title: Experimental conditions can obscure the second high-affinity site in LeuT.
abstract: Neurotransmitter:Na(+) symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na(+)-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na(+) ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na(+)-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na(+)-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.
authors.affiliation: Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, New York, USA.
authors.forename: Matthias
authors.initials: M
authors.lastname: Quick
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Britta
authors.initials: B
authors.lastname: Zehnpfennig
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
mesh_terms: D016477:Artifacts; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003902:Detergents / Q000378:metabolism; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D012964:Sodium / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D012964:Sodium; D007930:Leucine