Sodium-independent Currents of Opposite Polarity Evoked by Neutral and Cationic Amino Acids in Neutral and Basic Amino Acid Transporter cRNA-injected Oocytes

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 mM 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 (Km = 0.34 +/- 0.02 mM, Imax = 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-beta-phenylalanine. Superfusion of L-arginine evoked a saturable (Km = 0.09 +/- 0.02 mM, Imax = -29.2 +/- 1.3 nA) inward current; L-lysine and D-arginine also evoked inward currents. L-Glutamate and beta-alanine failed to evoke any currents. Effluxes of L-[3H]phenylalanine and L-[3H]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.

The rBAT Gene Is Responsible for L-Cystine Uptake via the bo,+-like Amino Acid Transport System in a “Renal Proximal Tubular” Cell Line (OK Cells)

“…3 and 6B). The b 0,ϩ -like transport activity expressed by rBAT cRNA in oocytes shows hetero-exchange between L-arginine and L-leucine (3)(4)(5). This has also been suggested for this transport activity in OK cells in the present study (Fig.…”

“…The exchange of amino acids via system b 0,ϩ -like in oocytes expressing rBAT (3)(4)(5), and also suggested for this transport activity in OK cells by the results presented here, offers an additional mechanism of accumulation for cystine and dibasic amino acids in the brush border membranes of the epithelial cells of the S3 segment of the nephron; exchange with intracellular neutral amino acids could drive the active reabsorption of cystine and dibasic amino acids. Indeed, rBAT-specific accumulation of cystine occurs in oocytes where reduction to cysteine is prevented by diamide.…”

“…Recently, it has been reported that system b 0,ϩ -like expressed by rBAT in oocytes shows trans-stimulation, suggesting that this transport system is an amino acid exchanger (3)(4)(5). To provide further evidence for the presence of system b 0,ϩ -like transport activity in OK cells, we searched for transstimulation of L-arginine efflux by different amino acids.…”

“…Heteroexchange diffusion of these amino acids has been reported for this transport activity expressed by rabbit (3,4), rat (5), and human 1 rBAT cRNA in oocytes. rBAT protein is expressed in the brush border plasma membrane of both the proximal straight tubules of the nephron and the small intestine (6,7).…”

Epithelia & Membrane Transport

“…We hypothesized that for an obligatory exchange the affinity for substrate amino acids on either side of the membrane is likely to be similar. We tested this hypothesis by measuring the efflux of L_[3H] arginine from oocytes (3-4 days after injection of NBAT-cRNA) in the presence of L-arginine and D-Ieucine in the external medium, using methods described previously (Ahmed et al 1995). Values are means ± S.E.M.…”