ABSTRACT
The neutral amino acid transporter ASCT2 catalyses uncoupled anion flux across the cell membrane in the presence of transported substrates, such as alanine. Here, we report that ASCT2 conducts anions already in the absence of transported substrates through a leak anion-conducting pathway. The properties of this leak anion conductance were studied by electrophysiological recording from ASCT2-expressing HEK293 cells. We found that the leak anion conductance was inhibited by the binding of the newly characterized inhibitors benzylserine and benzylcysteine to ASCT2. These inhibitors competitively prevent binding of transported substrates to ASCT2, suggesting that they bind to the ASCT2 binding site for neutral amino acid substrates. The leak anion conductance exhibits permeation properties that are similar to the substrate-activated anion conductance of ASCT2, preferring hydrophobic anions such as thiocyanate. Inhibition of the leak anion conductance by benzylserine requires the presence of extracellular, but not intracellular Na(+). The apparent affinity of ASCT2 for extracellular Na(+) was determined as 0.3 mm. Interestingly, a Na(+)-dependent leak anion conductance with similar properties was previously reported for the related excitatory amino acid transporters (EAATs), suggesting that this leak anion conductance is highly conserved within the EAAT protein family.
Subject(s)
Amino Acid Transport System ASC/antagonists & inhibitors , Anions/metabolism , Sodium/physiology , Amino Acid Transport System ASC/genetics , Amino Acid Transport System X-AG/metabolism , Animals , DNA, Complementary/genetics , Electric Stimulation , Electrophysiology , Glutamate Plasma Membrane Transport Proteins , Ion Channels/drug effects , Ion Channels/metabolism , Kinetics , Membrane Potentials/physiology , Minor Histocompatibility Antigens , Patch-Clamp Techniques , Protein Binding , Rats , Sodium/metabolism , Structure-Activity Relationship , Symporters/metabolismABSTRACT
Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp, Gln, Ala), Asp804 (Glu, Asn, Ala), and Asp808 (Glu, Asn, Ala). Electrogenic cation transport properties of these 12 mutants were analyzed in two-electrode voltage-clamp experiments on Xenopus laevis oocytes by measuring the voltage dependence of K+-stimulated stationary currents and pre-steady-state currents under electrogenic Na+/Na+ exchange conditions. Whereas mutants D804N, D804A, and D808A hardly showed any Na+/K+ pump currents, the other constructs could be classified according to the [K+] and voltage dependence of their stationary currents; mutants N776A and E779Q behaved similarly to the wild-type enzyme. Mutants E779D, E779A, D808E, and D808N had in common a decreased apparent affinity for extracellular K+. Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the current-voltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na+/Na+ exchange currents are rather voltage-independent and at potentials above -60 mV faster than the wild type. Thus, the characteristic voltage-dependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn776 and Asp804, respectively, decreases the extracellular Na+ affinity.
Subject(s)
Sodium-Potassium-Exchanging ATPase/physiology , Amino Acid Substitution , Animals , Binding Sites , Electrophysiology , Kinetics , Membrane Potentials , Mutation , Oocytes , Potassium/metabolism , Rats , Sodium/metabolism , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolism , Xenopus laevisABSTRACT
Steady-state and pre-steady-state currents of Asn(776) mutants of Na,K-ATPase are presented. The stationary current generated by N776Q strongly depends on the membrane potential, but has a negative slope, opposite to that of the wild-type enzyme. The apparent rate constant of the reaction sequence E(1)P(Na(+)) <--> E(2)P + Na(+) of this mutant is rather independent of the membrane potential and is at resting and depolarizing membrane potential higher than that of the wild-type enzyme. Thus, the voltage-dependent increase of the rate coefficient of the wild type that is associated with extracellular Na(+) rebinding is almost absent in the N776Q mutant. These findings indicate that dislocating the carboxamide group of Asn(776) decreases the affinity of sodium at its extracellular binding site.
