Abstract
Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.
Highlights
Reorientation of the binding sites of the glutamate transporter requires Kϩ translocation
Kϩ-induced Relocation of the Transporter Is Associated with Charge Movement—We first tested whether Kϩ-dependent reaction steps of EAAC1 are electrogenic, by locking the transporter in the Kϩ exchange mode (Fig. 1A, top)
The most important conclusion from this work is that transport of glutamate and the co-transported Naϩ ions is based on a charge compensation mechanism, in which intrinsic negative charge of the transporter binding site partially compensates for the three positive charges of the bound cations/substrate in the fully loaded transporter in the translocation step, and overcompensates for the single positive charge of the bound Kϩ ion in the relocation step of the empty transporter
Summary
Reorientation of the binding sites of the glutamate transporter requires Kϩ translocation. Because the movement of 3 Naϩ ions across the hydrophobic barrier of the membrane is expected to be unfavorable, it has been suggested that the positive charge of the cations is at least partially compensated for by negative charge of the binding site(s) (10 –12) Consistent with this suggestion, several negatively charged amino acid residues, which are highly conserved within the SLC1 family and sensitive to mutation, are located in the C-terminal transport domain [13,14,15,16]. Based on indirect evidence from the voltage dependence of steady-state glutamate-induced transport currents [10, 19], as well as measurements on fluorescently labeled transporters [11], it was speculated that the Kϩ relocation step(s) is associated with net negative charge movement, despite the positive charge of the transported Kϩ ion. The results are consistent with a multistep charge compensation mechanism, in which fast cation binding precedes electrogenic cation exchange through an overall negatively charged transport domain
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