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 homologue LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded centrally-located (S1) binding site next to two Na+. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a novel 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 brings to light the caution needed in the selection of experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.
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