K+ amino acid transporter KAAT1 mutant Y147F has increased transport activity and altered substrate selectivity
1 The Whitney Laboratory, University of Florida, St Augustine, FL 32080,
USA
2 Department of Physiology and Functional Genomics, University of Florida
College of Medicine, Gainesville, FL 32610, USA
3 Harvard Institutes of Medicine, Harvard Medical School, Boston, MA 02115,
USA
* Present address: Research Department, Shriners Hospital for Children of
Northern California, Sacramento, CA 95817, USA.
Author for correspondence (e-mail:
wharvey{at}whitney.ufl.edu)
Accepted 10 October 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The multiple mutant exhibited no amino-acid-induced currents, indicating that one or more of the mutated residues are crucial for function. W75L and R76E mutations in the first transmembrane helix of KAAT1 led to results equivalent to those observed in the corresponding mutants of GAT1; namely, substrate (leucine) uptake and substrate-evoked net inward current were severely curtailed. The KAAT1 A523S mutant, which corresponds to a serotonin transporter mutant that is thought to render Li+ equivalent to Na+ as a co-transported ion, functioned no differently to WT.
The effects of mutation Y147F in the third transmembrane helix of KAAT1 were dramatically different from the equivalent mutation, Y140F, in GAT1. Although kinetic characteristics, expression levels and plasma membrane localization were all similar in Y147F and WT, the Y147F mutant exhibited a sevenfold increase in labeled leucine uptake by Xenopus oocytes in Na+ buffer. This increase is in sharp contrast to the complete loss of uptake activity in the GAT1 Y140F mutant. KAAT1 Y147F also differed from WT in cation selectivity and substrate spectrum, as revealed by amino-acid-induced net inward currents that were measured with a two-electrode voltage clamp.
Amino-acid-independent currents induced by Li+ and Na+ chloride salts were observed in both WT and the Y147F mutant. The Li+-induced current was 30% higher in Y147F than in WT, whereas no substrate-independent K+-induced currents above control levels were detected either in WT or Y147F. These results suggest that transport of K+, the physiological co-substrate in insect midgut, is tightly coupled to that of amino acids in KAAT1, in contrast to the independence of cation and amino acid transport in the closely related cation amino acid transporter channel, CAATCH1.
Key words: CAATCH1, KAAT1, GAT1, potassium, sodium, amino acid, transporter, leakage current
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amino acid residues in highly conserved regions within the 12 transmembrane
domains of SNF transporters have been investigated intensively, especially in
GAT1. All charged and aromatic residues of GAT1 have been mutated to determine
if they react in a critical way with Na+, Cl- or amino
or carboxyl groups of GABA. In an effort to abolish the K+, but not
the Na+, recognition site of KAAT1, nine residues were mutated back
to the superfamily's conserved form in a multiple mutant. In addition, noting
that mutating serine 545 to alanine (S545A) in the serotonin transporter,
SERT, renders Li+ equal to Na+ as a co-substrate
(Sur et al., 1997), we mutated
the corresponding KAAT1 residue, alanine 523 to serine (A523S) and examined
its effects on cation selectivity.
Finally, we noted that, among 10 tryptophan residues, 12 tyrosine residues
and five charged residues that were mutated in GAT1, only tryptophan 68,
arginine 69, and tyrosine 140, which are conserved in the SNF family, were
functionally critical. A single substitution in any of these highly conserved
residues resulted in a complete loss of GABA transport activity
(Pantanowitz et al., 1993;
Kleinberger-Doron and Kanner,
1994
; Bismuth et al.,
1997
). Residues that correspond to these three critical residues
in GAT1 are also conserved in KAAT1. These three residues were mutated to
yield W75L, R76E and Y147F (Fig.
1). The results of these mutations provide fresh insight into
binding sites for ions and amino acids in SNF transporters.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Site-directed mutagenesis and cRNA transcription of KAAT1
Mutagenesis was performed using the QuikChangeTM Site-Directed
Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Mutagenic primers were
synthesized by Midland Certified Reagent Co. (Midland, TX, USA). The `domain
swapping' method was applied to eliminate the occasional involvement of
undesired random mutations. After an EcoRI site of the pSport vector
was removed, the resulting structure has four unique sites that are located in
the KAAT1 coding region from nucleotides 1 to 1902. They are EcoRI
175, NcoI 500, XhoI 1366 and StuI 1738, which
divide the entire coding region into four domains. Mutants were verified by
sequencing, and the mutation-containing domain, flanked by two unique sites,
was cut and pasted into the corresponding domain of wild-type KAAT1. Capped
cRNA transcripts were synthesized using the mMESSAGE mMACHINETM kit
(Ambion, Austin, TX, USA). The quality of the transcripts was routinely
monitored on formaldehyde denaturing agarose gels
(Lehrach et al., 1977).
Radiolabeled amino acid uptake
[3H]L-leucine uptake was measured 2-3 days after injection with
100 ng of cRNA. Oocytes (10 per assay) were exposed to
1.9x105 Bq ml-1 [3H]L-leucine
(Amersham, Piscataway, NJ, USA) at 22°C for 30 min or 60 min. Data were
collected and analyzed as previously described
(Stevens, 2001;
Castagna et al., 1998
;
Feldman et al., 2000
).
Electrophysiology
Electrical determinations were carried out as described earlier
(Feldman et al., 2000).
Briefly, injected oocytes were superperfused (22°C) with modified ND96
media, containing 98 mmol l-1 NaCl, 2 mmol l-1 KCl, 1
mmol l-1 MgCl2, 1 mmol l-1 CaCl2,
10 mmol l-1 Taps/NMG+ buffer
{3-[tris(hydroxymethyl)methyl] amino propanesulphonic acid/N-methyl
glucamine, pH 8.0}, using a peristaltic pump. In some experiments,
Na+ was completely replaced by K+, Li+ or
NMG+. In experiments designed to determine anionic specificity,
Cl- was completely replaced by gluconate-. Transmembrane
currents were measured in intact oocytes using a two-electrode voltage clamp
(Warner model OC725-B, Hamden, CT, USA) with agar-bridged bath electrodes.
Current/voltage relations were generated using voltage steps or ramps (36 mV
s-1, 1.8 mV per point) between -150 mV to +30 mV from a holding
potential of -60 mV. The protocol to measure steady-state currents minimized
or eliminated the rapidly decaying transient currents that were previously
reported for KAAT1 (Bossi et al.,
1999
). Substrate-dependent, current/voltage data were obtained by
subtracting control current values (i.e. those in the absence of substrate)
from those in the presence of substrate.
Immunofluorescent microscopy
Stage VVI Xenopus oocytes were injected with 50-100 ng of
Y147F or wild-type (WT) cRNA or water. Injected oocytes were incubated in
Barth's solution (88 mmol l-1 NaCl, 1 mmol l-1 KCl, 2.4
mmol l-1 NaHCO3, 15 mmol l-1 Hepes, 0.32 mmol
l-1 CaNO3, 0.4 mmol l-1 CaCl2 and
0.81 mmol l-1 MgSO4) at 18°C for 3 days. After
incubation in 20% sucrose containing phosphate-buffered saline (PBS)
overnight, the oocytes were fixed in 5% paraformaldehyde for 60 min. The fixed
oocytes were then embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek USA,
Inc., Torrance, CA, USA). Frozen sections were cut using a Microm HM 500
microtome (Microm, Walldorf, Germany). The sections were incubated with
polyclonal primary antibody, which was prepared in rabbits against the amino
terminus of KAAT1 (a gift from Paul Linser). Sections were stained with
2x10-3 mg ml-1 affinity-purified antibody at room
temperature for 30 min, washed three times with PBS, then incubated with
secondary, tetramethylrhodamine isothiocyanate-labeled, goat-anti-rabbit
immunoglobulin G (Sigma) for 30 min and again washed three times with PBS.
Images were observed with a fluorescence microscope (Axioplan, Zeiss,
Germany).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
[3H]L-leucine uptake by oocytes expressing mutants of
KAAT1
Several mutations have been shown to affect transporter function in GAT1
(see reviews by Bismuth et al.,
1997; Kanner,
1994
). RNA from a multiple mutant of KAAT1 that included nine of
these mutations was expressed in Xenopus oocytes. Critical residues
of the mammalian GABA transporter (GAT1) and serotonin transporter (SERT) that
have been identified previously (Sur et
al., 1997
) were mutated singly in KAAT1. Uptake of
[3H]L-leucine was measured for 30 min in oocytes that had been
injected previously with cRNA. The multiple mutant had a much lower uptake
rate than WT (data not shown). Mutations W75L and R76E of KAAT1 abolished
transporter-mediated uptake (Fig.
3), as they do in GAT1. The mutation S523A had no discernible
effect (data not shown). In sharp contrast to these results,
[3H]L-leucine uptake was sevenfold higher in the Y147F mutant than
in KAAT1 WT (Fig. 3).
|
Amino acid modulation of currents in KAAT1 Y147F- and WT-injected
oocytes
The effects of amino acid substrates on KAAT1 WT currents are different in
K+ than in Na+ media, and the difference is pH dependent
(Castagna et al., 1998).
Currents with K+ are much lower than those with Na+ for
almost all amino acids, despite K+ being the predominant cation in
the midgut lumen in vivo. For WT at pH 8 in K+ buffer, the
selectivity sequence is Met>Leu>Phe>Thr>Gly>>Pro
(Fig. 4, inset), whereas in
Na+ buffer it is Pro>Thr>Gly=Met>Leu>Phe
(Fig. 4, inset). By contrast,
for the Y147F mutant in K+ buffer the sequence is Leu>Met>Phe
with Thr, Gly and Pro being barely detectable, whereas in Na+
buffer it is Met>Leu>Phe>Thr>Gly>Pro
(Fig. 4). Confirming the
results from tracers, the transporter net currents with both K+ and
Na+ were usually greater in Y147F than in WT; thus, the change in
substrate-evoked net inward currents measured in Y147F ranged from 10 nA to
1000 nA (Fig. 4), whereas for
WT they ranged from 10 nA to 225 nA (Fig.
4, inset). For methionine in Na+ medium, the Y147F
value was 1000 nA compared with <100 nA for WT. Proline uptake in
Na+ was a notable exception; the Y147F value was 10 nA compared
with 225 nA for WT.
|
Kinetics of [3H]L-leucine uptake by KAAT1 WT and
Y147F
The concentration dependence of labeled leucine uptake in Na+
medium was measured in oocytes incubated with [3H]L-leucine for
Y147F and WT (Fig. 5A; WT data
are enlarged in Fig. 5B). The
Vm was much higher in Y147F than in WT but the
Km was the same in mutant and WT.
|
Activation of leucine-associated currents by leucine and
K+
The L-leucine dependence of the current that flows into oocytes clamped at
-60 mV was measured in the presence of 100 mmoll-1 K+ at
pH 7.6 and 25°C. For Y147F, the Vmax was 755 nA, the
apparent Km was 3.0 mmoll-1 L-leucine and the
apparent Hill coefficient () was 1.0
(Fig. 6A;
Table 1). The WT
Vmax (734 nA) was nearly the same value
(Fig. 6B;
Table 1). Thus, paradoxically,
the maximal leucine-associated current was unchanged by the mutation, whereas,
as discussed above, the Vmax for [3H ]L-leucine
uptake was increased by the mutation.
|
|
The ability of K+ to activate L-leucine-evoked current was
measured in oocytes that were clamped at -60 mV in the presence of 0.2
mmoll-1 L-leucine at pH 7.6 and 25°C. For Y147F, the change in
net inward current Vmax was 1103 nA, the Km was 93
mmoll-1 K+ and was 1.9
(Fig. 7A;
Table 1). In WT, values were
estimated by non-linear regression analysis of the obtained [K+]
value up to the physiologically plausible [K+] limit of 100
mmoll-1. In this case, the estimated Vmax (1136
nA) was similar to that of Y147F, the apparent Km (245
mmoll-1) was somewhat higher and the apparent Hill coefficient
(
=1.2) was less than that of the mutant
(Fig. 7B;
Table 1).
|
Amino-acid-independent Li+ and Na+ currents in
Y147F- and WT-injected oocytes
GAT1 exhibits GABA-independent, chloride-enhanced, Cs+- and
Li+-activated currents (Mager
et al., 1996). Similarly, Li+- and
Na+-activated currents, which were greater than those in controls,
were exhibited in both KAAT1 Y147F and WT. However, no K+ currents
were observed in oocytes expressing Y147F or WT in the absence of amino acids
(Fig. 8). Thus, even though
KAAT1 is <40% identical to GAT1 (whereas other neurotransmitter
transporters are >80% identical to each other), it appears to retain many
aspects of the molecular mechanism for co-transport that are observed in GAT1.
The Cl--dependent Li+ current is approximately 30%
higher in the KAAT1 mutant than in WT (Fig.
8). The Li+ current appears to vary directly with the
transport activity in both KAAT1 and GAT1; thus, both Li+ current
and amino acid transport are increased in the KAAT1 mutant, whereas the
Li+ current decreases by 30% when GABA transport is lost in the
GAT1 mutant.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
KAAT1 is a classical nutrient, neutral amino acid transporter
Our hypothesis that KAAT1 is a System B type co-transporter
(Stevens, 1992) is supported
by the interaction between lysine and KAAT1 at pH values of 7.5 and 10.
Classical studies on whole cells
(Christensen, 1984
) and
membrane vesicles (Liu and Harvey,
1996
) had shown that the electrical charge on an amino acid plays
a critical role in its interaction with transporters, but the co-existence of
heterogeneous transporters in vivo complicated the analysis. The
overexpression of the cloned KAAT1 transporter in Xenopus eggs
largely eliminated the complication. At pH 7.5, where lysine is mainly
cationic, few lysine-evoked inward currents beyond controls were observed; by
contrast, at pH 10, where lysine is mainly zwitterionic, lysine-associated
current was substantial (Fig.
9, upper panel). The same result was obtained by measuring
lysine-induced currents at the two pH values
(Fig. 9, lower panel).
|
Amino-acid- and cation-binding sites
The most striking effects of the KAAT1 mutation Y147F are the enhanced
leucine uptake and the altered pattern of aminoacid-evoked currents in
voltage-clamped oocytes (Fig.
4). These effects contrast sharply to the complete loss of
transport activity in the equivalent position 140 of GAT1. Noting that Y140 is
conserved in all SNF transporters and that the mutation at position 140 from
tyrosine to phenylalanine is essentially the loss of a hydroxyl group, Bismuth
et al. (1997) suggested that
Y140 may be the amino-binding residue of GAT1. They speculated that an H-bond
may form between the oxygen atom of tyrosine 140 and an amine hydrogen of
GABA. KAAT1 Y147 is equivalent to GAT1 Y140 because the amino terminus of
KAAT1 is seven amino acids longer than that of GAT1. However, if leucine acts
on KAAT1, as GABA does on GAT1, then our finding of a sevenfold increase in
transporter activity in KAAT1 Y147F would be contrary to this hypothesis
there is no longer a tyrosine oxygen atom present to bind to the amino
hydrogen of leucine yet the transport is increased dramatically (Figs
5,
10). Despite the increased
[3H]L-leucine transport (Figs
3,
5) and leucine-associated net
inward current in the Y147F mutant over WT (Figs
4,5,6,7,8),
the impact on proline-associated net inward current is similar to that
observed for GABA in GAT1 and the primary substrates of other SNF
transporters. Proline transport in KAAT1 exclusively requires Na+
in the manner that GABA requires Na+ in GAT1. Notably, the mutant
loses the capability for proline-evoked currents
(Fig. 4).
|
Nevertheless, the two dramatic effects loss of GABA transport in
the GAT1 mutant, Y140F, and gain of leucine transport in the KAAT1 mutant,
Y147F support the hypothesis that this site is crucial for solute
transport. As Na+ is a substrate for all SNF transporters, perhaps
the binding of Na+ (or K+) at KAAT1 Y147 or the
regulation of amino acid or ion binding at a distant site, is dependent on
events at KAAT1 Y147. The presence of electronegative cysteine residues at
positions C151 and C154, near Y147, in KAAT1
(Fig. 1, lower box) but not at
the corresponding locations of GAT1 are consistent with this view. The
corresponding Y147 site is also crucial in CAATCH1, which has both transporter
and channel functions (Feldman et al.,
2000) but is not a co-transporter
(Quick and Stevens, 2001
). The
mutation Y147F also enhanced transporter activity and altered the amino acid
current-inducing spectrum in CAATCH1, as well as abolishing its channel
function (Stevens et al.,
2002
).
Neurotransmitter vs nutrient amino acid transporters
A comparison of the neurotransmitter transporter GAT1 with the two nutrient
amino acid transporters, KAAT1 and CAATCH1, demonstrates some fundamental
differences between nutrient and neurotransmitter amino acid transporters
(Fig. 10).
GAT1, KAAT1 and CAATCH1 are similar in that they are all activated by
alkaline metal cations. GAT1 and KAAT1, but not CAATCH1, are also activated by
Cl-. GAT1 has 599 amino acid residues, whereas KAAT1 has 634
residues and CAATCH1 has 633 residues, with moderately high sequence identity
(30%) between GAT1 and the nutrient transporters. All three transporters have
12 transmembrane domains and fit a common template, with minimal insertions or
deletions of residues (Fig. 1;
see also Nelson and Lill,
1994). All three transporters are energized directly by
H+ V-ATPase-generated voltages and possibly indirectly by
K+ or Na+ gradients that are secondary to the primary
proton transport (Moriyama et al.,
1992
; Nelson and Harvey,
1999
).
The three types of transporter differ in several important ways:
Implications of oocyte current data for living insects
Neither the Na+ nor Li+ currents reported here in
oocytes are likely to have immediately obvious physiological significance for
caterpillars because the [Na+] is extremely low in these
plant-eating insects (Harvey et al.,
1975) and Li+ is not detectable. However, an
Na+ channel may be important in mosquito larvae that have
moderately high [Na+] in the hemolymph
(Edwards, 1982
).
Transmembrane currents in Xenopus oocytes that are overexpressing
xenic transporters are commonly thought to be carried by ions that are
co-transported with an organic substrate. For example, Gaustella et al. (1990)
initially interpreted inward currents measured in GAT1-injected oocytes as the
co-transport of Na+, Cl- and GABA into the oocytes, and
this interpretation has been experimentally proven by Loo et al.
(2000). Similarly, currents
measured in KAAT1-injected oocytes
(Castagna et al., 1997
) and
CAATCH1-injected oocytes (Feldman et al.,
2000
) were interpreted as the co-transport of Na+ and
amino acids into the oocytes. However, Quick and Stevens
(2001
) showed that
3H-labeled amino acid uptake has no stoichiometric relationship to
inward currents measured simultaneously with a two-electrode voltage clamp in
the same CAATCH1-expressing oocyte. They concluded that amino acid uptake and
ion fluxes are not coupled in the oocytes. This finding was unexpected because
the magnitude of the inward current depends upon the amino acid that is
present (Feldman et al.,
2000
).
The implication that cation flux and amino acid transport are not coupled
in living caterpillar midgut, from which CAATCH1 was cloned, is even more
puzzling, because the only driving force for amino acid uptake in
vivo is the transapical membrane voltage, which is thought to drive
cation-coupled amino acid uptake (reviewed by
Castagna et al., 1997;
Harvey et al., 1998
). If
cation flux and amino acid uptake are not coupled then the apically localized,
electrogenic H+ V-ATPase would have no obvious function, and the
source of energy for driving the amino acid uptake would be unknown.
Three solutions to this puzzle come to mind. First, as the lipid
composition of plasma membranes in oocytes is different from that in posterior
midgut columnar cells, transport and uptake may be uncoupled in
CAATCH1-expressing oocytes but not in midgut. As a precedent, Ca+
transport and Ca2+-ATPase activity become uncoupled when `boundary
lipids' are removed from the membrane containing the Ca2+ pump
(Hidalgo, 1982). Second,
CAATCH1 may possess a coupling `slip'
(Gerencser and Stevens, 1994
),
i.e. the stoichiometric coupling of amino acid uptake and ionic fluxes may
vary, from say 1:1 to zero. Such a slip is well documented in the coupling of
ATP hydrolysis to proton transport by H+ V-ATPases
(Moriyama and Nelson, 1988
).
Finally, perhaps CAATCH1 has other functions than cation:amino acid
co-transport. For example, it may function as a channel that mediates fluxes
that alkalinize the midgut.
On the other hand, KAAT1 appears to be a cation:neutral amino acid
co-transporter, as suggested by Castagna et al.
(1998). Thus, leucine induces
large increases in K+ and Na+ currents in
KAAT1-expressing oocytes (Bossi et al.,
1999
). Moreover, K+ and Na+, but not
Li+, currents coincide with leucine uptake, suggesting to Bossi et
al. (2000
) that there are two
populations of transporters present, one in which amino acid uptake and cation
flux are coupled and another in which they are not. Leucine uptake was maximal
near the physiological pH of 10 (Vincenti
et al., 2000
). Acidic pH led to complete inhibition of coupled
currents in the presence of either Na+ or K+
(Peres and Bossi, 2000
); these
results suggested that: "the operation of the transporter is maximal in
the physiologically alkaline native environment". The dependence of
ionic current upon amino acid identity
(Fig. 4) and the absence of
K+ current in both Y147F and WT in the absence of amino acids
(Fig. 7) are consistent with
this view. Taken together, these studies suggest that KAAT1 functions as a
co-transporter in living insects, where it couples the fluxes of the
predominant cation, K+, to the uptake of neutral amino acids.
Conclusions
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bismuth, Y., Kavanaugh, M. P. and Kanner, B. I.
(1997). Tyrosine 140 of the gamma-aminobutyric acid transporter
GAT-1 plays a critical role in neurotransmitter recognition. J.
Biol. Chem. 272,16096
-16102.
Bossi, E., Sacchi, V. F. and Peres, A. (1999). Ionic selectivity of the coupled and uncoupled currents carried by the amino acid transporter KAAT1. Pflugers Arch. 438,788 -796.[CrossRef][Medline]
Bossi, E., Vincenti, S., Sacchi, V. F. and Peres, A. (2000). Simultaneous measurements of ionic currents and leucine uptake at the amino acid cotransporter KAAT1 expressed in Xenopus laevis oocytes. Biochim. Biophys. Acta 1495,34 -39.[CrossRef][Medline]
Castagna, M., Shayakul, C., Trotti, D., Sacchi, F., Harvey, W.
R. and Hediger, M. A. (1997). Molecular characteristics of
mammalian and insect amino acid transporters: implications for amino acid
homeostasis. J. Exp. Biol.
200,269
-286.
Castagna, M., Shayakul, C., Trotti, D., Sacchi, V. F., Harvey,
W. R. and Hediger, M. A. (1998). Cloning and characterization
of KAAT1, a potassium-coupled amino acid transporter. Proc. Natl.
Acad. Sci. USA 95,5395
-5400.
Christensen, H. N. (1984). Organic ion transport during seven decades. The amino acids. Biochim. Biophys. Acta 779,255 -269.[Medline]
Edwards, H. A. (1982). Ion concentration and activity in the haemolymph of Aedes aegypti larvae. J. Exp. Biol. 101,143 -151.
Feldman, D. H., Harvey, W. R. and Stevens, B. R.
(2000). A novel electrogenic amino acid transporter is activated
by K+ or Na+, is alkaline pH-dependent, and is
Cl- independent. J. Biol. Chem.
275,24518
-24526.
Gerencser, G. A. and Stevens, B. R. (1994).
Thermodynamics of symport and antiport catalyzed by cloned or native
transporters. J. Exp. Biol.
196, 59-75.
Guastella, J., Nelson, N., Nelson, H., Czycyk, L., Keynan, S., Miedel, M. C., Davidson, N., Lester, H. A. and Kanner, B. I. (1990). Cloning and expression of a rat brain GABA transporter. Science 249,1303 -1306.[Medline]
Harvey, W. R., Maddrell, S. H. P., Telfer, W. H. and Wieczorek, H. (1998). H+ V-ATPases energize animal plasma membranes for secretion and absorption of ions and fluids. Am. Zool. 38,426 -441.
Harvey, W. R., Wood, J. L., Quatrale, R. P. and Jungreis, A. M. (1975). Cation distributions across the larval and pupal midgut of the Lepidopteran, Hyalophora cecropia, in vivo. J. Exp. Biol. 63,321 -330.[Abstract]
Hidalgo, C. (1982). Lipidprotein interactions and calcium transport in sarcoplasmic reticulum Ann.N. Y. Acad. Sci. 402,561 -562.[Medline]
Kanner, B. I. (1994). Sodium-coupled neurotransmitter transport: structure, function and regulation. J. Exp. Biol. 96,237 -249.
Kleinberger-Doron, N. and Kanner, B. I. (1994).
Identification of tryptophan residues critical for the function and targeting
of the gammaaminobutyric acid transporter (subtype A). J. Biol.
Chem. 269,3063
-3067.
Lehrach, H., Diamond, D., Wozney, J. M. and Boedtker, H. (1977). RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16,4743 -4751.[Medline]
Liu, Z. and Harvey, W. R. (1996). Cationic lysine uptake by System R+ and zwitterionic lysine uptake by System B in brush border membrane vesicles from larval Manduca sexta midgut. Biochim. Biophys. Acta 1282,32 -38.[Medline]
Loo, D. D., Eskandari, S., Boorer, K. J., Sarkar, H. K. and
Wright, E. M. (2000). Role of Cl- in electrogenic
Na+-coupled cotransporters GAT1 and SGLT1. J. Biol.
Chem. 275,37414
-37422.
Mager, S., Kleinberger-Doron, N., Keshet, G. I., Davidson, N.,
Kanner, B. I. and Lester, H. A. (1996). Ion binding and
permeation at the GABA transporter GAT1. J. Neurosci.
16,5405
-5414.
Mbungu, D., Ross, L. S. and Gill, S. S. (1995). Cloning, functional expression, and pharmacology of a GABA transporter from Manduca sexta. Arch. Biochem. Biophys. 318,489 -497.[CrossRef][Medline]
Moriyama, Y., Maeda, M. and Futai, M. (1992).
The role of V-ATPase in neuronal and endocrine systems. J. Exp.
Biol. 172,171
-178.
Moriyama, Y. and Nelson, N. (1988). The vacuolar H+-ATPase, a proton pump controlled by a slip. Prog. Clin. Biol. Res. 273,387 -394.[Medline]
Nelson, N. and Harvey, W. R. (1999). Vacuolar
and plasma membrane proton-adenosinetriphosphatases. Physiol.
Rev. 79,361
-385.
Nelson, N. and Lill, H. (1994). Porters and
neurotransmitter transporters. J. Exp. Biol.
196,213
-228.
Pantanowitz, S., Bendahan, A. and Kanner, B. I.
(1993). Only one of the charged amino acids located in the
transmembrane alpha-helices of the gamma-aminobutyric acid transporter
(subtype A) is essential for its activity. J. Biol.
Chem. 268,3222
-3225.
Peres, A. and Bossi, E. (2000). Effects of pH
on the uncoupled, coupled and pre-steady-state currents at the amino acid
transporter KAAT1 expressed in Xenopus oocytes. J.
Physiol. 525,83
-89.
Quick, M. and Stevens, B. R. (2001). Amino acid
transporter CAATCH1 is also an amino acid-gated cation channel. J.
Biol. Chem. 276,33413
-33418.
Stevens, B. R. (1992). Amino acid transport in intestine. In Mammalian Amino Acid Transport: Mechanisms and Control (ed. M. S. Kilberg and D. Haussinger), pp.149 -164. New York: Plenum.
Stevens, B. R. (2001). Theory and methods in nutrient membrane transport. In Surgical Research (ed. W. W. Souba and D. W. Wilmore), pp. 845-856. San Diego: Academic Press.
Stevens, B. R., Feldman, D. H., Liu, Z. and Harvey, W. R.
(2002). Conserved tyrosine-147 plays a critical role in
ligand-gated current of the epithelial cation/amino acid transporter/channel
(CAATCH1). J. Exp. Biol.
205,2545
-2553.
Sur, C., Betz, H. and Schloss, P. (1997). A
single serine residue controls the cation dependence of substrate transport by
the rat serotonin transporter. Proc. Natl. Acad. Sci.
USA 94,7639
-7644.
Vincenti, S., Castagna, M., Peres, A. and Sacchi, V. F. (2000). Substrate selectivity and pH dependence of KAAT1 expressed in Xenopus laevis oocytes. J. Membr. Biol. 174,213 -224.[CrossRef][Medline]