Department of Pharmacology, Johannes Gutenberg University, 55101 Mainz, Germany
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ABSTRACT |
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Membrane potential and currents were investigated with the
two-electrode voltage-clamp technique in Xenopus laevis
oocytes expressing hCAT-2A or hCAT-2B, the splice variants of the human cationic amino acid transporter hCAT-2. Both hCAT-2A- and
hCAT-2B-expressing oocytes exhibited a negative extracellular
L-arginine concentration ([L-Arg]o)-sensitive membrane potential,
additive to the K+ diffusion potential, when cells were
incubated in Leibovitz medium (containing 1.45 mM L-Arg and
0.25 mM L-lysine). The two carrier proteins produced inward
and outward currents, which were dependent on the L-Arg
gradient and membrane potential. Ion substitution experiments showed
that the hCAT-induced currents were independent of external
Na+, K+, Ca2+, or Mg2+.
The apparent Michaelis-Menten constant values at 60 mV, obtained from
plots of L-Arg-induced currents against
[L-Arg]o, were 0.97 and 0.13 mM in oocytes
expressing hCAT-2A and hCAT-2B, respectively; maximal currents
amounted to
194 ± 8 and
84 ± 2 nA, respectively. At
saturating [L-Arg]o, the current-voltage
relationships of hCAT-2A-expressing oocytes became steeper, yielding an
additional conductance up to 2 µS/oocyte, whereas those of
hCAT-2B-expressing oocytes were simply shifted to the right, resulting
in voltage-independent difference currents. The distinct
electrochemical properties of the two isoforms of hCAT-2 are assumed to
contribute differentially to the membrane transport and the maintenance
of cationic amino acids in various tissues.
amino acid transporter; membrane current; two-electrode voltage clamp
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INTRODUCTION |
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CATIONIC AMINO ACIDS (CAA) are transported through biological membranes by various distinct transport systems, characterized by differences in structure, substrate specificity, site of expression, and regulation (for a review, see Refs. 12 and 22). The discovery that nitric oxide (NO) plays an important role as a mediator of both regulatory and cytotoxic functions (21) puts L-arginine (L-Arg), the immediate precursor of NO, into a new interesting context. The transport of L-Arg might influence the availability of substrate for NO synthesis in different cell types (8). Five transport systems for CAA have been identified; these are b+, y+, b0,+, y+L , and B0,+. Only system b+, described in preimplantation mouse blastocysts (28), seems to be specific for CAA, whereas the other systems also accept neutral amino acids (NAA). Systems y+L and B0,+ exhibit higher affinities for NAA than for CAA (12, 29). They are preferentially expressed in epithelial cells, in which they play a major role in reabsorption and transepithelial transport of CAA. System y+ is found almost ubiquitously and has been described as selective for CAA (12).
The transport of CAA by system y+ is Na+ and pH independent and sensitive to transstimulation (5). In recent years, several proteins, involved in the transport of CAA, have been identified on the molecular level. At least three related proteins (CAT-1, CAT-2B, and CAT-3, for cationic amino acid transporter) exhibit system y+-like activity when expressed in Xenopus laevis oocytes (6, 14-16, 19, 30). A fourth member of the CAT family, CAT-2A, demonstrates a much lower substrate affinity and less dependence on transstimulation than system y+-like CATs (7, 18). The two isoforms, CAT-2A and CAT-2B, result from alternative splicing of primary transcripts from the same gene. They only differ in a short stretch of 42 amino acids, demonstrated to be responsible for the differences in the transport properties of the CAT proteins (10). CAT-4 has only recently been identified, and its transport activity has not yet been characterized (24). Except for CAT-4, the various CAT isoforms were originally identified from mouse (mCAT) and rat.
The cloning of a cDNA that encodes for the human homologue of CAT-1 (hCAT-1) has been reported by two groups (2, 31). More recently, cDNAs encoding for the human homologues hCAT-2A and hCAT-2B have been cloned, and the function of their protein products has been studied in cRNA-injected oocytes and compared with hCAT-1 (9). The latter study, in which L-Arg fluxes were determined by monitoring either the uptake or the release of radioactively labeled substrate, showed that the transport activities of the various transporters differ in their apparent substrate affinity, sensitivity to transstimulation, and maximal velocity. However, flux studies cannot take into account that the transport of charged amino acids is also determined by the membrane voltage (Em), as shown for mCAT-1 (17).
In this study, we aimed to clarify this point by the application of the two-electrode voltage-clamp method in Xenopus laevis oocytes, which were injected with the cRNAs of either hCAT-2A or hCAT-2B. This study shows that the two related carrier proteins produce inward and outward currents, which are dependent on the concentration gradient and Em. Both hCAT-2A and hCAT-2B generated a negative extracellular L-Arg concentration ([L-Arg]o)-sensitive membrane potential, additive to the K+ diffusion potential, when the oocytes were loaded before with CAA. At saturating [L-Arg]o, hCAT-2A-expressing oocytes showed an additional conductance of up to 2 µS/oocyte, whereas the current-voltage relationships in hCAT-2B-expressing oocytes were simply shifted to the right, resulting in voltage-independent difference currents. The distinct electrochemical properties of the hCAT-2 isoforms may serve to regulate differentially the transport of CAA through biological membranes.
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MATERIALS AND METHODS |
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Preparation of cRNA for microinjection. The plasmids phCAT-2A 118 and phCAT-2B 181 were linearized with Sal I and then transcribed in vitro from the SP6 promoter as described previously (9).
Preparation of Xenopus laevis oocytes. Bags of the ovary of the African clawed frog Xenopus laevis (anesthetized with ice and 0.1% 3-aminobenzoic acid ethyl ester) were surgically removed and placed in nominally Ca2+-free saline solution (in mM: 96 NaCl, 2 KCl, 1 MgCl2, and 10 HEPES; pH 7.6). Oocytes were either mechanically singled out, using a platinum loop, or obtained by enzymatic treatment to remove their follicular envelopes (1.9 U/10 ml collagenase A, 30-40 min) and stored in a modified Leibovitz medium at 4°C for up to 7 days. The Leibovitz medium was modified by dilution with H2O (1:2) and the addition of 1 mM L-glutamine, 100 µg/ml gentamicin, and 15 mM HEPES (pH 7.6). The CAA content of the medium amounted to 1.45 mM L-Arg and 0.25 mM L-lysine. cRNA (25 ng/25 nl H2O) was injected into the oocytes under microscopic control using a pneumatic transjector (5246, Eppendorf-Netheler-Hinz, Hamburg, Germany) and a micromanipulator (MMJ, Märzhäuser, Wetzlar, Germany). The cRNA-injected oocytes were then incubated at 18°C for 48-72 h in the modified Leibovitz medium for translation, processing, and embedding of the mature proteins into the cell membrane before the electrophysiological experiments were carried out. H2O-injected or native oocytes were used as controls.
Measurement of membrane potential.
Single oocytes were placed on a plastic grid in an organ bath of ~1
ml, which was built into a Perspex block that also contained a main
reservoir of 100 ml of modified Ringer solution (in mM: 96 NaCl, 2 KCl,
1 MgCl2, 1.8 CaCl2, and 5 HEPES; pH was
adjusted to 7.5 by NaOH), which was cooled to 20°C. Communication
between the two compartments was provided by connecting bores through which the fluids were driven by gas pressure (O2).
Substances could thus be easily added or removed from the main
reservoir by a rapid fluid exchange without mechanically or
electrically disturbing the test compartment. The
Em of oocytes was recorded intracellularly with
conventional micropipettes made from borosilicate glass (Science
Products, Frankfurt, Germany) filled with 3 M KCl (resistances between
10 and 20 M); micropipettes were connected to a unity gain buffer
amplifier (current clamp). The oocytes were impaled with the help of a
micromanipulator under microscopic control, and the recorded signals
were displayed on a Nicolet 310 scope (Nicolet Instruments, Madison,
WI) and digitally stored on floppy disks in a DOS-compatible format.
Measurement of membrane currents.
In the same experimental setup as described above, two electrodes with
tips broken down to larger diameters, yielding resistances between 0.8 and 1.2 M, were inserted into the same oocyte. The two-electrode
voltage clamp was performed as described (25). All
measurements were carried out in conjunction with a TEC05 two-electrode
voltage-clamp amplifier (NPI Electronics, Tamm, Germany). Ion
substitution experiments were performed in which replacement of either
Na+ with K+ or Cl
with
gluconate
was performed in the test solution.
Mg2+ was omitted from the solution, or Ca2+ was
buffered by EGTA. In some experiments, all cations in the solution were replaced by choline chloride.
Chemicals. Collagenase A was obtained from Boehringer (Mannheim, Germany). All other chemicals used were at least of reagent grade and purchased from Sigma Chemical (Deisenhofen, Germany).
Evaluation of results. All data were stored and edited on Intel microprocessor-based desk computers using either DOS or Windows as the operating system. Data are shown as original recordings or means ± SE. Concentration-response curves for [L-Arg]o were fitted to rectangular hyperbolas with the use of GraphPad prism 3.0 (GraphPad Software, San Diego, CA), yielding maximal current (Imax) and apparent Michaelis-Menten constant (Km), under the assumption that the L-Arg-induced transporter currents obey Michaelis- Menten kinetics. Statistical analysis was performed using ANOVA followed by modified t-statistics according to Bonferroni. P values <0.05, <0.01, and <0.001 were considered as significant. Absence of significance was also reported [designated by NS (not significant)].
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RESULTS |
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Em of both untreated and
H2O-injected oocytes was around 50 mV, indicating that
the injection procedure did not significantly alter the membrane
properties of native oocytes. In contrast, oocytes injected with cRNA
coding for hCAT-2A or hCAT-2B were found to develop more negative
membrane potentials in L-Arg-free solution when the cells
were loaded before with CAA. This difference was more pronounced with
hCAT-2A than with hCAT-2B (Fig. 1). When control oocytes were exposed to [L-Arg]o of 1 mM, Em remained more or less unchanged (Fig.
2A), although minor
depolarizations of 1-2 mV were occasionally observed. In contrast,
oocytes expressing hCAT-2B were drastically depolarized in the presence
of 1 mM [L-Arg]o (Fig. 2B). The
L-Argo-induced depolarization was concentration dependent (not shown) and completely reversible after washout of the
amino acid by repeated exchange of the bath solution (Fig. 2B).
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A more detailed analysis of oocytes expressing hCAT-2B revealed that
Em was determined by two major components.
First, when NaCl (96 mM) was replaced by KCl in the extracellular
solution, the membrane was depolarized to about 50 mV. Second, the
addition of 1 mM L-Arg further depolarized the membrane
near to 0 mV (Fig. 3, C and
D). The corresponding changes in Em
were also observed in the reverse order of solution changes and again
completely reversible. In control oocytes, the
[L-Arg]o-sensitive component of
Em was absent (Fig. 3, A and
B). Similar results, as described for hCAT-2B, were also
obtained in hCAT-2A-injected oocytes (not shown).
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It was now of interest to relate the
[L-Arg]o-induced changes in
Em to charge movements across the cell membrane.
In a first series of experiments, Em was held
constant at 60 mV, and the membrane current was observed at
cumulatively increasing [L-Arg]o. [L-Arg]o induced concentration-dependent
inward currents in oocytes expressing either hCAT-2A (Fig.
4A) or hCAT-2B (Fig.
4B). Note the relaxation of effects at higher
concentrations, which was mainly observed in oocytes expressing
hCAT-2A. The effects of L-Argo were completely
reversed after washout, followed by a slight increase in outward
current, which probably resulted from further loading of the oocyte
with L-Arg during the experiment. In
H2O-injected oocytes, inward currents of <2 nA were
observed in response to 10 mM [L-Arg]o (not
shown). A plot of L-Arg-induced effects against [L-Arg]o was fitted to rectangular hyperbolas
(Fig. 4C) using nonlinear regression analysis
(R2 > 0.99). The original recordings and
the analysis of data show that hCAT-2B is saturated at lower
concentrations of L-Argo than hCAT-2A, whereas
the maximal effects of [L-Arg]o are smaller
with hCAT-2B (
84 ± 11 nA) than with hCAT-2A (
194 ± 18 nA). The apparent Km values amounted to 0.13 and
0.97 mM, respectively (Fig. 4C). There were no significant
differences in the results, when either one
[L-Arg]o or cumulatively increasing amounts
of [L-Arg]o were tested in each experiment. A
significant distortion of the concentration-response curves, due to the
intracellular accumulation of L-Arg when increased stepwise
in the bath fluid, is therefore excluded.
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In another series of experiments, current-voltage relationships were
obtained by the application of voltage-clamp steps to various
potentials between 140 and +60 mV, starting at a holding potential of
60 mV (10-mV voltage increment, 3-s pulse duration, 5-s pulse
interval). Figure 5 shows the original
current traces in H2O-injected, hCAT-2A- and
hCAT-2B-expressing oocytes in L-Arg-free solution. Inward
and outward currents were much larger in hCAT-2A- and
hCAT-2B-expressing than in H2O-injected oocytes. Figure
5D shows the current-voltage relationships of the
H2O-injected oocytes and those of the cRNA-injected oocytes
obtained from the current values at the end of each voltage-clamp
pulse. The subtraction of the control values (obtained from
H2O-injected oocytes) from the currents obtained from the
oocytes expressing hCAT-2A or hCAT-2B eliminates the background
currents, therefore reflecting the pure transporter currents (Fig.
5E) in L-Arg-free solution. The currents in Fig.
5E were either inward or outward, dependent on
Em, and reversed at
71 and
85 mV in oocytes
expressing hCAT-2A and hCAT-2B, respectively. From the nearly linear
relationship between current and voltage, in the range from
100 to 0 mV, similar conductances were calculated (1.5 and 1.1 µS/oocyte,
respectively) for the two transporters in either direction, according
to G =
I/
E, where
G is conductance.
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In the next set of experiments, current-voltage relationships were
obtained under control conditions and in the presence of 10 and 1 mM
L-Argo in oocytes expressing hCAT-2A (Fig.
6A) and hCAT-2B (Fig.
6B), respectively. The difference between the currents in
the presence and absence of L-Argo reveals the
[L-Arg]o induced current changes (Fig.
6C). [L-Arg]o shifted the reversal
potential of the current-voltage curves of both transporters to more
positive values (Fig. 6, A and B). However, the
current-voltage curves in hCAT-2A-expressing oocytes became steeper in
the presence of [L-Arg]o (Fig.
6A), whereas those of hCAT-2B-expressing oocytes were simply
shifted to the right (Fig. 6B). From the nearly linear relationship between the change in membrane current and voltage (in the range from 100 to 0 mV), an additional ohmic conductance of
1.2 µS was calculated for hCAT-2A, which was induced by
[L-Arg]o (Fig. 6C). In contrast,
the change in membrane currents of hCAT-2B-expressing oocytes was
nearly independent of Em. Therefore, the
L-Arg-induced current changes in hCAT-2B-expressing oocytes
were not accompanied by a change in conductance of the transporter. A
further examination of the voltage dependence of hCAT-2A at different
[L-Arg]o revealed different conductance
increments of the transporter (Fig.
7A), which were plotted
against [L-Arg]o (Fig. 7B).
The L-Arg-induced conductance changes increased steeply
toward 2 µS with increasing [L-Arg]o;
saturation was observed at ~3 mM.
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In the last series of experiments, the effects of ion substitutions on
the current-voltage relationships of oocytes expressing hCAT-2A or
hCAT-2B were investigated in the absence and presence of
L-Arg. Qualitatively, the same changes in currents were
observed with both transporters in Na+-free (Fig.
8, A and C) and
Cl-free (Fig. 8, B and D) solutions
and also when Mg2+ was omitted from the solution or when
Ca2+ was buffered by EGTA (not shown). Finally, to exclude
any relatively nonselective cation conductance except for CAA, all
cations in the test solution were replaced by choline. In this
set of experiments, hCAT-2A- and hCAT-2B-expressing oocytes were first
exposed to 10 and 1 mM L-Arg, respectively, in Ringer
solution. After washout, the preparations were again exposed to the
same concentrations of L-Arg in choline solution. The
effects of L-Arg on membrane currents in Ringer solution
were almost identical to those in choline solution (Fig.
9), which is further evidence for the
assumption that the L-Arg-induced currents are unrelated to
any other ion conductances.
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DISCUSSION |
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The intracellularly recorded Em of native
and H2O-injected oocytes from Xenopus laevis was
about 50 mV in normal Ringer solution. Em was
nearly abolished at an extracellular K+ concentration of 96 mM, which indicates that Em in oocytes, as in
most living cells, is mainly determined by the diffusion of K+. Em was more negative in
hCAT-2A-expressing or hCAT-2B-expressing oocytes that were previously
loaded with CAA. A negative Em of more than
100 mV was frequently recorded. The addition of
L-Arg to the test fluid and/or changing the extracellular
K+ concentration revealed that Em in
these oocytes resulted from two major components: a K+ as
well as a CAA diffusion potential. In CAA-free solution, hCAT-2A and hCAT-2B are likely to extrude CAA from intracellular pools when they are loaded first in Leibovitz medium, thereby driving Em of the oocytes to more negative values.
There is no wonder that the diffusion of CAA can alter
Em because the transport of charge is not
matched by a cotransport of another ion species. Whether this is just a
consequence of electrogenic transport or may also have a biological
function has not been determined. Läuger (20)
already raised this general question and pointed out that
electrogenicity of ion transport may be more than only an epiphenomenon
or an unavoidable by-product. For hCAT-2A- and hCAT-2B-mediated
transport, two aspects can be discussed. First, the efflux of CAA
drives Em to the reversal potential of the
transporter currents, depending on the ratio of loaded transporters on
either side of the membrane, thereby minimizing the loss of
intracellular CAA when the extracellular CAA concentration is low. The
K+ diffusion potential helps to trap intracellular CAA in a
similar way due to the additive effect on Em.
The electrochemical gradient for CAA predicts that CAA are concentrated
intracellularly in physiological conditions due to the negative
Em, which has been shown earlier by HPLC
analysis in Xenopus oocytes (9). Second, if
Em could be either decreased or increased by the
activity of CAT in native cells, many other physiological functions
that depend on Em could also theoretically be
influenced. Indeed, a significant contribution of CAA to the
Em has been demonstrated in mouse pancreatic -cells in which depolarization in response to
[L-Arg]o stimulated insulin secretion
(23).
In oocytes expressing hCAT-2A or hCAT-2B, large inward and outward
currents in CAA-free solution were detected that were dependent on
Em. From the analysis of current-voltage
relationships in CAA-free solution, it became clear that both
transporters, hCAT-2A and hCAT-2B, have similar ohmic conductances and
therefore may pass symmetrically CAA in either direction, depending on
Em and the concentration gradient. The
corresponding reversal potentials were negative to the K+
reversal in H2O-injected oocytes. Because oocytes
were generally incubated in CAA-containing solution and then first
examined in CAA-free solution, the outward currents are obviously due
to the efflux of CAA, which also explains the hyperpolarization
observed in the current-clamp mode. It is not quite clear why, at more negative potentials, substantial inward currents were also observed in
CAA-free solution. One explanation could be the existence of an
extracellular pool of CAA located in the space between the plasma
membrane and the vitelline envelope of the oocytes. Alternatively, hCAT-2A and hCAT-2B could also transport charged particles other than
CAA. In this context, the observation that the expression of different
heterologous membrane proteins in Xenopus laevis oocytes induced the expression of endogenous
hyperpolarization-activated cation channels is also of interest
(27). Our ion substitution experiments, however, indicate
that the observed transporter currents are solely dependent on CAA and
not dependent on Na+, Cl, Ca2+,
or Mg2+.
When L-Arg was added to the bath fluid, substantial current
changes were observed in hCAT-2A-expressing and hCAT-2B-expressing oocytes that were dependent on concentration. It is noteworthy that,
especially with hCAT-2A at saturating
[L-Arg]o, a relaxation of peak transporter
currents was observed. Such an inactivation process is similar to that
seen with many ionic channels (11, 13), which points to an
interesting similarity of currents arising from either channel or
transporter activity. The time-dependent reduction in the
transporter-induced current flow might also result from a local
intracellular accumulation of L-Arg near the membrane rather than from a change in the overall L-Arg
concentration gradient. This becomes clear from a simple calculation.
Given a peak L-Arg current of 96 nA, 6 × 1011 charged particles would pass the oocyte membrane per
second, corresponding to 1 pmol L-Arg. At 10 mM
[L-Arg]o, assuming an oocyte volume of 0.5 µl (26) and an intracellular L-Arg
concentration of ~2 mM in hCAT-expressing oocytes (1 nmol/oocyte,
Ref. 9), it would take at least 1 h to align the intracellular to
the extracellular concentration of L-Arg. This corresponds
roughly to uptake measurements of radiolabeled L-Arg
(9). The situation may be different in smaller cells. In
mouse pancreatic
-cells (cell volume of ~1 pl) clamped at
70 mV,
it has been shown that 10 mM [L-Arg]o induced an inward current of ~3 pA (23). The intracellular
concentration would reach 10 mM within 6 min, which is in line with
uptake studies in intact islets (3).
At 60 mV, the Km values were 0.97 mM for
hCAT-2A and 0.13 mM for hCAT-2B. These findings confirm that hCAT-2A is
a transporter with lower substrate affinity compared with hCAT-2B, as
shown by Closs et al. (9) with radiolabeled
L-Arg. The L-Arg-induced currents were
generally larger in hCAT-2A- than those in hCAT-2B-expressing oocytes.
Whether this is due to differences in the transport capacity of the
transporters or reflects different quantities of expressed or
functional proteins has yet to be determined. This question may be
adequately resolved when researchers are able to analyze transporter
currents on a microscopic level, as currently possible with ion
channels by patch-clamp or noise analysis (13). There are
other methods available that can count the number of transport proteins, like antibody binding or freeze fracture; however, these methods cannot determine the number of functional transporters.
Another difference between hCAT-2A and hCAT-2B, seen in our experiments, seems to be substantial. The slopes of the current-voltage relationships of oocytes expressing hCAT-2A were dependent on [L-Arg]o, revealing increases in ohmic conductances between 0 and 2 µS/oocyte. In contrast, the current-voltage curves in oocytes expressing hCAT-2B were simply shifted to the right at 1 mM [L-Arg]o, resulting into a negative change in current, which was independent of Em. This does not imply that the transporter current is inward at all voltages. At potentials negative to the reversal, the observed current changes probably reflect an increase in inward current; at potentials positive to the reversal, the observed current changes probably reflect a decrease in outward current. Because of the shift by [L-Arg]o in the reversal potential, there is a middle portion along the voltage axis in which both an increase in inward and a decrease in outward current can be observed.
The conductance of hCAT-2B is therefore obviously unchanged by [L-Arg]o. One possible explanation for the difference may be that the occupancy by CAA of the transporters inside and/or outside the cell membrane is a determining factor. If the substrate affinity of hCAT-2B is larger than that of hCAT-2A at the cytoplasmic sites of the transporters (which is unknown at present), hCAT-2B may also be saturated inside the cell membrane (when the cells are previously loaded with CAA), in contrast to hCAT-2A, which may need higher concentrations. In the case of saturation, no further increase in transporter conductance may be expected with the addition of L-Arg, as seen with hCAT-2B. Further experiments in which both the intracellular and the extracellular concentrations will be controlled, either with the use of the cut-open method or by studying the transport activity in giant patches, may resolve this question.
L-Arg-induced currents have been described previously for mCAT-1, expressed in Xenopus oocytes (17), with Km and Imax values similar to those determined for hCAT-2B (about 0.1 mM and 100 nA, respectively); however, the voltage dependence of mCAT-1 was more comparable to that of hCAT-2A. In addition, voltage-dependent L-Arg-induced currents have also been described in oocytes expressing proteins related to b0,+ amino acid transporter (1, 4), probably by activating endogenous transporters in the oocytes. The diversity of transport systems, with distinct electrochemical properties, for the same substrate may help to regulate precisely the concentration of intracellular L-Arg at varying extracellular concentrations of CAA in various tissues.
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the Umweltministerium Rheinland-Pfalz (to H. Nawrath) and the Deutsche Forschungsgemeinschaft (Cl100/3-2 and the Collaboration Research Center SFB553,B4 to E. I. Closs).
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Nawrath (Electrophysiology) or E. I. Closs (Molecular Biology), Pharmakologisches Institut der Universität Mainz, Obere Zahlbacher Str. 67, D-55101 Mainz, Germany (E-mail: nawrath{at}mail.uni-mainz.de or closs{at}mail.uni-mainz.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.
Received 3 December 1999; accepted in final form 1 June 2000.
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