Dataset Information

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh.

ABSTRACT: GltPh from Pyrococcus horikoshii is a homotrimeric Na(+)-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na(+) and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na(+) binds with low affinity to the apoprotein (Kd 120 mm), with a particularly low kon value (5.1 m(-1)s(-1)). At least two sodium ions bind before aspartate. The binding of Na(+) requires a very high activation energy (Ea 106.8 kJ mol(-1)) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na(+) concentration present. Binding of aspartate was not observed in the absence of Na(+), whereas in the presence of high Na(+) concentrations (above the Kd for Na(+)) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (kon of 1.4 × 10(5) m(-1)s(-1)), with low Ea and Q10 values (42.6 kJ mol(-1) and 1.8, respectively). We conclude that Na(+) binding is most likely the rate-limiting step for substrate binding.

SUBMITTER: Hanelt I

PROVIDER: S-EPMC4481202 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh.

Hänelt Inga I Jensen Sonja S Wunnicke Dorith D Slotboom Dirk Jan DJ

The Journal of biological chemistry 20150428 26

GltPh from Pyrococcus horikoshii is a homotrimeric Na(+)-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na(+) and aspartate binding to GltPh. Trypto ...[more]

PMID: 25922069

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Project description: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.

Dataset Information

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh.

Publications

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh.

Similar Datasets

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