A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop

Neuronal and glial glutamate transporters remove the excitatory neurotransm itter glutamate from the synaptic cleft. The proteins belong to a large fam ily of secondary transporters, which includes bacterial glutamate transport ers. The C-terminal half of the glutamate transporters is well conserved an d thought to contain the translocation path and the binding sites for subst rate and coupling ions. A serine-rich sequence motif in this part of the pr oteins is located in a putative intracellular loop. Cysteine-scanning mutag enesis was applied to this loop in the glutamate transporter GltT of Bacill us stearothermophilus. The loop was found to be largely intracellular, but three consecutive positions in the conserved serine-rich motif (S269, S270, end E271) are accessible from both sides of the membrane. Single-cysteine mutants in the serine-rich motif were still capable of glutamate transport, but modification with N-ethylmaleimide blocked the transport activity in s ix mutants (T267C, A268C, S269C, S270C, E271C, and T272C), Two milimolars L -glutamate effectively protected against the modification of the cysteines at position 269-271 from the periplasmic side of the membrane but was unabl e to protect cysteine modification from the cytoplasmic side of the membran e. The results indicate that the conserved serine-rich motif in the glutama te transporter forms a reentrant loop, a structure that is found in several ion channels but is unusual for transporter proteins. The reentrant loop i s of crucial importance for the function of the glutamate transporter.