©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sodium-independent Currents of Opposite Polarity Evoked by Neutral and Cationic Amino Acids in Neutral and Basic Amino Acid Transporter cRNA-injected Oocytes (*)

Aamir Ahmed (§) , George J. Peter , Peter M. Taylor , Alexander A. Harper , Michael J. Rennie

From the (1) Department of Anatomy and Physiology, University of Dundee, Dundee, DD1 4HN, Scotland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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= 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.


INTRODUCTION

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/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 blike amino acid transport could contribute toward absorption of amino acid.

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.


EXPERIMENTAL PROCEDURES

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 ( Vwas 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.


RESULTS

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 Vof -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 Iand Iwere 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 Vof -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 Iand Iwere dependent upon Vat 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 10Cmol). 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 Vof -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 Vof -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% minat an external phenylalanine concentration of 0.05 m M.

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 0.0034 = 0.59 pmol/oocytemin. 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.

Under normal experimental conditions the potassium cation has an outwardly directed electrochemical gradient (since [K]) = 89 ± 2 m M in NBAT cRNA injected oocytes (and 88 ± 3 m M in HO injected oocytes) n = 5 oocytes, compared to [K]= 2 m M). We tested the hypothesis that the outward Imight 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 Iby more than 50% (Fig. 4) in NBAT-expressing oocytes voltage clamped at a Vof -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 BaClin the superfusion medium during superfusion with 0.2 m M L-phenylalanine reduced the Ito 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.




DISCUSSION

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) 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 Iand Iwere voltage-dependent and sodium- and chloride-independent which for Iindicates 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) .

Phenylalanine:cationic amino acid exchange may account for a minor portion (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 Iwas 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.

It has been suggested that NBAT protein promotes the uptake of neutral and cationic amino acids by a single transport activity (system b) (6) in Xenopus oocytes (2, 3) . Amino acid transport by a system blike 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 blike transport activities may be distinct from one another.

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-like transport reflects the activity of more than a single protein product.

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.

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

Values are means ± S.E. for 3-10 oocytes for each measurement. A small inward current (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



FOOTNOTES

*
This work was supported in part by the Medical Research Council, UK and the University of Dundee. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: 44-382-344574; Fax: 44-382-345514; E-mail: aahmed@anatphys.dundee.ac.uk.

The abbreviations used are: NBAT, neutral and basic amino acid transporter; rBAT, rabbit basic amino acid transporter; TMA, tetramethylammonium.

A. Ahmed, G. J. Peter, P. M. Taylor, A. A. Harper, and M. J. Rennie, unpublished data.


ACKNOWLEDGEMENTS

We thank S. Grant for technical assistance.


REFERENCES
  1. Christensen, H. N. (1990) Physiol. Rev. 70, 43-77 [Free Full Text]
  2. 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. U. S. A. 91, 7548-7552 [Abstract]
  3. Wells, R. G., and Hediger, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5596-5600 [Abstract]
  4. Bertran, J., Werner, A., Moore, M. L., Stange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5601-5605 [Abstract]
  5. Magagnin, S., Bertran, J., Werner, A., Markovich, D., Biber, J., Palacin, M., and Murer, H. (1992) J. Biol. Chem. 267, 15384-15390 [Abstract/Free Full Text]
  6. Van Winkle, L. J., Campione, A. L., and Gorman, J. M. (1988) J. Biol. Chem. 263, 3150-3163 [Abstract/Free Full Text]
  7. Pickel, V. M., Nirenberg, M. J., Chan, J., Mosckovitz, R., Udenfriend, S., and Tate, S. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7779-7783 [Abstract/Free Full Text]
  8. Busch, A. E., Herzer, T., Waldegger, S., Schmidt, F., Palacin, M., Biber, J., Markovich, D., Murer, H., and Lang, F. (1994) J. Biol. Chem. 269,25581-25586 [Abstract/Free Full Text]
  9. Tate, S., Yan, N., and Udenfriend, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1-5 [Abstract]
  10. Closs, E. I., Albritton, L. M., Kim, J. W., and Cunningham, J. M. (1993) J. Biol. Chem. 268, 7538-7544 [Abstract/Free Full Text]
  11. Taylor, M. A., and Smith, D. (1987) Dev. Biol. 124, 287-290 [Medline] [Order article via Infotrieve]
  12. Van Winkle, L. J. (1993) Biochim. Biophys. Acta 1154, 157-172 [Medline] [Order article via Infotrieve]
  13. Veyhl, M., Spangenberg, J., Puschel, B., Poppe, R., Dekel, C., Fritzsch, G., Haase, W., and Koepsell, H. (1993) J. Biol. Chem. 268, 25041-25053 [Abstract/Free Full Text]
  14. Mosckovitz, R., Yan, N., Heimer, E., Felix, A., Tate, S. S., and Udenfriend, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4022-4026 [Abstract]
  15. Furriols, M., Chillaron, J., Mora, C., Castello, A., Bertran, J., Camps, M., Testar, X., Vilaro, S., Zorzano, A., and Palacin, M. (1993) J. Biol. Chem. 268, 27060-27068 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.