Two conformational changes are associated with glutamate translocation by the glutamate transporter EAAC1.
Glutamate is transported across membranes by means of a carrier mechanism that is thought to require conformational changes of the transport protein. In this work, we have determined the thermodynamic parameters of glutamate and the Na+ binding steps to their extracellular binding sites along with the activation parameters of rapid, glutamate-induced processes in the transport cycle by analyzing the temperature dependence of glutamate transport at steady state and pre-steady state. Our results suggest that glutamate binding to the transporter is driven by a negative reaction enthalpy (DeltaH0 = -33 kJ/mol), whereas the tighter binding of the non-transportable inhibitor TBOA is caused by an additional increase in entropy. Processes linked to the binding of glutamate and Na+ to the transporter are associated with low activation barriers, indicative of diffusion-controlled reactions. The activation enthalpies of two processes in the glutamate translocation branch of the transport cycle were DeltaH++ = 95 kJ/mol and DeltaH++ = 120 kJ/mol, respectively. Such large values of DeltaH++ suggest that these processes are rate-limited by conformational changes of the transporter. We also found a large activation barrier for steady-state glutamate transport, which is rate-limited by the K+-dependent relocation of the empty transporter. Together, these results suggest that two conformational changes accompany glutamate translocation and at least one conformational change accompanies the relocation of the empty transporter. We interpret the data with an alternating access model that includes the closing and opening of an extracellular and an intracellular gate, respectively, in analogy to a hypothetical model proposed previously on the basis of the crystal structure of the bacterial glutamate transporter GltPh.
Mim
Carsten
C
Department of Physiology, University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, Florida 33136, USA.
Tao
Zhen
Z
Grewer
Christof
C
D000465:Algorithms; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D050580:Excitatory Amino Acid Transporter 3 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D050581:Excitatory Amino Acid Transporter 4 / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D008958:Models, Molecular; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014162:Transfection
C440256:4-methoxy-7-nitroindolinyl-glutamate; D050580:Excitatory Amino Acid Transporter 3; D050581:Excitatory Amino Acid Transporter 4; D005971:Glutamates; D007211:Indoles; C493400:SLC1A1 protein, human; C493391:SLC1A6 protein, human; C120673:benzyloxyaspartate; D001224:Aspartic Acid; D018698:Glutamic Acid; D012964:Sodium