title: The discovery of slowness: low-capacity transport and slow anion channel gating by the glutamate transporter EAAT5.
abstract: Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na(+) and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na(+) and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a K(m)= 61 ± 11 μM. Binding of Na(+) to the empty transporter is associated with a K(m) = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a K(m) = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na(+) binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system.
authors.affiliation: Department of Chemistry, Binghamton University, Binghamton, New York, USA.
authors.forename: Armanda
authors.initials: A
authors.lastname: Gameiro
authors.forename: Simona
authors.initials: S
authors.lastname: Braams
authors.forename: Thomas
authors.initials: T
authors.lastname: Rauen
authors.forename: Christof
authors.initials: C
authors.lastname: Grewer
mesh_terms: D001692:Biological Transport; D004553:Electric Conductivity; D050582:Excitatory Amino Acid Transporter 5 / Q000378:metabolism*; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000378:metabolism; D015640:Ion Channel Gating; D007700:Kinetics; D007834:Lasers; D010782:Photolysis; D012964:Sodium / Q000378:metabolism
chemical_list: C440256:4-methoxy-7-nitroindolinyl-glutamate; D050582:Excitatory Amino Acid Transporter 5; D005971:Glutamates; D007211:Indoles; D012964:Sodium