Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity.

The sodium-potassium-chloride transporter NKCC1 of the SLC12 family performs Na+ -dependent Cl- - and K+ -ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na+ , K+ , and 2 Cl- ) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- -ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl- -sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.

ATP13A2 deficiency disrupts lysosomal polyamine export.

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome-a parkinsonism with dementia1-and early-onset Parkinson's disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson's disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system.

Transport unplugged: KCCs are regulated through an N-terminal plug of the ion pathway.

The ability to regulate transmembrane ion transport in response to various cues is vital to any living cell. In neurons, one key example of critical ion control relates to the extrusion of chloride mediated by the potassium-chloride-cotransporters (KCC1-4). In a recent hallmark study, Chi et␣al (2021) report cryo-EM structures of human KCC1 and KCC3b, delineating in detail how regulation by phosphorylation inhibits the transport activity. The authors also identify a stabilizing binding site for nucleotides and speculate on its functional role.

A non-helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition.

MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+ -dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.

Structure and autoregulation of a P4-ATPase lipid flippase.

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.

Poly(Sitosterol)-Based Hydrophobic Blocks in Amphiphilic Block Copolymers for the Assembly of Hybrid Vesicles.

Amphiphilic block copolymer and lipids can be assembled into hybrid vesicles (HVs), which are an alternative to liposomes and polymersomes. Block copolymers that have either poly(sitostryl methacrylate) or statistical copolymers of sitosteryl methacrylate and butyl methacrylate as the hydrophobic part and a poly(carboxyethyl acrylate) hydrophilic segment are synthesized and characterized. These block copolymers assemble into small HVs with soybean L-α-phosphatidylcholine (soyPC), confirmed by electron microscopy and small-angle X-ray scattering. The membrane's hybrid nature is illustrated by fluorescence resonance energy transfer between labeled building blocks. The membrane packing, derived from spectra when using Laurdan as an environmentally sensitive fluorescent probe, is comparable between small HVs and the corresponding liposomes with molecular sitosterol, although the former show indications of transmembrane asymmetry. Giant HVs with homogenous distribution of the block copolymers and soyPC in their membranes are assembled using the electroformation method. The lateral diffusion of both building blocks is slowed down in giant HVs with higher block copolymer content, but their permeability toward (6)-carboxy-X-rhodamine is higher compared to giant vesicles made of soyPC and molecular sitosterol. This fundamental effort contributes to the rapidly expanding understanding of the integration of natural membrane constituents with designed synthetic compounds to form hybrid membranes.

Accuracy of self-reported opioid use in orthopaedic trauma patients.

PURPOSE: Opioids have long been a mainstay of treatment for pain in patients with orthopaedic injuries, but little is known about the accuracy of self-reported narcotic usage in orthopaedic trauma. The purpose of this study is to evaluate the accuracy of self-reported opioid usage in orthopaedic trauma patients. METHODS: A retrospective review of all new patients presenting to the orthopaedic trauma clinic of a level 1 trauma centre with a chief complaint of recent orthopaedic-related injury over a 2-year time frame was conducted. Participants were administered a survey inquiring about narcotic usage within the prior 3 months. Responses were cross-referenced against a query of a statewide prescription drug monitoring program system. RESULTS: The study comprised 241 participants; 206 (85.5%) were accurate reporters, while 35 (14.5%) were inaccurate reporters. Significantly increased accuracy was associated with hospital admission prior to clinic visit (ß = - 1.33; χ2 = 10.68, P < 0.01; OR: 0.07, 95% CI 0.01-0.62). Decreased accuracy was associated with higher pre-visit total morphine equivalent dose (MED) (ß = 0.002; χ2 = 11.30, P < 0.01), with accurate reporters having significantly lower pre-index visit MED levels compared to underreporters (89.2 ± 208.7 mg vs. 249.6 ± 509.3 mg; P = 0.04). An Emergency Department (ED) visit prior to the index visit significantly predicted underreporting (ß = 0.424; χ2 = 4.28, P = 0.04; OR: 2.34, 95% CI 1.01-5.38). CONCLUSION: This study suggests that most new patients presenting to an orthopaedic trauma clinic with acute injury will accurately report their narcotic usage within the preceding 3 months. Prior hospital admissions increased the likelihood of accurate reporting while higher MEDs or an ED visit prior to the initial visit increased the likelihood of underreporting.

GluN3-Containing NMDA Receptors in the Rat Nucleus Accumbens Core Contribute to Incubation of Cocaine Craving.

Cue-induced cocaine craving progressively intensifies (incubates) after withdrawal from cocaine self-administration in rats and humans. In rats, the expression of incubation ultimately depends on Ca2+-permeable AMPARs that accumulate in synapses onto medium spiny neurons (MSNs) in the NAc core. However, the delay in their accumulation (∼1 month after drug self-administration ceases) suggests earlier waves of plasticity. This prompted us to conduct the first study of NMDAR transmission in NAc core during incubation, focusing on the GluN3 subunit, which confers atypical properties when incorporated into NMDARs, including insensitivity to Mg2+ block and Ca2+ impermeability. Whole-cell patch-clamp recordings were conducted in MSNs of adult male rats 1-68 d after discontinuing extended-access saline or cocaine self-administration. NMDAR transmission was enhanced after 5 d of cocaine withdrawal, and this persisted for at least 68 d of withdrawal. The earliest functional alterations were mediated through increased contributions of GluN2B-containing NMDARs, followed by increased contributions of GluN3-containing NMDARs. As predicted by GluN3-NMDAR incorporation, fewer MSN spines exhibited NMDAR-mediated Ca2+ entry. GluN3A knockdown in NAc core was sufficient to prevent incubation of craving, consistent with biotinylation studies showing increased GluN3A surface expression, although array tomography studies suggested that adaptations involving GluN3B also occur. Collectively, our data show that a complex cascade of NMDAR and AMPAR plasticity occurs in NAc core, potentially through a homeostatic mechanism, leading to persistent increases in cocaine cue reactivity and relapse vulnerability. This is a remarkable example of experience-dependent glutamatergic plasticity evolving over a protracted window in the adult brain.SIGNIFICANCE STATEMENT "Incubation of craving" is an animal model for the persistence of vulnerability to cue-induced relapse after prolonged drug abstinence. Incubation also occurs in human drug users. AMPAR plasticity in medium spiny neurons (MSNs) of the NAc core is critical for incubation of cocaine craving but occurs only after a delay. Here we found that AMPAR plasticity is preceded by NMDAR plasticity that is essential for incubation and involves GluN3, an atypical NMDAR subunit that markedly alters NMDAR transmission. Together with AMPAR plasticity, this represents profound remodeling of excitatory synaptic transmission onto MSNs. Given the importance of MSNs for translating motivation into action, this plasticity may explain, at least in part, the profound shifts in motivated behavior that characterize addiction.

Screw Thread Configuration Has No Effect on Outcomes of In Situ Fixation for Stable Slipped Capital Femoral Epiphysis.

BACKGROUND: No consensus exists regarding the optimal surgical management of slipped capital femoral epiphysis (SCFE). Treatment goals include avoiding slip progression and sequelae such as avascular necrosis (AVN). Factors associated with surgical implants merit further research. This study investigates the effect of screw thread configuration and the number of screws on surgical outcomes. METHODS: A total of 152 patients undergoing cannulated, stainless steel, in situ screw fixation of SCFE between January 2005 and April 2018 were included. Procedure laterality, screw number and thread configuration (partially threaded/fully threaded), bilateral diagnosis, Loder classification, final follow-up, patient demographics, and endocrinopathy history were analyzed. Primary outcomes were return to the operating room (ROR), AVN, hardware failure/removal, and femoroacetabular impingement (FAI). RESULTS: Most patients received a single (86.2%), partially threaded (81.6%) screw; most were unilateral (67.8%) and stable (79.6%). Mean follow-up was 2.0±2.7 years, with a 15.8% rate of ROR, 5.3% exhibiting AVN, 6.6% exhibiting FAI, and 9.2% experiencing hardware failure/removal. Number of screws was the sole predictor of ROR [odds ratio (OR)=3.35, 95% confidence interval (CI): 1.18-9.49]. Unstable SCFE increased the odds of AVN (OR=38.44; 95% CI: 4.35-339.50) as did older age (OR=1.43, 95% CI: 1.01-2.03). Female sex increased risk for FAI (OR=4.87, 95% CI: 1.20-19.70), and bilateral SCFE elevated risk for hardware failure/removal versus unilateral SCFE (OR=4.41, 95% CI: 1.39-14.00). Screw thread configuration had no significant effect on any outcome (for each, P ≥0.159). CONCLUSIONS: Rates of ROR, AVN, FAI, and hardware failure/removal did not differ between patients treated with partially threaded or fully threaded screws. The use of 2 screws was associated with an increased likelihood of ROR. These findings suggest that screw thread configuration has no impact on complication rates, whereas screw number may be an important consideration in SCFE fixation. LEVEL OF EVIDENCE: Level III-retrospective cohort study.

The Effect of Asset Degradation on Trust in Swarms: A Reexamination of System-Wide Trust in Human-Swarm Interaction.

OBJECTIVE: The effects of asset degradation on trust in human-swarm interaction were investigated through the lens of system-wide trust theory. BACKGROUND: Researchers have begun investigating contextual features that shape human interactions with robotic swarms-systems comprising assets that coordinate behavior based on their nearest neighbors. Recent work has begun investigating how human trust toward swarms is affected by asset degradation through the lens of system-wide trust theory, but these studies have been marked by several limitations. METHOD: In an online study, the current work manipulated asset degradation and measured trust-relevant criteria in a within-subjects design and addressed the limitations of past work. RESULTS: Controlling for swarm performance (i.e., target acquisition), asset degradation and trust (i.e., reliance intentions) in swarms were negatively related. In addition, as degradation increased, perceptions of swarm cohesion, obstacle avoidance, target acquisition, and terrain exploration efficiency decreased, the latter two of which (coupled with the reliance intentions criterion) support the tenets of system-wide trust theory as well as replicate and extend past work on the effects of asset degradation on trust in swarms. CONCLUSION: Human-swarm interaction is a context in which system-wide trust is relevant, and future work ought to investigate how to calibrate human trust toward swarm systems. APPLICATIONS: Based on these findings, design professionals should prioritize ways to depict swarm performance and system health such that humans do not abandon trust in systems that are still functional yet not over-trust those systems which are indeed performing poorly.

Trusting Autonomous Security Robots: The Role of Reliability and Stated Social Intent.

OBJECTIVE: This research examined the effects of reliability and stated social intent on trust, trustworthiness, and one's willingness to endorse use of an autonomous security robot (ASR). BACKGROUND: Human-robot interactions in the domain of security is plausible, yet we know very little about what drives acceptance of ASRs. Past research has used static images and game-based simulations to depict the robots versus actual humans interacting with actual robots. METHOD: A video depicted an ASR interacting with a human. The ASR reviewed access credentials and allowed entrance once verified. If the ASR could not verify one's credentials it instructed the visitor to return to the security checkpoint. The ASR was equipped with a nonlethal device and the robot used this device on one of the three visitors (a research confederate). Manipulations of reliability and stated social intent of the ASR were used in a 2 × 4 between subjects design (N = 320). RESULTS: Reliability influenced trust and trustworthiness. Stated social intent influenced trustworthiness. Participants reported being more favorable toward use of the ASR in military contexts versus public contexts. CONCLUSION: The study demonstrated that reliability of the ASR and statements regarding the ASR's stated social intent are important considerations influencing the trust process (inclusive of intentions to be vulnerable and trustworthiness perceptions). APPLICATION: If robotic systems are authorized to use force against a human, public acceptance may be increased with availability of the intent-based programming of the robot and whether or not the robot's decision was reliable.

Crystal structure of the integral membrane diacylglycerol kinase.

Diacylglycerol kinase catalyses the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria. For half a century, this 121-residue kinase has served as a model for investigating membrane protein enzymology, folding, assembly and stability. Here we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are, however, at variance with a published solution NMR model in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.

High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions.

P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N and C termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p-Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than 1 order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol 4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate. Using thrombin as the protease, Cdc50p remains intact and in complex with Drs2p, which is cleaved at two positions, namely after Arg104 and after Arg 1290, resulting in a homogeneous sample lacking 104 and 65 residues from its N and C termini, respectively. Removal of the 1291-1302-amino acid region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, whereas the 1-104 N-terminal residues have an additional but more modest significance for activity. The present results therefore reveal that trimming off appropriate regions of the terminal extensions of Drs2p can greatly increase its ATPase activity in the presence of PI4P and demonstrate that relief of such auto-inhibition remains compatible with subsequent regulation by PI4P. These experiments suggest that activation of the Drs2p-Cdc50p flippase follows a multistep mechanism, with preliminary release of a number of constraints, possibly through the binding of regulatory proteins in the trans-Golgi network, followed by full activation by PI4P.

Structural insights into electron transfer in caa3-type cytochrome oxidase.

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36 Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.

The effect of propensity to trust and perceptions of trustworthiness on trust behaviors in dyads.

Research on trust has burgeoned in the last few decades. Despite the growing interest in trust, little is known about trusting behaviors in non-dichotomous trust games. The current study explored propensity to trust, trustworthiness, and trust behaviors in a new computer-mediated trust relevant task. We used multivariate multilevel survival analysis (MMSA) to analyze behaviors across time. Results indicated propensity to trust did not influence trust behaviors. However, trustworthiness perceptions influenced initial trust behaviors and trust behaviors influenced subsequent trustworthiness perceptions. Indeed, behaviors fully mediated the relationship of trustworthiness perceptions over time. The study demonstrated the utility of MMSA and the new trust game, Checkmate, as viable research methods and stimuli for assessing the loci of trust.

On the molecular mechanism of flippase- and scramblase-mediated phospholipid transport.

Phospholipid flippases are key regulators of transbilayer lipid asymmetry in eukaryotic cell membranes, critical to many trafficking and signaling pathways. P4-ATPases, in particular, are responsible for the uphill transport of phospholipids from the exoplasmic to the cytosolic leaflet of the plasma membrane, as well as membranes of the late secretory/endocytic pathways, thereby establishing transbilayer asymmetry. Recent studies combining cell biology and biochemical approaches have improved our understanding of the path taken by lipids through P4-ATPases. Additionally, identification of several protein families catalyzing phospholipid 'scrambling', i.e. disruption of phospholipid asymmetry through energy-independent bi-directional phospholipid transport, as well as the recent report of the structure of such a scramblase, opens the way to a deeper characterization of their mechanism of action. Here, we discuss the molecular nature of the mechanism by which lipids may 'flip' across membranes, with an emphasis on active lipid transport catalyzed by P4-ATPases. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Crystal structure of the ß2 adrenergic receptor-Gs protein complex.

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The ß(2) adrenergic receptor (ß(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric ß(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the ß(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the ß(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

Structure and function of an irreversible agonist-ß(2) adrenoceptor complex.

G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human ß(2) adrenergic receptor (ß(2)AR) as a guide, we designed a ß(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent ß(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound ß(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 µs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters.

An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di- and tripeptide-bound complexes of a bacterial homologue of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co-crystal structures combined with functional studies reveal that biochemically distinct peptide-binding sites likely operate within the POT/PTR family of proton-coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.