The Function And Conformational Changes Of 4b-4c Loop Of EAAT1 During The Transport Cycle

Glutamate is the major excitatory neurotransmitter in thebrain.The synaptic actions of this neurotransmitter are terminatedby glutamate transporters,which move the transmitter away from the synapse and back into the cells.Mammalian excitatory amino acid transporters(EAATs)reside on the plasma membrane of neurons and glia and are responsible for removing glutamate from the extracellular space,maintaining its concentration below neurotoxic levels.Disruption of this process is associated with several pathological conditions,ncluding ischemia,stroke,and amyotrophic lateral sclerosis.This transporter family is comprised of five EAAT subtypes EAAT1(excitatory amino acid transporter-1),GLT-1(glial glutamate transporter type one),EAAC1(excitatory amino acid carrier-1,EAAT3),EAAT4 and EAAT5.In eukaryotes,the transport of glutamate against its concentration gradient is driven by the co-transport of three sodium ions and a proton,and the counter transport of a potassium ion,resulting in a stoichiometric transport current.Several crystal structures of a glutamate transporter homolog,Gltph,from the archaeon Pyrococcus horikoshii are nowavailable.The structure reveals a trimer with a permeation pathway through each of the protomers,indicating that the protomer is the functional unit.This is also the case for the eukaryotic glutamate transporters.The protomer contains eight transmembrane domains and two oppositely orientedreentrant loops,one between domains 6 and 7(HP1)and the other between domains 7 and 8(HP2).Each subunit is composed of two domains:a scaffold domain(ScaD),including transmembrane helices TM1～TM2 and TM4～TM5;and a transport domain(TranD),including TM3,TM6～TM8 and re-entrant helical loops 1～2(HP1～HP2).The Gltph structures represent excellent models for the brain transporters.Despite these structural similarities,however,bacterial and mammalian transportersshare less than 40%sequence homology.One particular region of discrepancy is found in transmem branedomain(TM)4.This domain is comprised of three separate helices:4A,4B and 4C.In mammalian EAATs,a span of over 50amino acids(hereby called the 4b-4c loop),whichis absent from the bacterial sequence,extendsfrom between TM 4b and 4c.It is unclear,however,where these 50 extraresidues are located within the context of thebacterial crystal structure;that is,it is unknown whether they fill the vestibule or whether the reside outside the perimeter of the crystallized transporter protein.We use site-directed mutagenesis,substituted cysteine accessibility and cysteines cross linking to determine the location and functionality of the 4B-4C loop.The finding of this project may provide theoretical basis to illuminate the transport mechanism of EAATs.Part Ⅰ.Investigating the relationship between EAAT1 4b-4c loop and substrate transport channelTo probe the structural role of the TM4b-4c loop of EAATs in glutamate transport,each of its 57 amino acid residues were mutated to cysteine.Thirteen of the single mutants have very low transport activity.Aqueous accessibility of the introduced cysteines from the remaining mutants was then explored with membrane-permeant and membrane-impermeant sulfhydryl reagents under different conditions.F190C,V238C and A243C were affected by MTSET while Q189C,F190C,V238C,A243C and L244C were sensitive to MTSEA.Q189C and L244C transport activity was diminished in the presence of potassium,which is expected to favor the outward-facing conformation of the transporter.Inversely,L244C was protected by glutamate.The modification of A243C by MTSEA was enhanced by either potassium and glutamate or DL-TBOA.From these results,we suggest residues F190C,V238C,A243C may be located near the extracellular surface and TM4b-4c loop forms multiple reentrant membrane loops at the cell surface.Alternatively,F190C,V238C,and A243C may function in the transport pathway which is exposed to MTSET.In addition,Q189C,A243C and L244C are conformationally sensitive and may play arole in the transport cycle.Part Ⅱ.Investigating substrate-induced motion betwee 4b-4c loop and transmembrane domain 7 of the glutamate transporter EAAT1Mammalian EAATs contain a stretch of over 50 amino acids in TM4b-4c loop that is absent in the bacterial protein.To investigate the spatial proximity and functional significance of residues in glutamate transporter,cysteine pairs were introduced at positions A243 of TM4b-4c loop and T396 or A414 of TM7,respectively.An inhibition of transportby Cu(Ⅱ)(1,10-phenanthroline)3 and cadmium ions is observed in the double mutants A243C/T396C and A243C/S414C,but not inthe corresponding single mutants.Treatment with dithiothreitol after CuPh restores much of the transport activity.Inhibition by Cu(II)(1,10-phenanthroline)3 and cadmium is only observed when the cysteine pairs are introduced in the same polypeptide.Therefore,the formation of disulfide bonds occurs intra-molecularly.Glutamate,potassium and DL-TBOA facilitates cross-linking in the A243C/T396C transporter and it suggests that TM4b-4c loop and β-bridge region in TM7come into close proximity to each other in the inward-facing and outward-facing conformation of EAAT1.However,glutamate and potassium prevent cross-linking in A243C/A414C and it suggests that TM4b-4c loop and thetipof TM7 assume descend proximity in the inward-facing conformation of the transporter.These data provide evidence that substrate-induced structural rearrangements occur between TM4b-4c loop and TM7 during the transport cycle.