Cooperation of the conserved aspartate 439 and bound amino acid substrate is important for high-affinity Na+ binding to the glutamate transporter EAAC1.
The neuronal glutamate transporter EAAC1 contains several conserved acidic amino acids in its transmembrane domain, which are possibly important in catalyzing transport and/or binding of co/countertransported cations. Here, we have studied the effects of neutralization by site-directed mutagenesis of three of these amino acid side chains, glutamate 373, aspartate 439, and aspartate 454, on the functional properties of the transporter. Transport was analyzed by whole-cell current recording from EAAC1-expressing mammalian cells after applying jumps in voltage, substrate, or cation concentration. Neutralization mutations in positions 373 and 454, although eliminating steady-state glutamate transport, have little effect on the kinetics and thermodynamics of Na(+) and glutamate binding, suggesting that these two positions do not constitute the sites of Na(+) and glutamate association with EAAC1. In contrast, the D439N mutation resulted in an approximately 10-fold decrease of apparent affinity of the glutamate-bound transporter form for Na(+), and an approximately 2,000-fold reduction in the rate of Na(+) binding, whereas the kinetics and thermodynamics of Na(+) binding to the glutamate-free transporter were almost unchanged compared to EAAC1(WT). Furthermore, the D439N mutation converted l-glutamate, THA, and PDC, which are activating substrates for the wild-type anion conductance, but not l-aspartate, into transient inhibitors of the EAAC1(D439) anion conductance. Activation of the anion conductance by l-glutamate was biphasic, allowing us to directly analyze binding of two of the three cotransported Na(+) ions as a function of time and [Na(+)]. The data can be explained with a model in which the D439N mutation results in a dramatic slowing of Na(+) binding and a reduced affinity of the substrate-bound EAAC1 for Na(+). We propose that the bound substrate controls the rate and the extent of Na(+) interaction with the transporter, depending on the amino acid side chain in position 439.
Tao
Zhen
Z
University of Miami School of Medicine, Miami, FL 33136, USA.
Grewer
Christof
C
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics; D002460:Cell Line; D017124:Conserved Sequence; D004563:Electrochemistry; D050580:Excitatory Amino Acid Transporter 3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D051381:Rats; D012964:Sodium / Q000378:metabolism*
D000838:Anions; D050580:Excitatory Amino Acid Transporter 3; C493398:Slc1a1 protein, rat; D001224:Aspartic Acid; D018698:Glutamic Acid; D012964:Sodium