Transmembrane Movement Research Articles

The gene set enrichment analysis of BRAF V600E -mutated HT-29 colorectal cancer cells treated of Selumetinib

Objective: BRAF activating mutations represents approximately 10% colorectal cancer (CRC) patients who are associated with a poor prognosis in stage II, III and IV. Therefore, to better understand the antitumor effects of Selumetinib in BRAF V600E -mutated CRC, gene set enrichment analysis (GSEA) of gene expression profile of BRAFV 600E - mutated HT-29 cells treated of Selumetinib is proposed in this study. Methods: The differentially expressed genes across the comparison of groups of non- treated and treated of Selumetinib in BRAF V600E -mutated HT-29 cells were analyzed by GSEA. Results: We identified the significant pathways of DNA dependent DNA replication, DNA replication, regulation of DNA replication, cell activation, leukocyte activation, receptor complex, endoplasmic reticulum membrane, RNA processing, RRNA processing, amino acid transmembrane transporter activity, nucleolar part, ribonucleoprotein complex, ribosome biogenesis and assembly and regulation of protein modification process for the upregulated genes and the significant pathways of response to hypoxia, response to extracellular stimulus, ATPase coupled to transmembrane movement of ions, ATPase coupled to transmembrane movement of ions phosphorylative mechanism, lipase activity, MAP kinase activity, phospholipase activity and actin filament for the downregulated genes relevant to resistance of Selumetinib in BRAF V600E - mutated HT-29 cells. Our data revealed that GLI2, IL8, LAT2, ICOSLG, TLR4, SPINK5, HDAC4, LAT and TGFB1 are the upregulated important genes and PDIA2, TGFB2, GIPR and GCGR are the importantly downregulated genes associated with resistance of BRAF V600E -mutated HT-29 cells to Selumetinib. Conclusion: We identified the upregulated and/or downregulated genes significantly associated with the resistance of Selumetinib in BRAF V600E -mutated HT-29 cells. These top upregulated and/or downregulated genes have significant value in prediction of sensitivity of CRC patients to Selumetinib and in identification of effective combination strategy for treatment of CRC patients.

Organoplatinum Compounds as Anion‐Tuneable Uphill Hydroxide Transporters

AbstractActive transport of ions uphill, creating a concentration gradient across a cell membrane, is essential for life. It remains a significant challenge to develop synthetic systems that allow active uphill transport. Here, a transport process fuelled by organometallic compounds is reported that creates a pH gradient. The hydrolysis reaction of PtII complexes results in the formation of aqua complexes that established rapid transmembrane movement (“flip‐flop”) of neutral Pt−OH species, leading to protonation of the OH group in the inner leaflet, generating OH− ions, and so increasing the pH in the intravesicular solution. The organoplatinum complex effectively transports bound hydroxide ions across the membrane in a neutral complex. The initial net flow of the PtII complex into the vesicles generates a positive electric potential that can further drive uphill transport because the electric potential is opposed to the chemical potential of OH−. The OH− ions equilibrate with this transmembrane electric potential but cannot remove it due to the relatively low permeability of the charged species. As a result, effective hydroxide transport against its concentration gradient can be achieved, and multiple additions can continuously drive the generation of OH− against its concentration gradient up to ΔpH>2. Moreover, the external addition of different anions can control the generation of OH− depending on their anion binding affinity. When anions displayed very high binding affinities towards PtII compounds, such as halides, the external anions could dissipate the pH gradient. In contrast, a further pH increase was observed for weak binding anions, such as sulfate, due to the increase of positive electric potential.

Conformational changes of a phosphatidylcholine flippase in lipid membranes.

Type 4 P-type ATPases (P4-ATPases) actively and selectively translocate phospholipids across membrane bilayers. Driven by ATP hydrolysis, P4-ATPases undergo conformational changes during lipid flipping. It is unclear how the active flipping states of P4-ATPases are regulated in the lipid membranes, especially for phosphatidylcholine (PC)-flipping P4-ATPases whose substrate, PC, is a substantial component of membranes. Here, we report the cryoelectron microscopy structures of a yeast PC-flipping P4-ATPase, Dnf1, in lipid environments. In native yeast lipids, Dnf1 adopts a conformation in which the lipid flipping pathway is disrupted. Only when the lipid composition is changed can Dnf1 be captured in the active conformations that enable lipid flipping. These results suggest that, in the native membrane, Dnf1 may stay in an idle conformation that is unable to support the trans-membrane movement of lipids. Dnf1 may have altered conformational preferences in membranes with different lipid compositions.

Molecular insight into coordination sites for substrates and their coupling kinetics in Na+ /HCO3- cotransporter NBCe1.

The secondary active transporter NBCe1 couples the transmembrane movement of Na+ and carbonate species with an apparent stoichiometry of 1Na+ :2HCO3- (the 'influx' mode) or 1Na+ :3HCO3- (the 'efflux' mode). Here, we employed molecular biology, electrophysiology and structural biology approaches to investigate the molecular mechanism for the transport coupling of Na+ and HCO3- in NBCe1. In Xenopus oocytes, decreasing extracellular [HCO3- ] from 66 to 4mm progressively decreases the Na+ affinity of NBCe1. However, decreasing [Na+ ] from 96 to 35mm has little effect on the HCO3- affinity. The residues responsible for the coordination of Na+ and HCO3- in the substrate pocket of NBCe1 were respectively determined by mutational and molecular simulation studies. Mutation to the residues for HCO3- coordination decreased the affinities of NBCe1 for both Na+ and HCO3- . However, mutation to the residues for Na+ coordination decreased the affinity for Na+ but had little effect on the affinity for HCO3- . Molecular simulation showed that NBCe1 has the capacity to coordinate only two ions of HCO3- or CO32- . We propose that (1) NBCe1 has an ordered substrate-binding kinetics with the binding of HCO3- preceding that of Na+ ; (2) NBCe1 operating in the influx mode moves 1Na+ +2HCO3- , whereas NBCe1 in the efflux mode moves 1Na+ +1HCO3- +1CO32- . The substrate-binding kinetics of NBCe1 is distinct from the known kinetics models of many other Na+ -coupled transporters with Na+ binding preceding the driven solute. KEY POINTS: Under physiological conditions, the secondary active transporter NBCe1 can operate in the 'influx' mode with an apparent stoichiometry of 1Na+ :2HCO3- or in the 'efflux' mode with an apparent stoichiometry of 1Na+ :3HCO3- . NBCe1 has an ordered substrate-binding kinetics with HCO3- preceding the binding of Na+ . The kinetics of NBCe1 is distinct from the known kinetics of many other Na+ -driven cotransporters for which the binding of Na+ usually precedes the driven substrate. The residues responsible for the coordination of Na+ and those for carbonate species in the substrate-binding pocket of NBCe1 were determined by mutation and molecular simulation studies. The substrate-binding pocket of NBCe1 contains just two coordination sites for HCO3- or CO32- . It is proposed that NBCe1 in the influx mode moves 1Na+ +2HCO3- across the plasma membrane, whereas NBCe1 in the efflux mode moves 1Na+ +1HCO3- +1CO32- .

Salt\u2011responsive transcriptome analysis of canola roots reveals candidate genes involved in the key metabolic pathway in response to salt stress

Salinity is a major constraint on crop growth and productivity, limiting sustainable agriculture in arid regions. Understanding the molecular mechanisms of salt-stress adaptation in canola is important to improve salt tolerance and promote its cultivation in saline lands. In this study, roots of control (no salt) and 200 mM NaCl-stressed canola seedlings were collected for RNA-Seq analysis and qRT-PCR validation. A total of 5385, 4268, and 7105 DEGs at the three time points of salt treatment compared to the control were identified, respectively. Several DEGs enriched in plant signal transduction pathways were highly expressed under salt stress, and these genes play an important role in signaling and scavenging of ROS in response to salt stress. Transcript expression in canola roots differed at different stages of salt stress, with the early-stages (2 h) of salt stress mainly related to oxidative stress response and sugar metabolism, while the late-stages (72 h) of salt stress mainly related to transmembrane movement, amino acid metabolism, glycerol metabolism and structural components of the cell wall. Several families of TFs that may be associated with salt tolerance were identified, including ERF, MYB, NAC, WRKY, and bHLH. These results provide a basis for further studies on the regulatory mechanisms of salt stress adaptation in canola.

An electrodiffusive neuron-extracellular-glia model for exploring the genesis of slow potentials in the brain.

Within the computational neuroscience community, there has been a focus on simulating the electrical activity of neurons, while other components of brain tissue, such as glia cells and the extracellular space, are often neglected. Standard models of extracellular potentials are based on a combination of multicompartmental models describing neural electrodynamics and volume conductor theory. Such models cannot be used to simulate the slow components of extracellular potentials, which depend on ion concentration dynamics, and the effect that this has on extracellular diffusion potentials and glial buffering currents. We here present the electrodiffusive neuron-extracellular-glia (edNEG) model, which we believe is the first model to combine compartmental neuron modeling with an electrodiffusive framework for intra- and extracellular ion concentration dynamics in a local piece of neuro-glial brain tissue. The edNEG model (i) keeps track of all intraneuronal, intraglial, and extracellular ion concentrations and electrical potentials, (ii) accounts for action potentials and dendritic calcium spikes in neurons, (iii) contains a neuronal and glial homeostatic machinery that gives physiologically realistic ion concentration dynamics, (iv) accounts for electrodiffusive transmembrane, intracellular, and extracellular ionic movements, and (v) accounts for glial and neuronal swelling caused by osmotic transmembrane pressure gradients. The edNEG model accounts for the concentration-dependent effects on ECS potentials that the standard models neglect. Using the edNEG model, we analyze these effects by splitting the extracellular potential into three components: one due to neural sink/source configurations, one due to glial sink/source configurations, and one due to extracellular diffusive currents. Through a series of simulations, we analyze the roles played by the various components and how they interact in generating the total slow potential. We conclude that the three components are of comparable magnitude and that the stimulus conditions determine which of the components that dominate.

Molecular mechanisms of brain water transport.

Our brains consist of 80% water, which is continuously shifted between different compartments and cell types during physiological and pathophysiological processes. Disturbances in brain water homeostasis occur with pathologies such as brain oedema and hydrocephalus, in which fluid accumulation leads to elevated intracranial pressure. Targeted pharmacological treatments do not exist for these conditions owing to our incomplete understanding of the molecular mechanisms governing brain water transport. Historically, the transmembrane movement of brain water was assumed to occur as passive movement of water along the osmotic gradient, greatly accelerated by water channels termed aquaporins. Although aquaporins govern the majority of fluid handling in the kidney, they do not suffice to explain the overall brain water movement: either they are not present in the membranes across which water flows or they appear not to be required for the observed flow of water. Notably, brain fluid can be secreted against an osmotic gradient, suggesting that conventional osmotic water flow may not describe all transmembrane fluid transport in the brain. The cotransport of water is an unconventional molecular mechanism that is introduced in this Review as a missing link to bridge the gap in our understanding of cellular and barrier brain water transport.

Differential expression of hydroxyurea transporters in normal and polycythemia vera hematopoietic stem and progenitor cell subpopulations

Polycythemia vera (PV) is a myeloproliferative neoplasm marked by hyperproliferation of the myeloid lineages and the presence of an activating JAK2 mutation. Hydroxyurea (HU) is a standard treatment for high-risk patients with PV. Because disease-driving mechanisms are thought to arise in PV stem cells, effective treatments should target primarily the stem cell compartment. We tested for the antiproliferative effect of patient treatment with HU in fluorescence-activated cell sorting-isolated hematopoietic stem/multipotent progenitor cells (HSC/MPPs) and more committed erythroid progenitors (common myeloid/megakaryocyte-erythrocyte progenitors [CMP/MEPs]) in PV using RNA-sequencing and gene set enrichment analysis. HU treatment led to significant downregulation of gene sets associated with cell proliferation in PV HSCs/MPPs, but not in PV CMP/MEPs. To explore the mechanism underlying this finding, we assessed for expression of solute carrier membrane transporters, which mediate transmembrane movement of drugs such as HU into target cells. The active HU uptake transporter OCTN1 was upregulated in HSC/MPPs compared with CMP/MEPs of untreated patients with PV, and the HU diffusion facilitator urea transporter B (UTB) was downregulated in HSC/MPPs compared with CMP/MEPs in all patient and control groups tested. These findings indicate a higher accumulation of HU within PV HSC/MPPs compared with PV CMP/MEPs and provide an explanation for the differential effects of HU in HSC/MPPs and CMP/MEPs of patients with PV. In general, the findings highlight the importance of transporter expression in linking therapeutics with human disease.

Carbon dioxide transport across membranes.

Carbon dioxide (CO2) movement across cellular membranes is passive and governed by Fick's law of diffusion. Until recently, we believed that gases cross biological membranes exclusively by dissolving in and then diffusing through membrane lipid. However, the observation that some membranes are CO2 impermeable led to the discovery of a gas molecule moving through a channel; namely, CO2 diffusion through aquaporin-1 (AQP1). Later work demonstrated CO2 diffusion through rhesus (Rh) proteins and NH3 diffusion through both AQPs and Rh proteins. The tetrameric AQPs exhibit differential selectivity for CO2 versus NH3 versus H2O, reflecting physico-chemical differences among the small molecules as well as among the hydrophilic monomeric pores and hydrophobic central pores of various AQPs. Preliminary work suggests that NH3 moves through the monomeric pores of AQP1, whereas CO2 moves through both monomeric and central pores. Initial work on AQP5 indicates that it is possible to create a metal-binding site on the central pore's extracellular face, thereby blocking CO2 movement. The trimeric Rh proteins have monomers with hydrophilic pores surrounding a hydrophobic central pore. Preliminary work on the bacterial Rh homologue AmtB suggests that gas can diffuse through the central pore and three sets of interfacial clefts between monomers. Finally, initial work indicates that CO2 diffuses through the electrogenic Na/HCO3 cotransporter NBCe1. At least in some cells, CO2-permeable proteins could provide important pathways for transmembrane CO2 movements. Such pathways could be amenable to cellular regulation and could become valuable drug targets.

Diagnóstico tardio de fibrose cística em criança com teste de triagem neonatal negativo

Cystic fibrosis (CF) is an autosomal recessive genetic disease in which CFTR (cystic fibrosis transmembrane conductance regulator) gene dysfunction, related to the transmembrane chlorine movement. This leads to the production of thicker secretions that occlude the light of the involved structures, causing inflammation and compromising the function of the affected organs. The respiratory and digestive systems are the main affected by this pathology, generating growth deficits, chronic respiratory infections, progressive damage to the lung tissue. Because it is a multisystem disease, early treatment aided by the screening test showed a better outcome when compared to treatment initiated after late diagnosis. The various neonatal screening tests (TTN) for cystic fibrosis have high sensitivity and specificity, but false positives and false negatives may occur. In this case report, we describe a preschooler who had a negative neonatal screening test and his diagnosis occurred after 2 years of age in the investigation of a hepatomegaly. However, the child presented pulmonary symptoms from the first days of life that were being treated as bronchial hyperresponsiveness and, later, as asthma. In the diagnostic investigation, the first dosage of chlorine in sweat has already shown significant elevation. Enzymatic treatment was started with slow and progressive improvement of respiratory symptoms. And although the child responded to the start of treatment, it already had significant impairment of lung function. Thus, although TTN for cystic fibrosis has high sensitivity, it does not replace sweat chlorine dosage in children with very suggestive symptoms.

Recombinant production of membrane proteins in yeast.

Biochemical pathways are compartmentalized in living cells. This permits each cell to maintain chemical compositions that differ between the cytosol, intracellular organelles and the external environment. Achieving this requires each compartment to be very selective in what is allowed to enter and leave. Nature has solved this by surrounding each cell and each organelle with a virtually solute impermeable lipid membrane, embedded with integral membrane proteins that mediate strictly controlled trans-membrane movement of matter and information. Access to pure and active integral membrane proteins is therefore required to comprehend membrane biology, ultimately through high-resolution structures of the membrane proteome and, therefore, also for our understanding of physiology. Unfortunately, apart from a few exceptions, membrane proteins cannot be purified from native tissue but need to be produced recombinantly, which is eminently challenging. This chapter shows how we have engineered yeast to provide high levels of prime quality membrane proteins of prokaryotic, archaeal or eukaryotic origin for structural biology.

Effect and mechanism of phospholipid scramblase 4 (PLSCR4) on lipopolysaccharide (LPS)-induced injury to human pulmonary microvascular endothelial cells.

BackgroundPrevious experiments revealed phospholipid scramblase 4 (PLSCR4) mRNA to be significantly increased in a lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) model of human pulmonary microvascular endothelial cells (HPMECs); however, the effect of PLSCR4 and its mechanism have not been reported to date. The PLSCR family is thought to mediate the transmembrane movement of phospholipids (PS), and has been found to be involved in pyroptosis through combing with gasdermin D (GSDMD). We therefore speculated that PLSCR4 may contribute to cell death via pyroptosis.MethodsTo investigate the effect and mechanism of PLSCR4 in ARDS, we constructed an in vitro model of LPS-induced ARDS in HPMECs transfected with PLSCR4 small interfering RNA (siRNA) or scramble siRNA (sc siRNA). After 4 h of LPS stimulation, western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), tracer flux assays, and fluorescence assays were used to study the relationship between PLSCR4 and pyroptosis with regards to their impact on ARDS. We also established an ARDS mouse model which was pretreated with a liquid complex of PLSCR4 siRNA/sc siRNA-lipofectamine 2000 through the fundus venous plexus. Finally, we used DNA pull-down and protein profiling to study the potential transcription factor of PLSCR4.ResultsIt was found that when the expression of PLSCR4 was elevated, the concentration of interleukin 1 beta (IL-1β) and IL-18 decreased, along with barrier damage (P<0.05). Furthermore, HPMEC injury was reduced with more distribution of PS and N-terminal cleavage product (GSDMD-NT) of GSDMD on the external side of cell membrane. However, the pyroptosis-relevant proteins of GSDMD and caspase-1 were not obviously changed (P<0.05); we further found that when PLSCR4 was depressed, the lung injury was aggravated in the mice. In the DNA pull-down assay, P62280 remarkably increased, which suggested that P62280 might be the transcription factor for PLSCR4.ConclusionsPLSCR4 alleviated pyroptosis by transporting PS to the outside of the membrane, blocking the formation of pyroptosis pores composed of GSDMD. Moreover, P62280 might be the transcription factor of PLSCR4. These insults may provide useful insights into the clinical treatment of ARDS.

Structural basis of human monocarboxylate transporter 1 inhibition by anti-cancer drug candidates

Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer treatment because of their enhanced expression in various tumors. Here, we report five cryo-EM structures, at resolutions of 3.0-3.3Å, of human MCT1 bound to lactate or inhibitors in the presence of Basigin-2, a single transmembrane segment (TM)-containing chaperon. MCT1 exhibits similar outward-open conformations when complexed with lactate or the inhibitors BAY-8002 and AZD3965. In the presence of the inhibitor 7ACC2 or with the neutralization of the proton-coupling residue Asp309 by Asn, similar inward-open structures were captured. Complemented by structural-guided biochemical analyses, our studies reveal the substrate binding and transport mechanism of MCTs, elucidate the mode of action of three anti-cancer drug candidates, and identify the determinants for subtype-specific sensitivities to AZD3965 by MCT1 and MCT4. These findings lay out an important framework for structure-guided drug discovery targeting MCTs.

The transport pathway in the ABCG2 protein and its regulation revealed by molecular dynamics simulations

Atomic-level structural insight on the human ABCG2 membrane protein, a pharmacologically important transporter, has been recently revealed by several key papers. In spite of the wealth of structural data, the pathway of transmembrane movement for the large variety of structurally different ABCG2 substrates and the physiological lipid regulation of the transporter has not been elucidated. The complex molecular dynamics simulations presented here may provide a breakthrough in understanding the steps of the substrate transport process and its regulation by cholesterol. Our analysis revealed drug binding cavities other than the central binding site and delineated a putative dynamic transport pathway for substrates with variable structures. We found that membrane cholesterol accelerated drug transport by promoting the closure of cytoplasmic protein regions. Since ABCG2 is present in all major biological barriers and drug-metabolizing organs, influences the pharmacokinetics of numerous clinically applied drugs, and plays a key role in uric acid extrusion, this information may significantly promote a reliable prediction of clinically important substrate characteristics and drug-drug interactions.

Abstract P092: Palmitoylation Controls Cell Surface Abundance Of Trpm7

Magnesium regulates numerous cellular functions and enzymes and abnormal magnesium homeostasis contributes to vascular dysfunction and the development of hypertension. The transient receptor potential melastatin 7 (TrpM7) has emerged as a key player in cardiovascular magnesium homeostasis. This bifunctional channel/kinase is ubiquitously expressed and regulates embryonic development. Its integral membrane ion channel domain regulates transmembrane movement of divalent cations, primarily Ca 2+ , Mg 2+ and Zn 2+ , and its kinase domain controls gene expression via histone phosphorylation. TrpM7 not only localizes on the cell surface to serve as a critical regulator of transmembrane Mg 2+ flux, but also forms an intracellular Zn 2+ release channel in vesicles of unknown origin. Palmitoylation is a dynamic reversible posttranslational modification, which regulates ion channel activity, stability, and subcellular localization. We found that TrpM7 is palmitoylated in multiple cell types. Here we sought to identify palmitoylated cysteines and the functional consequences of TrpM7 palmitoylation in HEK cells. Mutation of Cysteines 1143, 1144 and 1146 on TrpM7 (TrpM7-AAA) to alanines reduced its palmitoylation by 68.4±8% (n=13; P &lt;0.05), identifying this cluster of cysteines as the principal sites of palmitoylation. Fluorescent microscopy indicated that TrpM7-AAA was retained in the endoplasmic reticulum (ER), but when the palmitoylated cysteines in TrpM7 were replaced with the analogous regions of TrpM2 (M7/M2) or TrpM5 (M7/M5), the chimaeric proteins could exit the ER despite being 80.6±13% (n=3; P &lt;0.05) and 70.6±17% (n=3; P &lt;0.05) less palmitoylated than wild type respectively. Using membrane-impermeable biotinylation reagents we found that palmitoylation alters the balance of distribution of TrpM7 between vesicular and cell surface pools. The proportion of M7/M2 and M7/M5 reaching the cell surface membrane was decreased by 69.0±14% (n=3; P &lt;0.05) and 51.4±9% (n=3; P &lt;0.05) respectively, compared to wild type. Therefore, inhibiting TrpM7 palmitoylation reduced its cell surface abundance. The impact of palmitoylation on TrpM7 channel activity and its contributions to hypertension and vascular pathologies need further investigation.

Hydrogen sulfide decreases Cd translocation from root to shoot through increasing Cd accumulation in cell wall and decreasing Cd2+ influx in Isatis indigotica

Hydrogen sulfide (H2S), a small gaseous signalling molecule, plays a pivotal role in the plant response to heavy metal stress. Here, we revealed a novel mechanism of Isatis indigotica resistance to cadmium (Cd) stress, in which H2S promotes Cd accumulation in the root and decreases the long-distance transport of Cd from the root to shoot. Cd significantly inhibited Isatis indigotica growth and induced the endogenous H2S level. Application of NaHS (a H2S donor) alleviated the effects of Cd. NaHS restriction of the translocation factor of Cd, elevated the Cd content in roots and depressed the Cd content in shoots. Cd stress decreased the cellulose and pectin contents in the cell wall, but NaHS restored the effect of Cd on the cell wall components. The Cd2+ fluxes were detected by noninvasive microtest technology (NMT). The data showed that NaHS pretreatment decreased the Cd2+ influx and proportion of the Cd content in organelles. We analyzed the effect of NaHS on the metallothionein and phytochelatin (PC) contents in roots and found that the PC and metallothionein1A (MT1A) contents were induced by NaHS. Additionally, the chemical forms of Cd2+ were changed by NaHS. Thus, H2S alters the content of cell wall component, improves Cd accumulation in the cell wall, depresses Cd2+ transmembrane movement, induces the synthesis of metallothioneins and decreases the toxicity of intracellular Cd. Our finding has great value to reduce the loss of Isatis indigotica resulted by heavy metals stress.

SSO and other putative inhibitors of FA transport across membranes by CD36 disrupt intracellular metabolism, but do not affect FA translocation

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Quantitation of a neutral‐buoyancy assay (NBA) to estimate transmembrane N2 flux

Several assays exist for CO2, O2 and NH3 permeability, but a major technical void has been the absence of an assay for the transmembrane movement of N2 and other relatively inert gases. Here, we report the quantitation of a novel and simple yet powerful assay based on the neutral buoyancy of a Xenopus oocyte injected with a bubble of nitrogen (N2) gas. Briefly, we inject a precise volume of N2 (200 nl)—with a known number of gas molecules (nGas)—creating a bubble that thereby lowers cell density (ρCell) sufficiently for the oocyte to float. After injection, we transfer the oocyte to a glass tube (rated to 10 ATM), nearly filled with saline. We then pressurize the atmosphere above the saline solution sufficiently for the bubble inside the oocyte to constrict, thereby raising ρCell (decreasing buoyancy) and causing the oocyte to descend to a neutrally buoyant depth of 5 cm. As N2 diffuses out of the bubble, dissolves in the surrounding cytoplasm, and eventually exits the oocyte, the tendency is for the bubble to collapse gradually (increasing ρCell) and for the oocyte to sink. Rather than allowing the oocyte to sink substantially, we developed a neutral buoyancy clamp system (high‐resolution camera, software developed in‐house, digitally controlled pressure regulator) to adjust and record continuously the pressure in the air phase (PNB) required to maintain neutral buoyancy at an oocyte depth of 5 cm. The pressure inside the bubble (PBubble &gt; PNB by a fixed amount) falls approximately exponentially over time, and proportionally with the decreasing nGas. In order to calculate nGas from the recorded PNB, we performed a series of experiments in which—for each oocyte—we measured the spontaneous initial oocyte volume before bubble injection (Vi,Xo), injected a known volume of N2 (VGas), and then measured initial PNB for that combination of Vi,Xo and VGas. We repeated this procedure for 295 oocytes with Vi,Xo ranging from 812 to 1590 nl, and VGas ranging from 50 to 350 nl. For VGas from 100 and 300 nl, we observed a linear dependence of PNB on VGas/Vi,Xo, with the best‐fit equation: PNB = 11.28*(VGas/Vi,Xo) – 0.05 (Eq.1), R2 = 0.95. Here, the units of PNB, the slope, and the intercept are ATA. Recalling the ideal gas law and inserting the expression for VGas from Eq.1, we conclude that the apparent nGas = PB*Vi,Xo*(PNB + 0.05)/(11.28*RT) (Eq.2). Here, PB is room barometric pressure, and all units are consistent with an R of 0.0821 L atm K–1 mol–1. Because of unavoidable experimental error in Vi,Xo and VGas, and variability in oocyte density, the apparent nGas at time = 0 does not always match 8.26 nmol, the value computed for 200 nl of injected gas in our standard assays. We therefore rearranged Eq.2 to replace the best‐fit y‐intercept (i.e., 0.05 ATA) with an adjusted y‐intercept (b) that is characteristic for each oocyte. Using the revised relationship, nGas= PB*Vi,Xo*(PNB + b)/(11.28*RT) (Eq.3), we are now able to obtain the actual decay in nGas during our standard NBA and estimate absolute transmembrane N2 fluxes. While specifically developed for N2 fluxes, the NBA should be applicable to measuring transmembrane fluxes of other gasses.Support or Funding InformationOffice of Naval Research (N00014‐15‐1‐2060 &amp; N00014‐16‐1‐2535)

Optimizing a Cell-Based Assay for Fluorescent Phospholipid Scrambling

Phospholipid scramblases are membrane proteins that facilitate transmembrane movement of phospholipids and disruption of membrane lipid asymmetry. The anionic phospholipid phosphatidylserine (PS) is typically sequestered on the intracellular leaflet of the plasma membrane but scramblase activity can translocate PS to the outer surface, where its charged headgroup plays a signaling role in processes such as apoptosis or blood clotting. A series of recent structures of phospholipid scramblases have heightened interest and controversy over their function and physiology.

Life with Bacterial Mechanosensitive Channels, from Discovery to Physiology to Pharmacological Target.

General principles in biology have often been elucidated from the study of bacteria. This is true for the bacterial mechanosensitive channel of large conductance, MscL, the channel highlighted in this review. This channel functions as a last-ditch emergency release valve discharging cytoplasmic solutes upon decreases in osmotic environment. Opening the largest gated pore, MscL passes molecules up to 30 Å in diameter; exaggerated conformational changes yield advantages for study, including in vivo assays. MscL contains structural/functional themes that recur in higher organisms and help elucidate how other, structurally more complex, channels function. These features of MscL include (i) the ability to directly sense, and respond to, biophysical changes in the membrane, (ii) an α helix ("slide helix") or series of charges ("knot in a rope") at the cytoplasmic membrane boundary to guide transmembrane movements, and (iii) important subunit interfaces that, when disrupted, appear to cause the channel to gate inappropriately. MscL may also have medical applications: the modality of the MscL channel can be changed, suggesting its use as a triggered nanovalve in nanodevices, including those for drug targeting. In addition, recent studies have shown that the antibiotic streptomycin opens MscL and uses it as one of the primary paths to the cytoplasm. Moreover, the recent identification and study of novel specific agonist compounds demonstrate that the channel is a valid drug target. Such compounds may serve as novel-acting antibiotics and adjuvants, a way of permeabilizing the bacterial cell membrane and, thus, increasing the potency of commonly used antibiotics.