Experimental conditions can obscure the second high-affinity site in LeuT

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.