the interpretation that the MMTS-induced increase in Imax(choline) in rOCT1 wild-type is due to cysteine modification of C322 and C451 [MMTS activation of Imax(choline): rOCT1(8{Delta}C) > rOCT1(9{Delta}C), no activation of rOCT1(10{Delta}C)].

Parallel measurements of choline (10 mM)-induced capacitance changes [Cmax(choline)] and choline-induced inward currents [Imax(choline)] (Table 3) revealed a significantly higher ratio between Imax(choline) and Cmax(choline) (in nA/nF) for rOCT1(10{Delta}C), 112 ± 9, compared with rOCT1 wild-type, rOCT1(9{Delta}C), or rOCT1(8{Delta}C), 36 ± 25, 56 ± 12, and 59 ± 14, respectively (P < 0.01) (Table 3). Because Imax reflects transport rate whereas choline-induced capacitance changes reflect the number of transporters in the membrane, the increased Imax(choline)/Cmax(choline) ratio after mutation of cysteines 322 and 451 [rOCT1(10{Delta}C)] suggests an increased turnover number. MMTS increased the Imax(choline)/Cmax(choline) ratios for rOCT1 wild-type, rOCT1(8{Delta}C), and rOCT1(9{Delta}C) by 2.6-, 1.9-, and 1.6-fold, respectively, but had no effect on rOCT1(10{Delta}C). This suggests that the increase in turnover number of rOCT1 was due to MMTS modification of cysteines C322 and C451.

Effects of MMTS on transport by rOCT2 mutant in which cysteine 451 was exchanged by methionine. In oocytes expressing rOCT2(C451M), transport activity was measured that was reduced with borderline significance compared with wild-type (data not shown). Thirty-second incubation with 5 mM MMTS reduced the Imax(choline) for rOCT2 by 70 ± 17% (n = 21) but had no effect on rOCT2(C451M) (activation factor 0.98 ± 0.09, n = 17). The I0.5(choline) for rOCT2(C451M) was significantly higher than for wild-type [0.62 ± 0.04 mM (n = 6) vs. 0.38 ± 0.14 (n = 7) P < 0.05] but was not altered by treatment with MMTS (0.79 ± 0.11 mM, n = 6). The data suggest that the effects of MMTS on activity and affinity of rOCT2 are due to modification of cysteine 451, and that cysteine 451 is critical for the affinity of choline for rOCT2.

Effect of MMTS on purified and reconstituted rOCT1 protein. Previously, we showed that rOCT1-mediated MPP uptake into the proteoliposomes is potential dependent and saturable, with an apparent Km value of 30 µM (15). Here we investigated the effect of MMTS on MPP uptake into proteoliposomes containing purified rOCT1 protein (Fig. 9). After incubation of the proteoliposomes for 1 min with 1 mM MMTS, we obtained an apparent Km of 37 ± 12 µM, indicating that MMTS does not change the affinity for MPP. Incubation of the proteoliposomes for 1 min with 0.1 or 1 mM MMTS increased the MPP uptake rates of 0.1 µM [3H]MPP or 250 µM [3H]MPP by 60–70% (P < 0.001). The data indicate that modification of rOCT1 by MMTS increases the Vmax and that this effect does not require regulation or accessory proteins. The different degrees of activation by MMTS observed in proteoliposomes and oocytes may be explained by different conformational states of rOCT1 or by modulatory functions of additional proteins in oocytes.


Figure 9
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  		Fig. 9. Increase in transport activity of purified and reconstituted rOCT1 protein after modification with MMTS. rOCT1 was expressed in Sf9 cells, purified, and reconstituted into proteoliposomes containing 100 mM potassium cyclamate. The proteoliposomes were incubated for 1 min in the absence of MMTS, with 0.1 mM MMTS, or with 1 mM MMTS. After 10-fold dilution uptake of 0.1 µM [3H]MPP (open columns) or 250 µM [3H]MPP (shaded columns) was measured in the presence of valinomycin. Proteoliposomes were incubated for 1 s with [3H]MPP in the presence of 90 mM sodium cyclamate and 10 mM potassium cyclamate. Uptake measurements were performed in the absence and presence of 100 µM quinine. Mean values ± SD of quinine-inhibited MPP uptake from 4 parallel measurements are shown. The data indicate an increase of Vmax of MPP uptake in proteoliposomes containing purified rOCT1. ***P < 0.001 for effect of MMTS on transport activity.

 
Cysteine 451 in rOCT1 is critical for affinity of choline to inhibit MPP uptake. We expressed rOCT1 wild-type, rOCT1(C322S), rOCT1(C451M), or rOCT1 (C322S, C451M) in oocytes and measured the uptake of 0.1 µM [3H]MPP in the presence of various concentrations of choline. From individual fits of the Hill equation to three independent experiments, we determined the following IC50(choline) values (means ± SD): rOCT1 wild-type 0.41 ± 0.19 mM, rOCT1(C322S) 0.64 ± 0.07 mM, rOCT1(C451M) 2.02 ± 0.91 mM, and rOCT1(C322S, C451M) 1.91 ± 0.62 mM. Compared with rOCT1 wild-type, the IC50(choline) values were about five times higher in rOCT1(C451M) and rOCT1(C322S, C451M) (P < 0.05 for difference). At variance, no significant difference of IC50(choline) was detected between rOCT1(C322S) and rOCT1 wild-type. Note the difference between tracer uptake and current measurements; the IC50(choline) value for choline inhibition of MPP uptake in rOCT1(C451M) was higher compared with rOCT1 wild-type whereas the I0.5(choline) values for choline-induced currents were similar (Table 3). This will be discussed below.


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This study shows that cation-induced currents by rOCT1 can be increased within seconds by modification of cysteines 322 and 451 with the lipid-permeant sulfhydryl reagent MMTS, whereas cation-induced currents by rOCT2 are decreased by modification of cysteine 451. Our data suggest that the stimulation of rOCT1 is due to an increase in transport activity within the membrane. We also provide evidence that cysteines 451 in rOCT1 and rOCT2 are critical for the affinity of choline. In addition, data are presented that indicate an interaction between cysteine 451 of rOCT1 with the large intracellular loop that has an effect on the affinity for choline.

When oocytes expressing rOCT1 were incubated for 30 s with 5 mM MMTS and washed for 1 or 2 min, the expressed uptake of organic cations was increased four- to ninefold. The increase in rOCT1-mediated uptake stayed after prolonged washing and did not lead to a significant increase in rOCT1 protein within the plasma membrane. This indicates that MMTS changes the transport activity of rOCT1 rather than the posttranscriptional regulation of rOCT1 expression. This interpretation was validated by the findings 1) that the activation of rOCT1-expressed cation transport by MMTS was not altered when exocytosis was blocked by BTB and 2) that MMTS also increased the Vmax of organic cation transport in proteoliposomes containing purified rOCT1.

The effects of MMTS on the function of rOCT1 expressed in oocytes were investigated in detail. We showed that the activation of rOCT1 by MMTS changed the substrate selectivity. It increased the I0.5(choline) about fivefold whereas it only slightly changed the half-maximal activation of currents that were induced by TEA or guanidine (see Table 2). At variance, the MMTS-mediated activation of currents induced by saturating substrate concentrations (Imax) was identical using choline, TEA, or guanidine as a substrate. For rOCT1-mediated transport of TEA, we showed that the stoichiometry between the number of translocated positive charges and the number of translocated TEA molecules was not increased by >30% after modification of rOCT1 with MMTS. This means that the observed increase in cation-mediated currents induced by MMTS cannot be due to an increase in ion slippage or to an increased flux of ions that are cotransported or antiported during substrate translocation. Taken together, the data suggest that MMTS increases the turnover of individual rOCT1 molecules within the plasma membrane; however, it is also possible that MMTS activates silent transporter molecules in the plasma membrane. The observation that the ratio between Imax(choline) and Cmax(choline) in rOCT1 was increased after MMTS treatment (see Table 3) is consistent with both interpretations.

The increase in turnover or activation of silent transporters by MMTS could be correlated with changes in the quaternary structure of rOCT1. Hong and coworkers (12) presented data that strongly suggest that human organic anion transporter OAT1, which is a member of the SLC22 family like rOCT1, forms homooligomers in the plasma membrane. Recently, we observed that purified OAT1 protein from rat (rOAT1) as well as purified rOCT1 protein form homooligomers whereas heterooligomerization could not be detected between rOAT1 and rOCT1 (T. Keller, F. Bernhard, C. Hunte, V. Gorboulev and H. Koepsell, unpublished observations). Since the homooligomerization of purified rOCT1 protein was also observed after modification of rOCT1 with MMTS, the effects of MMTS on the function of rOCT1 may not be due to an inhibition of oligomerization. Of course, these data do not exclude that modification of rOCT1 by MMTS induces subtle changes concerning the interaction of OCT1 monomers that lead to an activation of the complex.

Performing mutagenesis experiments in which cysteine residues of rOCT1 were replaced individually or in combinations, we demonstrated that the MMTS modification of two cysteine residues in rOCT1, cysteine 322 and cysteine 451, is responsible for the functional changes observed after modification of rOCT1 wild-type. First, the activation by MMTS was reduced when cysteine 322 was replaced by serine or when cysteine 451 was replaced by methionine, and the activation by MMTS was completely abolished when both cysteine residues were exchanged. Second, the effect of MMTS on the I0.5 value for choline-induced currents (5-fold increase) was abolished when cysteine 451 was replaced by methionine.

Functional characterization of the unmodified and MMTS-modified cysteine replacement mutants revealed that cysteine 451 is critical for cation affinity and substrate-induced ion conductivity. In addition, data were obtained that suggest interaction between the large intracellular loop and cysteine 451. The critical role of cysteine 451 for cation affinity of rOCT1 was shown as follows. First, in oocytes expressing rOCT1(C451M), I0.5(choline) was not influenced by MMTS whereas it was increased fivefold in oocytes expressing rOCT1 wild-type or rOCT1(C322S) that contains cysteine 451 (Table 3). Second, in oocytes expressing rOCT1(10{Delta}C), I0.5(choline) was not changed by MMTS whereas it was increased fourfold in oocytes expressing rOCT1(9{Delta}C) which contains cysteine 451 (Table 3). Interestingly, the same I0.5(choline) value of 1.4 mM was obtained for oocytes expressing rOCT1(9{Delta}C) that were modified with MMTS and for oocytes expressing rOCT1(10{Delta}C) where cysteine 451 is replaced by methionine. As mentioned above, there is only a small difference between the amino acid side chains of methionine (-CH2-CH2-S-CH3) and of MMTS-modified cysteine (-CH2-S-S-CH3).

The functional importance of cysteine 451 for substrate affinity of organic cation transporters of the SLC22 family is also indicated by the observation that 1) MMTS treatment increased I0.5(choline) in oocytes expressing rOCT2 whereas no effects of MMTS were observed when cysteine 451 was replaced by methionine and that 2) I0.5(choline) was significantly higher in rOCT2(C451M) compared with rOCT2 wild-type.

The observations that in rOCT1 neither the mutation of cysteine 322 to serine nor the mutation of cysteine 451 to methionine changed the I0.5(choline) value whereas the I0.5(choline) value was increased four- to fivefold when cysteines 322 and 451 were exchanged together (see Table 3), suggesting an interaction of the large intracellular loop with cysteine 451. In rOCT1, cysteines 322 and 451 do not form a disulfide bridge because these residues could be modified with MMTS. We speculate that the large intracellular loop with cysteine 322 between TMHs 6 and 7 comes into contact with cysteine 451. There may be functionally relevant structural differences between the environment of cysteine 451 in organic cation transporter orthologs and paralogs. Notably, stimulation by MMTS was observed for rOCT1, mOCT1, and hOCT1 although hOCT1 does not contain a cysteine residue at position 322. In addition, rOCT2 but not hOCT2 was inhibited by MMTS although both transporters contain a cysteine residue in position 451 and no cysteine in position 322.

Cysteine 451 is located at the transition of TMH 10 to the short cytosolic loop that connects TMHs 10 and 11 (Fig. 10). Recently, it was shown that cysteine 451 in human OCT2 was accessible to modification by HgCl2 that was added to the extracellular side of the plasma membrane (25). The authors concluded that cysteine 451 resides in the aqueous milieu. Note that cysteine 451 in rOCT1 is located close to glutamine 448 in TMH 10. Glutamine 448, leucine 447, and alanine 443 in TMH 10 belong to the binding domain of corticosterone that has been shown to be part of the substrate binding region of rOCT1 (7). Interestingly, cysteine 451 and the nine following COOH-terminal amino acids are largely conserved in the organic cation transporters, and the last five of these (ELYPT) are conserved throughout most members of the SLC22 family (20). For the sodium-carnitine cotransporter SLC22A5, data have been presented that suggest that a glutamate residue located five amino acids COOH terminal to cysteine 451 is involved in coupling between binding of sodium and translocation of carnitine (36, 37).


Figure 10
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  		Fig. 10. Structural models of rOCT1. Top: 2-dimensional model of membrane topology. Bottom: model of the tertiary structure that has been derived from the crystal structures of two transporters of the major facilitator superfamily (MFS; side view). Amino acids that have been previously localized within the substrate binding region and the two cysteine residues characterized in the present manuscript are indicated. Transmembrane {alpha}-helix (TMH) 4: tryptophane 218, tyrosine 222 and threonine 226 (grey); TMH 11: aspartate 475 (red); TMH 10: alanine 443 (cyan blue), leucine 447 (magenta), glutamine 448 (blue), cysteine 451 (yellow); large intracellular loop: cysteine 322 (yellow).

 
The functional data of the present paper together with the proposed localization of cysteine 451 in the tertiary structure of rOCT1 suggest that cysteine 451 is involved in recognition of organic cations and participates in structural changes that occur during translocation of organic cations. According to the modeled inward-facing conformation of rOCT1 (27), cysteine 451 is located at the rim of an inward open cleft containing the cation binding region. Since cysteine 451 is located at the beginning of the short intracellular loop connecting TMHs 10 and 11, and these TMHs contain amino acids that are involved in cation binding (7, 9), it can be easily imagined that modification or mutation of cysteine 451 changes both, affinity for cations and transport activity. Applying the putative rearrangement mechanism for lactose transport of LacY permease as proposed by Abramson et al. (1, 2) to our model of the inward-facing conformation of rOCT1, we also modeled its outward-facing conformation (D. Gorbunov, V. Gorboulev, N. Shatskaya, T. Müller, E. Bamberg, F. Friedrich; and H. Koepsell, unpublished observations). In this model, the orientations of TMHs 2, 4, 10, and 11 were largely different compared with the inward-facing conformation shown in Fig. 10. In the outward-facing conformation, the cytosolic thirds of TMHs 2, 4, 10, and 11 were close together. We know that these models are speculative; however, they provide reasonable working hypotheses and tentative explanations for functional effects of mutations. We speculate that cysteine 451 is part of the choline binding domain and is important for orientational changes in TMHs 10 and 11 during the transport cycle. This could explain why modification of cysteine 451 by MMTS or its exchange by methionine decreases the affinity for choline and may increase turnover. Concerning the effects on choline affinity, we cannot exclude that modification or mutation of cysteine 451 alters the position of TMH 10 and/or TMH 11 and thereby reduces the affinity for choline at a site distant from cysteine 451.

The observation that the rOCT1(C451M) mutant has an increased IC50(choline) whereas the I0.5(choline) for choline-induced current was not changed may be explained in two ways. It is possible that in the rOCT1(C451M) mutant at variance to rOCT1 wild-type, the affinity for choline is highly dependent on membrane potential. Since the I0.5(choline) was measured with a membrane potential clamped to –50 mV whereas the IC50 was obtained from transport measurements in nonclamped oocytes, electrical and tracer uptake experiments may have been performed at different membrane potentials. The second possibility is that the Cys451Met exchange generated or increased an allosteric interaction between the binding sites for MPP and choline, leading to decreased affinity for choline when inhibition of MPP uptake by choline was measured.

In summary, we presented data that highlight a key position of cysteine 451 in organic cation transporters for affinity and selectivity of cations as well as for translocation. A further functional characterization of cysteine 451 mutants and further modeling of tertiary structures may refine the present picture; however, crystallization of different conformations of rOCT1, of the rOCT1(C451M) mutant, of double mutant rOCT1(C322S/C451M), and of other mutants in which key positions for transporter function are modified will be necessary to understand how these polyspecific transporters work.


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This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB 487/A4.


   ACKNOWLEDGMENTS
 
The figures were prepared by Michael Christof.


   FOOTNOTES
 

Addresses for reprint requests and other correspondence: H. Koepsell, Institute of Anatomy and Cell Biology, Koellikerstr. 6, 97070 Würzburg, Germany (e-mail: Hermann{at}Koepsell.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* A. Sturm and V. Gorboulev contributed equally to this work. Back


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 REFERENCES
 

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