TY - JOUR
T1 - Mechanism of cation binding to the glutamate transporter EAAC1 probed with mutation of the conserved amino acid residue Thr101
AU - Tao, Zhen
AU - Rosental, Noa
AU - Kanner, Baruch I.
AU - Gameiro, Armanda
AU - Mwaura, Juddy
AU - Grewer, Christof
PY - 2010/6/4
Y1 - 2010/6/4
N2 - The glutamate transporter excitatory amino acid carrier 1 (EAAC1) catalyzes the co-transport of three Na+ ions, one H+ ion, and one glutamate molecule into the cell, in exchange for one K+ ion. Na + binding to the glutamate-free form of the transporter generates a high affinity binding site for glutamate and is thus required for transport. Moreover, sodium binding to the transporters induces a basal anion conductance, which is further activated by glutamate. Here, we used the [Na+] dependence of this conductance as a read-out of Na+ binding to the substrate-free transporter to study the impact of a highly conserved amino acid residue, Thr101, in transmembrane domain 3. The apparent affinity of substrate-free EAAC1 for Na+ was dramatically decreased by the T101A but not by the T101S mutation. Interestingly, in further contrast to EAAC1 WT, in the T101A mutant this [Na+] dependence was biphasic. This behavior can be explained by assuming that the binding of two Na+ ions prior to glutamate binding is required to generate a high affinity substrate binding site. In contrast to the dramatic effect of the T101A mutation on Na+ binding, other properties of the transporter, such as its ability to transport glutamate, were impaired but not eliminated. Our results are consistent with the existence of a cation binding site deeply buried in the membrane and involving interactions with the side chain oxygens of Thr101 and Asp367. A theoretical valence screening approach confirms that the predicted site of cation interaction has the potential to be a novel, so far undetected sodium binding site.
AB - The glutamate transporter excitatory amino acid carrier 1 (EAAC1) catalyzes the co-transport of three Na+ ions, one H+ ion, and one glutamate molecule into the cell, in exchange for one K+ ion. Na + binding to the glutamate-free form of the transporter generates a high affinity binding site for glutamate and is thus required for transport. Moreover, sodium binding to the transporters induces a basal anion conductance, which is further activated by glutamate. Here, we used the [Na+] dependence of this conductance as a read-out of Na+ binding to the substrate-free transporter to study the impact of a highly conserved amino acid residue, Thr101, in transmembrane domain 3. The apparent affinity of substrate-free EAAC1 for Na+ was dramatically decreased by the T101A but not by the T101S mutation. Interestingly, in further contrast to EAAC1 WT, in the T101A mutant this [Na+] dependence was biphasic. This behavior can be explained by assuming that the binding of two Na+ ions prior to glutamate binding is required to generate a high affinity substrate binding site. In contrast to the dramatic effect of the T101A mutation on Na+ binding, other properties of the transporter, such as its ability to transport glutamate, were impaired but not eliminated. Our results are consistent with the existence of a cation binding site deeply buried in the membrane and involving interactions with the side chain oxygens of Thr101 and Asp367. A theoretical valence screening approach confirms that the predicted site of cation interaction has the potential to be a novel, so far undetected sodium binding site.
UR - http://www.scopus.com/inward/record.url?scp=77952907248&partnerID=8YFLogxK
U2 - 10.1074/jbc.M110.121798
DO - 10.1074/jbc.M110.121798
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C2 - 20378543
AN - SCOPUS:77952907248
SN - 0021-9258
VL - 285
SP - 17725
EP - 17733
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 23
ER -