Nurunisa Akyuz

Transport dynamics in a glutamate transporter homologue.

Glutamate transporters are integral membrane proteins that catalyse neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons. Their mechanism of action involves transitions between extracellular (outward)-facing and intracellular (inward)-facing conformations, whereby substrate binding sites become accessible to either side of the membrane. This process has been proposed to entail transmembrane movements of three discrete transport domains within a trimeric scaffold. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods. We propose that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent transmembrane movements and may be rate determining in the transport cycle.

Transport domain unlocking sets the uptake rate of an aspartate transporter

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these ‘elevator-like’ transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the ‘humanizing’ mutations favour structurally ‘unlocked’ intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.

Structural brain imaging in children and adolescents following prenatal cocaine exposure: preliminary longitudinal findings.

The brain morphometry of 21 children, who were followed from birth and underwent structural brain magnetic resonance imaging at 8-10 years, was studied. This cohort included 11 children with prenatal cocaine exposure (CE) and 10 noncocaine-exposed children (NCE). We compared the CE versus NCE groups using FreeSurfer to automatically segment and quantify the volume of individual brain structures. In addition, we created a pediatric atlas specifically for this population and demonstrate the enhanced accuracy of this approach. We found an overall trend towards smaller brain volumes among CE children. The volume differences were significant for cortical gray matter, the thalamus and the putamen. Here, reductions in thalamic and putaminal volumes showed a robust inverse correlation with exposure levels, thus highlighting effects on dopamine-rich brain regions that form key components of brain circuitry known to play important roles in behavior and attention. Interestingly, head circumferences (HCs) at birth as well as at the time of imaging showed a tendency for smaller size among CE children. HCs at the time of imaging correlated well with the cortical volumes for all subjects. In contrast, HCs at birth were predictive of the cortical volume only for the CE group. A subgroup of these subjects (6 CE, 4 NCE) was also scanned at 13-15 years of age. In subjects who were scanned twice, we found that the trend for smaller structures continued into teenage years. We found that the differences in structural volumes between the CE and NCE groups are largely diminished when the HCs are controlled for or matched by study design. Participants in this study were drawn from a unique longitudinal cohort and, while the small sample size precludes strong conclusions regarding the longitudinal findings reported, the results point to reductions in HCs and in specific brain structures that persist through teenage years in children who were exposed to cocaine in utero.

The Outer Pore and Selectivity Filter of TRPA1

TRPA1 (transient-receptor-potential-related ion channel with ankyrin domains) is a direct receptor or indirect effector for a wide variety of nociceptive signals, and thus is a compelling target for development of analgesic pharmaceuticals such as channel blockers. Recently, the structure of TRPA1 was reported, providing insights into channel assembly and pore architecture. Here we report whole-cell and single-channel current recordings of wild-type human TRPA1 as well as TRPA1 bearing point mutations of key charged residues in the outer pore. These measurements demonstrate that the glutamate at position 920 plays an important role in collecting cations into the mouth of the pore, by changing the effective surface potential by ~16 mV, while acidic residues further out have little effect on permeation. Electrophysiology experiments also confirm that the aspartate residue at position 915 represents a constriction site of the TRPA1 pore and is critical in controlling ion permeation.

Plug-N-Play: Mechanotransduction Goes Modular

Mechanosensitive ion channels initiate sensory signals by converting mechanical information into electrochemical signals. In this issue of Neuron (Zhao et al., 2016), a data-rich structure-function study on mammalian mechanosensitive Piezo channels reveals a modular protein architecture that includes a central pore module surrounded by a force-sensing module.

A facile approach for the in vitro assembly of multimeric membrane transport proteins

Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter.

Dance Lessons for Proteins: The Dynamics and Thermodynamics of a Sodium/Aspartate Symporter

Secondary active transporters harvest the energy of the ionic gradients to drive concentrative uptake of their substrates. This process entails a series of protein conformational transitions that are coupled to binding and unbinding of ions and substrates on the extracellular and intracellular sides of the membrane. Over the last decade, crystallography has provided a growing number of structural snapshots of the transport cycle for several ion-driven transporters. Already these structures, although intrinsically static, have revealed a remarkable plasticity encoded in the architecture of these proteins. Because internal dynamics is an essential feature of transporters, it is necessary to complement crystallographic studies with other techniques that provide information on the ensemble properties of these proteins as well as on the conformational dynamics of individual molecules. Here, we will discuss the emerging approaches to obtain thermodynamic and dynamic information on transporters using a sodium/aspartate symporter from Pyrococcus horikoshii, GltPh, as a model system. GltPh is a bacterial homologue of the mammalian glutamate transporters, for which crystal structures of several states have been determined, providing a framework for further mechanistic studies. We will discuss how within this system the equilibrium and kinetic studies based on the isothermal titration calorimetry, fluorescence, and electron paramagnetic resonance spectroscopy inform us on the energetic relationship between the key functional states, and mechanisms of coupling between transport cycle and ionic gradients. We will further describe how single molecule studies open doors to a detailed characterization of the timing and order of the conformational transitions underlying transport processes.

Transport Dynamics in a Glutamate Transporter Homologue

Glutamate transporters are secondary active transporters, which mediate glutamate uptake from the synaptic cleft into glial cells and neurons allowing multiple rounds of neural transmission and preventing glutamate-mediated excitotoxicity. their structural dynamics is key to their function: during transport cycle, they alternate between outward facing and inward facing states, in which the substrate-binding sites are accessible from the extracellular and intracellular solutions, respectively. Crystallographic studies of a bacterial homologue GltPh revealed that this isomerization entails trans-membrane movements of three discrete transport domains within a trimeric scaffold. Here, using single-molecule fluorescence resonance energy transfer (smFRET) imaging, we report real time observations of these movements for the first time. We labeled GltPh with donor and acceptor fluorescent dye pairs at positions for which the inter-dye distances differ in the outward and inward states. Our observations reveal FRET states with efficiencies consistent with those predicted from the crystal structures. Remarkably, the rates of transitions between these states are modulated by substrate binding, as well as by lipid surroundings.