IDEN OF ANION PERMEATION PATHEWAY/BINDING SITES IN GLUTAMATE TRANSPORTERS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glutamate transporters are integral membrane proteins responsible for clearance of glutamate from the synaptic cleft following rounds of neurotransmission. Glutamate transporters are secondary transporters and couple substrate uptake to membrane gradients of sodium, potassium and protons. In addition to thermodynamically coupled ion fluxes they also demonstrate an uncoupled anion flux, which is gated by substrate binding. Recently we have determined the crystal structure of a glutamate transporter homologue from Pyroccocus horikoshii (GltPh) (Nature 431: 811-18, 2004; Nature 445: 387-93, 2007). It has been consequently demonstrated that this protein captures many functional features of its mammalian homologues, including anion permeation. We are planning to use anions containing heavy atoms such as Br-, SeCN- and AuCN- to attempt to locate anion permeation pathway/binding sites in the protein. GltPh crystals diffract routinely to 4-5 ¿ resolution with a small number diffracting to 3.2-3.5 ¿ . We are preparing crystals soaked in solutions that have anions replaced with the above compounds. Br- and AuCN- have been previously used by us for soaks and do not significantly affect quality of the crystals. However, in the past we have never collected rigorous data on these soaks and this is the gap we would like to fill during the proposed experiment. For each soak we plan to collect an energy scan and a full dataset at K edge for SeCN- and Br- soaks and at L-III edge for AuCN-. The work will also include screening crystals for higher diffraction quality for each condition. Because membrane crystals diffract weekly, we opt for screening directly at the synchrotron rather than at the in-house diffractometer.