Azmat Sohail

Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter

Neurotransmitter/sodium symporters (NSSs) are responsible for Na+-dependent reuptake of neurotransmitters and represent key targets for antidepressants and psychostimulants. LeuT, a prokaryotic NSS protein, constitutes a primary structural model for these transporters. Here we show that K+ inhibits Na+-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K+-induced conformational dynamics we measured fluorescence resonance energy transfer (FRET) between fluorescein site-specifically attached to inserted cysteines and Ni2+ bound to engineered di-histidine motifs (transition metal ion FRET). The measurements supported K+-induced closure of the transporter to the outside, which was counteracted by Na+ and substrate. Promoting an outward-open conformation of LeuT by mutation abolished the K+-effect. The K+-effect depended on an intact Na1 site and mutating the Na2 site potentiated K+ binding by facilitating transition to the inward-facing state. The data reveal an unrecognized ability of K+ to regulate the LeuT transport cycle.

Refinement of the Central Steps of Substrate Transport by the Aspartate Transporter GltPh: Elucidating the Role of the Na2 Sodium Binding Site.

Glutamate homeostasis in the brain is maintained by glutamate transporter mediated accumulation. Impaired transport is associated with several neurological disorders, including stroke and amyotrophic lateral sclerosis. Crystal structures of the homolog transporter GltPh from Pyrococcus horikoshii revealed large structural changes. Substrate uptake at the atomic level and the mechanism of ion gradient conversion into directional transport remained enigmatic. We observed in repeated simulations that two local structural changes regulated transport. The first change led to formation of the transient Na2 sodium binding site, triggered by side chain rotation of T308. The second change destabilized cytoplasmic ionic interactions. We found that sodium binding to the transiently formed Na2 site energized substrate uptake through reshaping of the energy hypersurface. Uptake experiments in reconstituted proteoliposomes confirmed the proposed mechanism. We reproduced the results in the human glutamate transporter EAAT3 indicating a conserved mechanics from archaea to humans.

The Environment Shapes the Inner Vestibule of LeuT

Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was crystallized in different conformations. Here, we investigated the inward-open state of LeuT. We compared LeuT in membranes and micelles using molecular dynamics simulations and lanthanide-based resonance energy transfer (LRET). Simulations of micelle-solubilized LeuT revealed a stable and widely open inward-facing conformation. However, this conformation was unstable in a membrane environment. The helix dipole and the charged amino acid of the first transmembrane helix (TM1A) partitioned out of the hydrophobic membrane core. Free energy calculations showed that movement of TM1A by 0.30 nm was driven by a free energy difference of ~15 kJ/mol. Distance measurements by LRET showed TM1A movements, consistent with the simulations, confirming a substantially different inward-open conformation in lipid bilayer from that inferred from the crystal structure.

Decrypting structural and functional changes in LeuTAa at atomic level employing LRET

Background Neurotransmitter:sodium symporters (NSS) are integral membrane proteins that mediate the reuptake of monoamine neurotransmitters previously released into the synaptic cleft. They are of pharmacological significance because they are the target of many clinically important drugs. LeuTAa, a leucine/alanine transporter is a bacterial homolog to NSS. Crystal structures of LeuTAa with open to outward, occluded and inward-facing states have already been resolved at high resolution. Hence, LeuTAa serves as a good paradigm for exploring the structurefunction relationship of NSS proteins.

Identifying forces that stabilize the oligomeric state of bacterial homologs of neurotransmitter transporters

Background Neurotransmitter transporters of the SLC1 and SCL6 family are found on presynaptic neurons and on glia cells. The function of these transporters is the termination of neurotransmission by the rapid removal of the neurotransmitter molecules from the synaptic cleft. These transporters couple substrate transport to ion gradients of sodium and chloride. Almost all of the eucaryotic transporters have been described to function as oligomers. However, the forces stabilizing the oligomeric state are not well understood. No crystal structures of eukaryotic transporters are available, but recently crystal structures of bacterial homologs thereof have been solved: GltPh (SLC 1 family) was found as a trimer, LeuT (SLC6 family) was crystallized as a dimer. These homologous crystal structures allow rationalizing on the driving forces that stabilize the eukaryotic counterparts.

Deciphering structural rearrangements during transport process in the bacterial transporter GltPh, homolog to mammalian glutamate transporter

Background Glutamate transporters are integral membrane proteins that catalyze the concentrative uptake of glutamate from the synapse by harnessing pre-existing ion gradients. In the central nervous system glutamate transporters are essential for normal development and function; they also are implicated in stroke, epilepsy and neurodegenerative diseases. The crystal structure of a eukaryotic glutamate transporter homologue from Pyrococcus horikoshii, is available at various conformations providing a structural framework for the determination of substrate and inhibitor binding to the transporter. In this study we aim to measure structural changes upon transport using lanthanide resonance energy transfer (LRET).

LRET-based distance measurements in the mammalian glutamate transporter EAAT3

Background EAAT3 (excitatory amino acid transporter 3) mediates the regulation of synaptic transmission by reuptake of glutamate in the synaptic cleft. It is distributed in neuronal membranes and is selectively enriched in the neurons of the hippocampus, cerebellum and the basal ganglia. It belongs to the family of soluble carrier family 1 member 1 (SLC1A1) and is expressed in kidney, a wide variety of epithelial tissues, brain and eyes.

LRET-based intramolecular distance measurements in LeuTAa

Background LeuTAa is a bacterial orthologue of mammalian Solute Carrier Class 6 (SLC6) neurotransmitter transporters from Aquifex aeolicus which transports leucine and alanine. SLC6 transporters are of great pharmacological interests because of their crucial role in neurotransmitter clearance. These proteins are also targets of many clinically relevant drugs. The crystal structure of LeuTAa has been resolved at atomic resolution of ~1.5 A. Although LeuT has a low overall sequence identity of about 20–25% to SLC6 members, the grade of conservation reaches around 55% in functionally critical transmembrane domains 1, 3, 6 and 8. For this very reason we are using LeuTAa as a good structural paradigm to explore the structural/functional information about SLC6-family members.

Luminescence resonance energy transfer-based intramolecular distance measurements in leucine transporter from Aquifex aeolicus

Background Solute carrier 6 (SLC6) membrane proteins are integral membrane proteins and of particular pharmacological interest because they are targets of many clinically important drugs. These SLC6 proteins play crucial roles ranging from nutrient uptake to neurotransmitter clearance. A leucine transporter LeuTAa from Aquifex aeolicus has been recognized as a bacterial orthologue of mammalian SLC6 family proteins. LeuTAa has been crystallized and its structure was resolved to high resolution. With respect to its kinship to other SLC6 transporters, though with low sequence identity (~20–25%), there are crucial regions in transmembrane segments 1, 3, 6 and 8 where conservation reaches ~50%. For this very reason LeuTAa provides a good structural paradigm to study homology models of SLC6 family members and learn more about the structure/function relationship in mammalian transporters.