Abstract
Recently, a new model for glutamate uptake by glutamate transporters was proposed based on crystal structures of the bacterial glutamate transporter homolog Glt
Ph
. It was proposed that hairpin two (HP2) functions as the extracellular gate and that Na
+
and glutamate binding closes HP2, thereby allowing for the translocation of the glutamate binding pocket across the membrane. However, the conformation of HP2 in the apo state and the Na
+
bound state is unknown. We here use double site-directed spin-labeling electron paramagnetic resonance spectroscopy on the bacterial transporter Glt
Ph
from
Pyrococcus horikoshi
to examine conformational changes in HP2. Surprisingly, the cotransported substrates Na
+
and aspartate induce opposite movements of HP2. We find that in the apo state, HP2 is in a similar conformation as in the aspartate-bound closed state. Na
+
binding to the apo state opens HP2, whereas the subsequent binding of aspartate closes HP2. Our findings show that Na
+
binding opens and stabilizes the extracellular gate, thereby allowing for amino acid substrate binding. In contrast, in the absence of Na
+
and aspartate, HP2 closes, suggesting a potential mechanism for the translocation of the empty binding pocket necessary to complete the transport cycle. The finding that physiological Na
+
concentrations stabilize the open HP2 state would ensure that the outward-facing conformation of the transporter is maintained in physiological solutions and that glutamate transporters are ready to quickly bind glutamate released from glutamatergic synapses.
