From the
To elucidate the electrical events associated with the movement
of amino acids by the neutral and basic amino acid transporter
(NBAT)-encoded protein (Yan, N., Mosckovitz, R., Gerber, L. D., Mathew,
S., Murty, V. V. V. S., Tate, S. S., and Udenfriend, S. (1994)
Proc. Natl. Acad. Sci. USA 91, 7548-7552), we have
investigated the membrane potential and current changes associated with
the increased transport of amino acids across the cell membrane of NBAT
cRNA-injected Xenopus laevis oocytes. Superfusion of 0.05
m
M
L-phenylalanine, in current-clamped NBAT-injected
oocytes, caused a hyperpolarization (8.5 ± 0.9 mV), but
superfusion of
L-arginine caused a depolarization (18.3
± 1.3 mV). In voltage-clamped (-60 mV) oocytes,
superfusion of
L-phenylalanine evoked a sodium- and
chloride-independent, saturable ( K
Transporter proteins located in the membranes of epithelial
cells play a vital role in absorption of amino acids from the lumen of
renal tubules and small intestine to the blood
(1) . A cDNA from
rat kidney cortex (NBAT
We
hypothesized that measurable inward currents would be generated in
response to the movement of cationic amino acids in NBAT-expressing
oocytes. We therefore examined the mechanisms of amino acid transport
in NBAT-expressing oocytes using a combination of two-electrode
voltage-clamp and radiotracer methodology. Our initial hypothesis
proved to be correct, as we report here, but to our surprise we also
found that outward currents were generated in response to the
superfusion of neutral amino acids. We further hypothesized that this
outward current would be (i) saturable and voltage-dependent and (ii)
due to either the inward movement of an anion or an outward movement of
a cation species. We discuss the evidence for the operation in
NBAT-expressing oocytes of a novel sodium- and chloride-independent but
potassium-linked transport mechanism which involves outward movement of
a positive charge associated with inward transport of neutral amino
acids. During the preparation of this manuscript Busch et al. (8) reported on the electrogenic properties of rBAT protein (a
rabbit glycoprotein with 80% predicted amino acid sequence homology to
NBAT) in Xenopus oocytes. These authors found consequent to
rBAT expression, inward and outward currents bearing some similarity to
those described here, but not linked to outward movement of potassium
as we discovered. The similarities and differences between the currents
evoked by NBAT- and rBAT-expressing oocytes raise important questions
which we discuss here.
Superfusion of
L-phenylalanine (0.05 m
M in
TMA chloride medium) in current-clamped NBAT-injected oocytes (resting
membrane potential -59.4 ± 5 mV) caused a
hyperpolarization (8.5 ± 0.9 mV) of the oocyte cell membrane,
whereas
L-arginine (0.05 m
M) caused a depolarization
(18.3 ± 1.3 mV). There was no detectable change in the membrane
potential of control (water-injected) oocytes (-63 ± 3 mV)
in response to superfusion of either
L-phenylalanine or
L-arginine (tested between 0.05 and 1 m
M).
Superfusion of NBAT-injected Xenopus oocytes, clamped at a
V
Assuming a single,
exchangeable oocyte pool of the major cationic amino acids (175 pmol of
arginine + lysine per oocyte)
(11) , the tracer efflux
represents 175
Under normal
experimental conditions the potassium cation has an outwardly directed
electrochemical gradient (since [K]
We have demonstrated electrogenic properties of both neutral
and cationic amino acid transport in NBAT-expressing oocytes. The
primary aim of this study was to elucidate the membrane currents
associated with the transport of cationic amino acids in
NBAT-expressing oocytes, and as expected the influx of the cationic
amino acid arginine produced an inward membrane current consistent with
1 arginine ion:1 charge movement stoichiometry; other cationic amino
acids also evoked inward currents. However, we also found that the
uptake of neutral (uncharged) amino acids (notably
L-phenylalanine,
L-glutamine, and
L-
Phenylalanine:cationic amino acid exchange may account for a minor
portion (
It has
been suggested that NBAT protein promotes the uptake of neutral and
cationic amino acids by a single transport activity (system
b
The molecular
architecture of the NBAT/D2 protein, which appears to have only one
membrane-spanning region (unlike 8-14 for conventional amino acid
transporters)
(3) and the presence of a leucine zipper motif at
its C terminus, raises the possibility that the protein may not
necessarily be a transporter in its own right but is instead associated
with a second catalytic ( i.e. transporting) subunit
(12) . Recently, Veyhl et al. (13) reported the
cloning of RS1, a 66-kDa membrane-associated protein with a single
transmembrane domain which activates sodium-
D-glucose
cotransport by SGLT1 when co-expressed in Xenopus oocytes. It
is therefore conceivable that NBAT cDNA encodes for an analogous
protein which stimulates the activity of amino acid transporters native
to Xenopus oocytes. An alternative possibility is that NBAT
encodes for a protein acting as an amino acid-activated pore
conductance which allows the movement of cations (such as potassium and
arginine) in conjunction with a wide variety of neutral amino acids.
The growing evidence that a multicomponent mechanism for neutral amino
acid transport is induced in NBAT cRNA injected oocytes may indicate
that the expression of system b
Neutral
amino acids evoke sodium- and chloride-independent outward currents in
oocytes expressing NBAT (present study) and rBAT protein (as
demonstrated by Busch et al. (8) ). Our results are
also consistent with that of Busch et al. (8) in so
far as both studies show that neutral amino acids are transported in
oocytes by obligatory exchange with a cation. The two studies differ in
the fact that we demonstrate directly, that only a minor
portion of the transport mechanism involves neutral:cationic amino acid
exchange, since the associated charge movements cannot account for the
evoked currents (at least in the case of NBAT-expressing oocytes).
Furthermore, our experimental evidence is consistent with the notion
that potassium is a major cation involved, although other small
intracellular cations (inorganic or organic) may also play a role in
exchange. In marked contrast there is no evidence for the involvement
of potassium in rBAT-induced neutral amino acid transport, even though
there is 80% homology between the predicted amino acid sequences of
NBAT
(9) and rBAT
(4) . The evidence of the involvement
of potassium in the transport of neutral amino acids in NBAT-expressing
oocytes (as presented in this study) and the lack of it in
rBAT-expressing oocytes
(8) may point toward important
differences in the structure-function relationships of two very similar
proteins.
Values are
means ± S.E. for 3-10 oocytes for each measurement. A
small inward current (
We thank S. Grant for technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 0.34 ± 0.02 m
M, I
= 31.3 ± 0.5 nA), outward current. This outward
current was reduced in the presence of high external [K] and
was barium-sensitive. Outward currents were also evoked by
L-leucine,
L-glutamine,
L-alanine,
D-phenylalanine, and
L-
-phenylalanine.
Superfusion of
L-arginine evoked a saturable
( K
= 0.09 ± 0.02
m
M, I
= -29.2 ± 1.3
nA) inward current;
L-lysine and
D-arginine also
evoked inward currents.
L-Glutamate and
-alanine failed
to evoke any currents. Effluxes of
L-[
H]phenylalanine and
L-[
H]arginine were trans-stimulated in
the presence of either amino acid. Flux-current comparisons indicated
amino acid:charge movement stoichiometry of 1:1 for both neutral and
cationic amino acids. These findings indicate that the amino acid
transport activity(ies) expressed in NBAT cRNA-injected oocytes is
electrogenic by a mechanism including the outward movement of a net
positive charge (potassium ion or cationic amino acid) in exchange for
uptake of a neutral amino acid.
/D2) (
)(2, 3) and a homologous rabbit cDNA termed rBAT
(4, 5) encode for membrane proteins which when expressed in
Xenopus laevis oocytes stimulate amino acid transport activity
resembling system b
(a sodium-independent neutral
and cationic amino acid transporter)
(6) . The NBAT protein is
located mainly in kidney and small intestinal epithelia
(7) where expression of system b
like amino
acid transport could contribute toward absorption of amino acid.
Expression of NBAT in Oocytes
The cDNA clone for
NBAT was a kind gift of Dr. S. Udenfriend, Roche Research Center,
Nutley, NJ. Synthetic cRNA was transcribed in vitro (transcription kit from Ambion Inc., Austin, TX) from NBAT cDNA
clone (in pSPORT 1 plasmid vector)
(9) , and 5 ng of cRNA in 50
nl of sterile water were injected into each defolliculated stage VI
X. laevis oocyte. All experiments were performed 3-5
days post cRNA injection. Resting membrane potential in current-clamped
mode and amino acid-evoked membrane currents were measured using a
two-electrode voltage-clamp recording system (GeneClamp 500, Axon
Instrs., Inc., Foster City, CA). Only oocytes with resting membrane
potential > -40 mV were used in this study. For
electrophysiological studies, oocytes were superfused with a medium
containing either 100 m
M NaCl, tetramethylammonium (TMA)
chloride, or sodium isethionate and including 2 m
M KCl, 1
m
M CaCl, 1 m
M MgCl
, and 10
m
M HEPES pH 7.5 with Tris-base (superfusion medium). Amino
acids at the indicated concentrations were added to this solution. The
temperature of the superfusion medium was controlled at 22 ± 1
°C. The potential-recording and current-passing electrodes were
filled with 1
M KCl. For experiments in which the chloride was
replaced with isethionate, the bath probe was isolated from the
superfusing solution using a 3
M KCl/agar bridge. Holding
potential ( V
was normally maintained at
-60 ± 1 mV in voltage-clamp experiments. Recordings of the
membrane current and potential were displayed on a chart recorder.
[K]
in oocytes was measured by standard
flame photometric methods. Amino acid fluxes were measured using
H-labeled
L-phenylalanine and
L-arginine
(Amersham Corp., UK) as tracers; amino acid influx to oocytes was
assessed as described previously
(9) , and efflux was estimated
(by a method described elsewhere
(10) ) from the appearance in
the medium (over successive 15-min periods) of tracer injected into
oocytes (0.1 kBq per oocyte in 50 nl of sterile water).
Electrophysiological Measurements
Measurements
were made in 3-10 individual oocytes (from three or more separate
batches in most cases) for each experimental maneuver unless otherwise
stated. Data were averaged from oocytes tested and are expressed as
means ± S.E. Transport and flame photometry data are expressed
as means ± S.E. for experiments performed on three to seven
oocyte batches (10 individual oocytes per batch). Analysis of variance
tests were performed where appropriate.
of -60 mV, with
L-phenylalanine in 100 m
M NaCl or TMA chloride
superfusion medium evoked similar reversible outward currents
( I
) ( and Fig. 1 A).
Under similar conditions superfusion of
L-arginine evoked a
reversible inward current ( I
) which was also
sodium-independent ( and Fig. 1 B). Both
I
and I
were also
chloride-independent since currents of equivalent magnitude were evoked
in 100 m
M NaCl or equimolar sodium isethionate medium
().
L-Phenylalanine and
L-arginine evoked
currents were saturable and exhibited Michaelis-Menten kinetics; at a
V
of -60 mV for one representative
batch of oocytes the K
(m
M) was
0.34 ± 0.02 and 0.09 ± 0.02 and I
(nA) was 31.3 ± 0.5 and -29.2 ± 1.3 for
L-phenylalanine and
L-arginine, respectively (values
are means ± S.E. where S.E. represents the error of the line
fitted to a Hane's transformation of data at six different amino
acid concentrations (0.05-1 m
M) with n =
3 oocytes at each concentration). Similar values were obtained in a
separate batch of oocytes. Outward currents were also observed for
other neutral amino acids, e.g.
L-glutamine,
L-alanine, and
L-
-phenylalanine (Table II).
Superfusion of the
D-isomers of phenylalanine and arginine
also induced outward and inward currents, respectively ().
The magnitudes of I
and I
were dependent upon V
at values
between -40 and -100 mV (Fig. 2). The null potential for
L-phenylalanine-induced current occurred between -100
and -110 mV.
Figure 1:
Representative current trace of
( A)
L-phenylalanine (0.1 m
M) outward current
directed upward and ( B)
L-arginine (0.05 m
M)
inward currents directed downward in 100 m
M NaCl and TMA
chloride medium at a V of -60 mV in NBAT-expressing
oocytes. A small inward current was evoked by
L-phenylalanine
and
L-arginine (both at 1 m
M) in control
HO-injected oocytes. Amino acids were superfused for the
duration indicated by the bar. Any short latency between
application and response reflects the time required for test
superfusate to reach the oocyte in the experimental
chamber.
Injection of oocytes with NBAT cRNA resulted in
increased uptake of a range of neutral and cationic amino acids
(including
L-arginine,
L-lysine,
L-phenylalanine,
L-alanine, and
L-glutamine)
as reported previously
(3, 9) . The increased uptake of
both
L-phenylalanine and
L-arginine tracers (0.05
m
M) by NBAT-injected oocytes was sodium-independent,
saturable, and inhibited by
D-leucine
(Fig. 3 A). The stoichiometry of
L-phenylalanine:charge movement (I) was
calculated by converting the current evoked by 0.05 m
M phenylalanine to a charge flux using Faraday's constant
( F = 9.65 10
C
mol
). Superfusion of 0.05 m
M
L-phenylalanine caused a shift of
10 mV in
current-clamped oocytes (see above), therefore the outward current
value used in this calculation was at a V
of -70 mV. This calculated charge flux represents a
stoichiometry for
L-phenylalanine:charge movement of 1:1
(I). The equivalent calculation for the inward arginine
evoked current (at a V
of -40 mV)
also represents a stoichiometry for
L-arginine:charge movement
of 1:1 (I).
Figure 3:
A, Expression of sodium-independent amino
acid uptake in NBAT cRNA-injected oocytes. A tracer amino acid
concentration of 0.05 m
M was used throughout. Values are means
± S.E. for four to seven oocyte batches. Open bars,
water injected; solid bars, NBAT cRNA injected. B,
trans-stimulation of
L-phenylalanine and
L-arginine
efflux by 1 m
M external amino acid in NBAT-expressing oocytes.
Values are means ± S.E. for 12-20 oocytes pooled from two
separate oocyte batches. Open bars, water injected; closed
bars, NBAT cRNA injected.
The identity of the ratios for
transport:charge movement during increased flux of both
L-phenylalanine and
L-arginine in NBAT cRNA-injected
oocytes raised the possibility that the transport activities expressed
in these oocytes could be the manifestation of a neutral-cationic amino
acid ( e.g. phenylalanine-arginine) exchanger. If this were the
case, then the two amino acids should exhibit reciprocal
trans-stimulation of transport, as indeed proved to be the case when
this was tested (Fig. 3 B): effluxes of both
L-[H]arginine and
L-[
H]phenylalanine were trans-stimulated
by addition of either amino acid on the trans (external) side
of the oocyte membrane (Fig. 3 B). Arginine tracer efflux
was 0.34 ± 0.05% min
at an external
phenylalanine concentration of 0.05 m
M.
0.0034 = 0.59 pmol/oocyte
min. This
rate is less than 15% of the measured phenylalanine influx and could
carry only an equivalent proportion of the associated outward current,
thus indicating that movement of other cations must be responsible for
the major part of the phenylalanine-evoked current.
)
= 89 ± 2 m
M in NBAT cRNA injected oocytes (and
88 ± 3 m
M in H
O injected oocytes) n = 5 oocytes, compared to [K]
= 2 m
M). We tested the hypothesis that the
outward I
might also be due to an outward
movement of potassium. Increasing the [K]
from 2 to 10 m
M (and thus reducing the outward potassium
gradient) in TMA chloride superfusion medium reduced the magnitude of
I
by more than 50% (Fig. 4) in NBAT-expressing
oocytes voltage clamped at a V
of
-60 mV; decreasing the [K]
from 2
to 1 m
M increased the magnitude of I
(Fig. 4). We also found that the
L-phenylalanine-evoked current was barium-sensitive since the
addition of 1 m
M BaCl
in the superfusion medium
during superfusion with 0.2 m
M
L-phenylalanine
reduced the I
to
60% of its value in the
absence of BaCl
(15.0 ± 1.4 versus 9.5
± 0.8 nA, n = 5 oocytes, p < 0.025).
Figure 4:
Effect of [K] (1-10
m
M) on the currents evoked by
L-phenylalanine (0.1
m
M) in TMA chloride superfusion medium at a V of
-60 mV in NBAT-expressing oocytes. Each point represents means
± S.E. from five oocytes. Abscissa indicates
[K] on a logarithmic scale.
-phenylalanine) in these oocytes also produced current
with 1 phenylalanine:1 charge movement stoichiometry but that this
current was in the opposite direction to that observed with the
cationic amino acids. Both I
and
I
were voltage-dependent and sodium- and
chloride-independent which for I
indicates
outward movement of a positive charge. These results are broadly
consistent with the recently published report for the amino acid
induced currents in rBAT-expressing oocytes
(8) .
15%) of phenylalanine transport but there appears to be an
additional (perhaps major) component of phenylalanine uptake associated
with the outward movement of another cation (most likely potassium).
This idea is consistent with the identification of at least two
kinetically distinct (high and low affinity) components of neutral
amino acid (
L-leucine) uptake induced in rBAT
(12) -
and NBAT
(
)
-expressing oocytes. The suggested
involvement of potassium is based on the following observations. (i)
The null potential for I
, calculated from
Fig. 2
, was -106 mV, a value close to the potassium
equilibrium potential in oocytes of -99 mV (where
[K]
) is 89 m
M) and (ii) the
magnitude of I
was reduced as a result of
decreased transmembrane potassium gradient. Moreover the potassium
conductance inhibitor barium significantly inhibits the
I
. All this evidence indicates that a component
of the phenylalanine-induced outward current is linked to the outward
movement of potassium which is barium-sensitive.
Figure 2:
Membrane potential dependence of
L-phenylalanine () (0.05 m
M)- and
L-arginine (
) (0.05 m
M)-induced currents in
NBAT-expressing oocytes. Values are means ± S.E. of three to
five oocytes for each point.
Most neutral amino
acids (both
L- and
D-isomers) tested induced an
outward current in NBAT-expressing oocytes, although -alanine and
the anionic
L-glutamate failed to evoke any current. The
acceptance of certain
D-amino acids as substrates by the
induced transport activities of NBAT/D2-expressing oocytes has been
reported previously
(3) (also shown here both by evoked
currents and by inhibition of transport with
D-leucine) and
confirms both the poor stereospecificity and the limited tolerance of
the
-amino group of the activated/expressed protein.
)
(6) in Xenopus oocytes
(2, 3) . Amino acid transport by a system b
like transporter has been previously reported in Xenopus oocytes using radiotracer methodology
(3) but in our
experiments the outward currents detected for
L-phenylalanine
or other neutral amino acids in NBAT-expressing oocytes were not
detected in control water injected oocytes, indicating that the major
native and expressed system b
like transport
activities may be distinct from one another.
-like transport
reflects the activity of more than a single protein product.
Functional Perspective
Epithelial tissues ( e.g. kidney and small intestine) are the major sites for the expression
of NBAT/rBAT-type
(4, 7, 14) glycoproteins
where they are known to be regulated during development
(15) .
In renal and intestinal epithelia the ability of transport processes to
exchange neutral for cationic amino acids may, for example, provide a
useful contribution to the processes by which absorbed arginine,
lysine, and ornithine are released into the blood, perhaps in exchange
for amino acids of nutritional importance to the epithelial cells such
as glutamine (a substrate for the NBAT-encoded protein). NBAT protein
is also expressed in neural tissue
(2, 7) , where the
electrogenic properties of associated amino acid transport activities
may provide an unexplored mechanism for modulating neuroexcitability.
Table:
Ion dependence of
L-phenylalanine and
L-arginine currents in
voltage-clamped (-60 mV) NBAT-expressing oocytes
0.3-1.0 nA) was observed in control
water injected oocytes for both
L-phenylalanine and
L-arginine.
Table: 0p4in
ND, not detected.
Table:
Charge:flux ratios for amino acid (AA)
transport activity in NBAT-expressing oocytes
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.