Identification And Functional Study Of The Critical Amino Acid Residues In The 4b-4c Loop Of Glutamate Transporter

In the central nervous system,glutamate is the most important excitatory amino acid transmitter.Since no enzyme systems in extracellular fluid can metabolize glutamate,the extracellular glutamate can only be removed by glutamate transporter for intracellular recycling.Otherwise,excessive glutamate between synapses will result to excitotoxic damage of nerve cells.Therefore,glutamate transporters actually paly a very important role in the prevention of neuroexcitotoxicity.Glutamate transporters of eukaryotes and prokaryotes are very similar in their structure,and both of them are comprised of three identical wedge-shaped subunits.However,the 4b-4c loop of eukaryotic glutamate transporter(EAAT)possesses more than 50 amino acid residues,which is much shorter in that of procaryotic glutamate transporter.It implys that the 4b-4c loop of EAATs probably undertake some special function.Recently,the crystal structure of EAATlcryst has been solved,however,26 of the original residues of the 4b-4c loop were deleted and 18 were mutated,so that it can not reflect the structure information of the native loop.Considering that it has been proved that the 4b-4c loop is closely implicated with the function and subcellular localization of EAATs,we speculate that there may be some critical residues involved in the loop.During the transport cycle,since the 4b-4c loop also support helical hairpin(HP)1,HP2 and transmembrane domain(TM)7 to complete local conformational changes,we conjecture that there may be a certain relative motion between the 4b-4c loop and TM8.Therefore,in order to verify our conjecture,we carried out the following two parts of the study.Part ?.Alanine scanning mutagenesis identifing critical amino acid residues in EAAT1 4b-4c loop involved in substrate transportTo investigate the role of the 4b-4c loop,we performed alanine-scanning mutagenesis on and observed dramatically decreased transport activity in T192A,Y194A,N242A,and G245A mutants.The surface expression of T192A and Y194A mutants even decreased by more than 80%,and most of them were detained in the cytoplasm.However,when T192 and Y194 were substituted with conservative residues,the transport activities and the surface expression of T192S and Y194F were largely recovered,and their Km values were comparable to the wild type EAAT1 as well.In contrast,N242 and G245 substituted with conservative amino acid could not rescue the uptake function,suggesting that N242 and G245 may play an irreplaceable role in the process of glutamate uptake.These results indicate that the 4b-4c loop of EAAT1 may not only affect glutamate uptake activity,but also influence the surface localization of EAAT1 by T192 and Y194.Part ?.Investigating substrate-induced motion between 4b-4c loop and TM8 of EAAT1To explore the spatial position and function of TM4 during the transport cycle,we introduced pairwise cysteine substitutions between the 4b-4c loop and TM8 in a cysteine-less version of EAAT1(CL-EAAT1).We observed pronounced inhibition of uptake activity by Cu(?)(1,10-phenanthroline)3(CuPh)for doubly substituted V238C/I469C and A243C/I469C mutants,but not for corresponding singly substituted CL-EAAT1 or for more than 20 other double-cysteine mutants.Dithiothreitol(DTT)treatment partially restored the uptake activity of the CuPh-treated V238C/1469C and A243C/I469C doubly substituted mutants,confirming that the effects of CuPh on these mutants were due to the formation of intramolecular disulfide bonds.Glutamate,KCl,and D,L-threo-?-benzyloxy-aspartate(DL-TBOA)weakened CuPh inhibition of the V238C/I469C mutant,but only KCl weakened CuPh inhibition of the V243C/I469C mutant,suggesting that the 4b-4c loop and TM8 are separated from each other in the inward-facing conformations of EAAT1.Our results suggest that the 4b-4c loop and TM8 are positioned in close proximity during the transport cycle,but are less closely spaced in the inward-facing conformation.In conclusion,this study found that there are four critical amino acid residues in the 4b-4c loop of EAAT1,which may significantly affect the uptake function of EAAT1 by altering the surface expression of EAAT1,substrate affinity and local conformational change.This study also proved that the 4b-4c loop and TM8a of EAAT1 can approach each other during the transport process,so then we reveal a wider range of interaction between the 4b-4c loop and the transport core of EAAT.