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 anionconducting 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.
؉ . 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 currentvoltage 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 voltagedependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn 776 and Asp 804 , respectively, decreases the extracellular Na ؉ affinity.
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.
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