Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT

Structure of the CLC-1 chloride channel from Homo sapiens.

CLC channels mediate passive Cl- conduction, while CLC transporters mediate active Cl- transport coupled to H+ transport in the opposite direction. The distinction between CLC-0/1/2 channels and CLC transporters seems undetectable by amino acid sequence. To understand why they are different functionally we determined the structure of the human CLC-1 channel. Its 'glutamate gate' residue, known to mediate proton transfer in CLC transporters, adopts a location in the structure that appears to preclude it from its transport function. Furthermore, smaller side chains produce a wider pore near the intracellular surface, potentially reducing a kinetic barrier for Cl- conduction. When the corresponding residues are mutated in a transporter, it is converted to a channel. Finally, Cl- at key sites in the pore appear to interact with reduced affinity compared to transporters. Thus, subtle differences in glutamate gate conformation, internal pore diameter and Cl- affinity distinguish CLC channels and transporters

Systems analysis of guard cell membrane transport for enhanced stomatal dynamics and water use efficiency

Stomatal transpiration is at the centre of a crisis in water availability and crop production that is expected to unfold over the next 20-30 years. Global water usage has increased 6-fold in the past 100 years, twice as fast as the human population, and is expected to double again before 2030, driven mainly by irrigation and agriculture. Guard cell membrane transport is integral to controlling stomatal aperture and offers important targets for genetic manipulation to improve crop performance. However, its complexity presents a formidable barrier to exploring such possibilities. With few exceptions, mutations that increase water use efficiency commonly have been found to do so with substantial costs to the rate of carbon assimilation, reflecting the trade-off in CO2 availability with suppressed stomatal transpiration. One approach yet to be explored in any detail relies on quantitative systems analysis of the guard cell. Our deep knowledge of transport and homeostasis in these cells gives real substance to the prospect for ‘reverse engineering’ of stomatal responses, using in silico design in directing genetic manipulation for improved water use and crop yields. Here we address this problem with a focus on stomatal kinetics, taking advantage of the OnGuard software and models of the stomatal guard cell (www.psrg.org.uk) recently developed for exploring stomatal physiology. Our analysis suggests that manipulations of single transporter populations are likely to have unforeseen consequences. Channel gating, especially of the dominant K+ channels, appears the most favorable target for experimental manipulation

SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure

SLC6A14 (ATB0,+) is a sodium- and chloride-dependent neutral and dibasic amino acid transporter that regulates the distribution of amino acids across cell membranes. The transporter is overexpressed in many human cancers characterized by an increased demand for amino acids; as such, it was recently acknowledged as a novel target for cancer therapy. The knowledge on the molecular mechanism of SLC6A14 transport is still limited, but some elegant studies on related transporters report the involvement of the 12 transmembrane \u3b1-helices in the transport mechanism, and describe structural rearrangements mediated by electrostatic interactions with some pivotal gating residues. In the present work, we constructed a SLC6A14 model in outward-facing conformation via homology modeling and used molecular dynamics simulations to predict amino acid residues critical for substrate recognition and translocation. We docked the proteinogenic amino acids and other known substrates in the SLC6A14 binding site to study both gating regions and the exposed residues involved in transport. Interestingly, some of these residues correspond to those previously identified in other LeuT-fold transporters; however, we could also identify a novel relevant residue with such function. For the first time, by combined approaches of molecular docking and molecular dynamics simulations, we highlight the potential role of these residues in neutral amino acid transport. This novel information unravels new aspects of the human SLC6A14 structure-function relationship and may have important outcomes for cancer treatment through the design of novel inhibitors of SLC6A14-mediated transport

Solute channels of the outer membrane: from bacteria to chloroplasts

Chloroplasts, unique organelles of plants, originated from endosymbiosis of an ancestor of today's cyanobacteria with a mitochondria-containing host cell. It is assumed that the outer envelope membrane, which delimits the chloroplast from the surrounding cytosol, was thus inherited from its Gram-negative bacterial ancestor. This plastid-specific membrane is thus equipped with elements of prokaryotic and eukaryotic origin. In particular, the membrane-intrinsic outer envelope proteins (OEPs) form solute channels with properties reminiscent of porins and channels in the bacterial outer membrane. OEP channels are characterised by distinct specificities for metabolites and a quite peculiar expression pattern in specialised plant organs and plastids, thus disproving the assumption that the outer envelope is a non-specific molecular sieve. The same is true for the outer membrane of Gram-negative bacteria, which functions as a permeability barrier in addition to the cytoplasmic membrane, and embeds different classes of channel pores. The channels of these prokaryotic prototype proteins, ranging from unspecific porins to specific channels to ligand-gated receptors, are exclusively built of P-barrels. Although most of the OEP channels are formed by P-strands as well, phylogeny based on sequence homology alone is not feasible. Thus, the comparison of structural and functional properties of chloroplast outer envelope and bacterial outer membrane channels is required to pinpoint the ancestral OEP `portrait gallery'

Anion Conducting States of Excitatory Amino Acid Transporters

Excitatory amino acid transporters (EAATs) are secondary active, electrogenic transporters which translocate L-glutamate (glu) against its concentration gradient using the co-transport of 3 Na+, 1 H+, and the counter-transport of 1 K+ ion. In addition, these carriers possess a thermodynamically uncoupled anion channel that fluxes Cl- but is promiscuous with several permeant anionic species. The roles of EAATs are to shape the spatio-temporal profile of released glu in both the synaptic cleft and extra-synaptic regions as well as maintaining a low ambient extracellular concentration of glu. This transport activity regulates activation of glu receptors and thus regulates excitatory neurotransmission. Using a combination of techniques, we were successful in identifying inward oriented transporter conformations which allow transitions to open channels states. This observation was enabled by our development of a novel method to isolate EAAT1 in the inward facing conformation. While constrained to these conformations, currents with the same macroscopic amplitudes as conducting states mediated by the outward facing, Na+ bound states were observed. The persistence of currents is indicative of a channel gating mechanism that is insensitive to transporter orientation and that the anion channel is open during the majority of the transport cycle. Additional conducting states allows for a larger contribution of the anion channel function of EAATs to shape cellular function then previously assumed. Next we investigated the gating mechanism of the anion channel. We assayed for the ability of Na+ to gate the anion channel in both glial (EAAT1 and EAAT2) and neuronal (EAAT3 and EAAT4) isoforms. We discovered that the glial isoforms are not gated by Na+ but are leak channels with an open probability and single channel conductance that is insensitive to Na+ concentrations. In contrast, neuronal EAAT isoforms EAAT3 and EAAT4 both display Na+ dependent channel activity. This is the first example of a significant functional difference between glial and neuronal transporter isoforms of the solute carrier 1 (SLC1) family. The research presented here allows for a greater understanding of low open probability channel states and the possible contributions of the EAAT anion channel to the functioning of the nervous system

Elevator-type mechanisms of membrane transport

Membrane transporters are integral membrane proteins that mediate the passage of solutes across lipid bilayers. These proteins undergo conformational transitions between outward- and inward-facing states, which lead to alternating access of the substrate-binding site to the aqueous environment on either side of the membrane. Dozens of different transporter families have evolved, providing a wide variety of structural solutions to achieve alternating access. A sub-set of structurally diverse transporters operate by mechanisms that are collectively named 'elevator-type'. These transporters have one common characteristic: they contain a distinct protein domain that slides across the membrane as a rigid body, and in doing so it 'drags" the transported substrate along. Analysis of the global conformational changes that take place in membrane transporters using elevator-type mechanisms reveals that elevator-type movements can be achieved in more than one way. Molecular dynamics simulations and experimental data help to understand how lipid bilayer properties may affect elevator movements and vice versa

Structural and biochemical characterization of the human neutral amino acid transporter ASCT2

The membrane transporter ASCT2 is a neutral amino acid exchanger that is in particular important to maintain glutamine homeostasis in human cells. ASCT2 has increasingly gained attention as a promising target for drug design in anti-cancer and anti-retroviral therapy. We used biochemical methods in combination with single-particle cryo-EM to functionally and structurally characterize human ASCT2. Structures of ASCT2 in inward- and outward-facing conformations, each in glutamine-free and -bound states, have revealed a one-gate elevator mechanism of transport, where the access to the binding side is controlled by the same flexible helical hairpin loop (the HP2 loop) on both sides of the membrane. Interestingly, ASCT2 in detergent micelle environment appears to favour inward-facing conformations, but reconstitution of ASCT2 in lipid nanodiscs promotes outward-facing states of the transporter, demonstrating a major effect of the bilayer environment on the energy landscape. Moreover, ASCT2 is an example, where an integrated approach using computer-aided compound design, functional testing, and structure determination with cryo-EM, was used to rationally design novel inhibitors for ASCT2. Our results provide a basis for future functional and structural characterisation of ASCT2