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The contamination of the aquatic ecosystem beyond tolerable limits may pose serious health challenges to its components. This study evaluated the toxic effects of a binary mixture of lead (Pb) and zinc (Zn) compounds on the activity of Na+/K+-ATPase in tissues of Clarias gariepinus in a controlled aquatic system. The study employed Box-Behnken Design (BBD) with 17 runs in which Pb and Zn concentrations were considered process variables in a time-dependent fashion. Metal exposure levels consisted of 0, 10 and 20% of 96 h-LC50 of Pb (55.12 mg/L) and Zn (32.15 mg/L) for three weeks. Thereafter, membrane-bound Na+/K+-ATPase activity was assessed in gill, hepatic and renal tissues, and data generated from the BBD were used for the development of models. Three regression models were obtained, for gill, hepatic and renal Na+/K+-ATPase activities with exposure to metals differ significantly (p 90%, and no significant lack of fit (p > 0.05) was observed in each case. Congruent to the synergistic interactions observed between Pb and Zn in the study, the gill and hepatic Na+/K+-ATPase activities were significantly inhibited, whereas renal Na+/K+-ATPase activity was significantly stimulated (p

A pleiotropic cytokine TNFα is an important inflammatory mediator of a number of diseases; its biological functions are fulfilled through two different receptors, TNFR1 and TNFR2. Changes in the ratio between these types of receptors shifting the balance between the pro-apoptotic and proliferation signaling pathways play a crucial role in eliciting the cell response to TNFα. The pathological processes in the body can alter the levels of TNFR1 and TNFR2 expression on the cells involved in disease development. Therefore, this study was aimed at investigating the level of co-expression of type 1 and 2 TNFα receptors in the major subpopulations of peripheral blood cells in patients with rheumatoid arthritis (RA) and bronchial asthma (BA). The greatest changes in the percentage of cells expressing TNFR1 and TNFR2 were revealed for the B-lymphocyte subpopulation. For the T-lymphocyte subpopulation, there were some differences in the percentage of cells expressing exclusively TNFR1 in RA and BA patients compared with those in healthy subjects, as well as between the RA and BA groups. A higher percentage of double-negative monocytes was observed in patients with BA and RA compared to healthy subjects. These findings indicate that the coexpression profile of TNFR1 and TNFR2 receptors in patients with RA and BA differ within these groups as well as compared to that in healthy subjects. These immune cell populations are actively involved in the pathogenesis of both rheumatoid arthritis and bronchial asthma, so the results may indicate that these cells might show different responses to TNFα as the percentage and the number of receptors on their surface vary.

Increasing interest has recently focused on determining whether quercetin may exert anti-aging properties or not? The objective of this study was determination of Na+, K+ -ATPase activity in quercetin-treated red blood cells during human aging. The study was carried out on human blood samples. The subjects were divided into different age groups, young, middle, and old. The effects of quercetin were evaluated by determining Na+, K+ -ATPase activity by co-incubating the red blood cells in presence of quercetin (10−6 M to 10−3 M final concentration). Quercetin causes 15% increase in Na+, K+ -ATPase activity at 10−4 M and 17% at 10−3 M as compared to the young control age group. The effect was insignificant at 10−5 M (7%) and 10−6 M (5%) in the young age group. Quercetin showed significant increase at 10−6 M to 10−3 M in Na+, K+ -ATPase activity as compared to the middle control age group. A significant increase in Na+, K+ -ATPase activity was observed at all concentrations [10−6 M (31%), 10−5 M (39%), 10−4 M (51%), and 10−3 M (61%)] in elderly population. We believe that these findings will help in further research against oxidative stress in red blood cells.

KRAS4B is a membrane-anchored signaling protein and a primary target in cancer research. Predictions from molecular dynamics simulations that have previously shaped our mechanistic understanding of KRAS signaling disagree with recent experimental results from neutron reflectometry, NMR, and thermodynamic binding studies. To gain insight into these discrepancies, we compare this body of biophysical data to back-calculated experimental results from a series of molecular simulations that implement different subsets of molecular interactions. Our results show that KRAS4B approximates an entropic ensemble of configurations at model membranes containing 30% phosphatidylserine lipids, which is not significantly shaped by interactions between the globular G-domain of KRAS4B and the lipid membrane. These findings revise our understanding of KRAS signaling and promote a model in which the protein samples the accessible conformational space in a near-uniform manner while being available to bind to effector proteins.

In the life sciences, including hemostasis and thrombosis, methods of structural biology have become indispensable tools for shedding light on underlying mechanisms that govern complex biological processes. Advancements of the relatively young field of computational biology have matured to a point where it is increasingly recognized as trustworthy and useful, in part due to their high space–time resolution that is unparalleled by most experimental techniques to date. In concert with biochemical and biophysical approaches, computational studies have therefore proven time and again in recent years to be key assets in building or suggesting structural models for membrane-bound forms of coagulation factors and their supramolecular complexes on membrane surfaces where they are activated. Such endeavors and the proposed models arising from them are of fundamental importance in describing and understanding the molecular basis of hemostasis under both health and disease conditions. We summarize the body of work done in this important area of research to drive forward both experimental and computational studies toward new discoveries and potential future therapeutic strategies.

Membrane-bound transcription factors (MTFs) differ from cytosolic transcription factors (TFs) and they innately bind to membranes. Under external stimuli, MTFs are released from various membranes, ...

With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membrane-bound complexes and an unsupervised classification (PySeg). Applications to intact cells and isolated endoplasmic reticulum (ER) allowed us to detect and classify small protein complexes. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Spatial distribution analysis showed that ER complexes have different localization patterns forming nanodomains. Therefore, this procedure allows a comprehensive detection and structural analysis of complexes in situ. A template-free image processing approach automatically detects and classifies membrane-bound protein complexes in cryo-electron tomograms of isolated endoplasmic reticulum and in intact cells.

Molecular dynamics (MD) simulations have become a widely used tool in the scientific community for understanding molecular scale phenomena that are challenging to address with wet-lab techniques. Coarse-grained simulations, in which multiple atoms are combined into single beads, allow for larger systems and longer time scales to be explored than atomistic techniques. Here, we describe the procedures and equipment required to set up coarse-grained simulations of membrane-bound proteins in a lipid bilayer that can mimic many membrane environments.

Graphical abstract, Highlights • The relative populations of residue types are shown for soluble and membrane proteins. • Populations within secondary structure bins vary with residue and protein solubility. • Solvent accessible surface area depends on secondary structure and protein solubility. • More exposed hydrophobic residues interact favorably with lipids in membrane proteins. • More buried polar residues make more hydrogen bonds within protein interiors., Knowledge of three-dimensional protein structure is integral to most modern drug discovery efforts. Recent advancements have highlighted new techniques for 3D protein structure determination and, where structural data cannot be collected experimentally, prediction of protein structure. We have undertaken a major effort to use existing protein structures to collect, characterize, and catalogue the inter-atomic interactions that define and compose 3D structure by mapping hydropathic interaction environments as maps in 3D space. This work has been performed on a residue-by-residue basis, where we have seen evidence for relationships between environment character, residue solvent-accessible surface areas and their secondary structures. In this graphical review, we apply principles from our earlier studies and expand the scope to all common amino acid residue types in both soluble and membrane proteins. Key to this analysis is parsing the Ramachandran plot to an 8-by-8 chessboard to define secondary structure bins. Our analysis yielded a number of quantitative discoveries: 1) increased fraction of hydrophobic residues (alanine, isoleucine, leucine, phenylalanine and valine) in membrane proteins compared to their fractions in soluble proteins; 2) less burial coupled with significant increases in favorable hydrophobic interactions for hydrophobic residues in membrane proteins compared to soluble proteins; and 3) higher burial and more favorable polar interactions for polar residues now preferring the interior of membrane proteins. These observations and the supporting data should provide benchmarks for current studies of protein residues in different environments and may be able to guide future protein structure prediction efforts.

Xiaoling Zhou1, Xiaoyuan Wang1, Yanzheng Gu2, Lan Chen3 , Yueping Shen4, Jingluan Tian1, Mingyuan Wang5, Shujun Chen1, Xiaoyu Duan1, Hanqing Gao1, Xiaopei Ji1, Qi Fang1,2, Xueguang Zhang2,3, Qun Xue*1,21. Department of Neurology, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006, China.2. Institute of Clinical Immunology, Jiangsu Key Laboratory of Clinical Immunology, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006, China.3. Department of Neurology, Nantong First People’s Hospital, Nantong, Jiangsu, 226000, China.4. Department of Epidemiology and Health Statistics, Soochow University, Suzhou, Jiangsu, 215006, China.5. Suzhou Red Cross Central Blood Station, Suzhou, Jiangsu, 215006, China.*Correspondence Author: Qun Xue (email: qxue_sz@163. com)

Summary Membrane-bound cargos in cells are generally transported by multiple kinesin motors. Quantifying the bimolecular on-rate of motors for their microtubule track is important for understanding of multi-motor transport but is complicated by diffusion of the motors in the plane of the lipid bilayer. Here, we describe a method to measure the kinesin on-rate that uses a modified microtubule gliding assay performed on a supported lipid bilayer and detects motor binding by a local increase in fluorescence. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2019)., Graphical abstract, Highlights • Measure binding kinetics of kinesin motor diffusing in a 2D membrane • Motor accumulation rates are measured by fluorescence microscopy • Accumulation rates are used to estimate motor on- and off-rates • Adaptable to any motor that moves along a cytoskeletal filament, Membrane-bound cargos in cells are generally transported by multiple kinesin motors. Quantifying the bimolecular on-rate of motors for their microtubule track is important for understanding of multi-motor transport but is complicated by diffusion of the motors in the plane of the lipid bilayer. Here, we describe a method to measure the kinesin on-rate that uses a modified microtubule gliding assay performed on a supported lipid bilayer and detects motor binding by a local increase in fluorescence.

The impacts of high hydrostatic pressure (HHP) treatments on metabolic functionality and quality of fruit and vegetables are still poorly understood. The effects of pressure intensity (100, 150 and 200 MPa) and holding time (5 and 10 min), and temperatures (20, 30 and 40 °C) during HHP treatment on fresh radish tubers has been studied. Colour, stiffness and turgor of the tubers were evaluated using colorimetry, texture analyses and direct turgor measurements with the cell pressure probe techniques. Comparison of turgor and stiffness losses, and colour changes, of skin and thin-walled parenchymatous inner radish tissue showed the effects of the various HHP treatment parameters on the permeability of biomembranes. HHP treatment resulted in a rapid reduction of cell turgor and, concomitantly of tuber stiffness, but also in a cellular redistribution of anthocyanins as indicated by colour changes. These responses were partially reversible up to 100 MPa applied pressure at 20-40 °C for 5–10 min, yielding a turgor threshold of 0.1 MPa. Beyond these limits, changes were irreversible, most probably due to the denaturation of membrane bound proteins such as ion channels and pumps. Overall, even short-term HHP treatments at room temperature are not suitable for sanitation by inactivating bacteria of perishable produce.

Borassus flabelifer peroxidase was ionically interacting with stone parts of its fruit. The apparently homogeneous membrane bound peroxidase was reversibly inhibited by various aromatic alcohols. Dixon plot clearly showed mixed type of inhibition. Ki values of peroxidase-inhibitor complexes were determined. The homogenous peroxidase had non-covalently interacting triglycerides or triglyceride esterified phytosterols. This Peroxidase was interacting with acid hydrolysable low density lipoprotein but not with high density lipoprotein. This may be one of the reasons for its stability and catalysis in organic solvents. Further studies may prove it as lipophilic enzyme. These waste stone parts may be utilized in extracting phytosterols and fatty acids which has medicinal value.

Human and mouse CD83 have been well characteized, however, the other mammalian CD83 genes have not been cloned and characterized. In this study, the porcine CD83 (pCD83) was cloned, expressed and characterized, and showed that the pCD83 gene has 81% and 74% homologies with humans and mice, respectively, which was identified to be glycosylated when expressed in eukaryotic cells, existing naturally in two forms: membrance-bound CD83 (mCD83) and soluble CD83 (sCD83), the latter was identified to be generated mainly from mCD83 by proteolytic shedding. The pCD83 was a dimmer mediated by intermolecular disulfide bond formed by the fifth cysteine in the exrtracellular domain. Functionally, the recombinant porcine sCD83 was preliminarily tested to have the ability to inhibit DC-mediated T cell activition. This study provided necessary fundation for further investigation on pCD83 functions.

Significance The stochastic nature of transcription/translation implies that the concentrations of cellular proteins are “noisy” and not constant in time or across cell populations. Liquid–liquid phase separation (LLPS) can reduce or “buffer” this noise by maintaining well-defined concentrations, even in the presence of concentration distributions. However, this idea was recently challenged experimentally in multicomponent systems. Our physical theory of LLPS in ternary systems (solutes ϕ and ψ in a solvent) predicts their LLPS properties as a function of the ϕ – ϕ (homotypic) and ϕ – ψ (heterotypic) interaction strengths. We show how buffering can be effective if the noise distribution aligns with the tie-lines of the phase diagram and suggest that evolution may optimize concentration buffering by selecting appropriate mutations.

Protein-rich droplets, such as stress granules, P-bodies, and the nucleolus, perform diverse and specialized cellular functions. Recent evidence has shown the droplets, which are also known as biomolecular condensates or membrane-less compartments, form by phase separation. Many droplets also contact membrane-bound organelles, thereby functioning in development, intracellular degradation, and organization. These underappreciated interactions have major implications for our fundamental understanding of cells. Starting with a brief introduction to wetting phenomena, we summarize recent progress in the emerging field of droplet–membrane contact. We describe the physical mechanism of droplet–membrane interactions, discuss how these interactions remodel droplets and membranes, and introduce "membrane scaffolding" by liquids as a novel reshaping mechanism, thereby demonstrating that droplet–membrane interactions are elastic wetting phenomena. “Membrane-less” and “membrane-bound” condensates likely represent distinct wetting states that together link phase separation with mechanosensitivity and explain key structures observed during embryogenesis, during autophagy, and at synapses. We therefore contend that droplet wetting on membranes provides a robust and intricate means of intracellular organization.

Membrane-bound and membraneless organelles/biomolecular condensates ensure compartmentalization into functionally distinct units enabling proper organization of cellular processes. Membrane-bound organelles form dynamic contacts with each other to enable the exchange of molecules and to regulate organelle division and positioning in coordination with the cytoskeleton. Crosstalk between the cytoskeleton and dynamic membrane-bound organelles has more recently also been found to regulate cytoskeletal organization. Interestingly, recent work has revealed that, in addition, the cytoskeleton and membrane-bound organelles interact with cytoplasmic biomolecular condensates. The extent and relevance of these complex interactions are just beginning to emerge but may be important for cytoskeletal organization and organelle transport and remodeling. In this review, we highlight these emerging functions and emphasize the complex interplay of the cytoskeleton with these organelles. The crosstalk between membrane-bound organelles, biomolecular condensates and the cytoskeleton in highly polarized cells such as neurons could play essential roles in neuronal development, function and maintenance.

Summary Phagocytes engulf pathogens into a membrane bound compartment called a phagosome, but what happens when engulfed pathogens start growing? In this issue of Cell Host & Microbe, Westman et al. (2020) show that lysosomes fuse with phagosomes to maintain phagosomal membrane integrity as the fungal pathogen Candida albicans expands.

The choline analog homocholine is not acetylated ~vitro by choline-0-acetyltransferase (ChAT, EC 2.3. 1.6) which is solubilized by 100 mM sodium phosphate buffer washes of a crude vesicular fraction of mouse forebrain. However, both homocholine and choline are acetylated by a form of ChAT which is non-ionically associated with a subcellular fraction of mouse forebrain containing membrane associated organelles and occluded ACh (P4 ). Acetylation of homocholine by membrane associated ChAT is saturable. 4-(l-Naphthylvinyl) pyridine (NVP) inhibits the acetylation of both choline (60%) and homocholine (40%) by membrane associated ChAT but reduces the acetylation of only choline by soluble ChAT (76%). Choline and homocholine serve as competitive alternative substrates for the same membrane associated ChAT whereas homocholine only acts as a competitive inhibitor of choline acetylation by soluble ChAT. Acetylhomocholine competitively inhibits the acetylation of choline by both soluble and membrane associated ChAT more dramatically than does the natural end product ACh.

The effect of the plasma membrane on the activity of angiotensin-I converting enzyme (ACE) plays a crucial role in the evaluation of food-derived ACE inhibitory peptides, although these peptides are commonly evaluated in the system with ACE in its free state. In this study, we constructed an in vitro membrane-bound ACE C domain system to simulate the presence of the plasma membrane. The resultant Km and Vmax suggested that the presence of the membrane reduced the affinity between ACE C domain and hippuryl-histidyl-leucine, while it increased the reaction velocity. The ACE inhibitory activity of four egg white peptides and five structurally modified peptides suggested that a moderate hydrophobicity/hydrophilicity of the peptide is beneficial for the improvement of their ACE inhibitory activity in a membrane-bound system. These results also indicated that the N terminal plays a significant role in the ACE inhibitory activity of peptides in the membrane-bound system.

Targeted DamID (TaDa) allows highly efficient cell-type-specific profiling of protein-DNA interactions. Cell-type-specificity, however, is governed by the GAL4/UAS system, which can exhibit differences in expression patterns depending upon the genomic insertion site and the UAS promoter strength. The TaDa system uses a bicistronic transcript to reduce the translation rates of Dam-fusion proteins, presenting the possibility of using the primary ORF within in the transcript to label expression domains and precisely identified the profiled cell populations in experimental samples. Here, we describe new TaDa vectors, pTaDaG, pTaDaG2 and pTaDaM, that use myristoylated-GFP or myristoylated-mCherry as the primary ORF. Differing lengths of the myristoylation sequence between the two GFP plasmids allows additional translational control. The mCherry plasmid allows profiled cells to be visualised when using the NanoDam system, in which an anti-GFP nanobody is fused to Dam to profile the localisation of GFP-fusion proteins. Fly lines created with this system allow easy visualisation of expression domains under both fluorescent dissecting and confocal microscopes without the use of antibody staining, whilst faithfully profiling protein-DNA interactions via Targeted DamID.

Background The circulating level of soluble lectin‐like oxidized low‐density lipoprotein receptor‐1 ( sLOX ‐1) is a valuable biomarker of acute myocardial infarction ( AMI ). The most electronegative low‐density lipoprotein, L5, signals through LOX ‐1 to trigger atherogenesis. We examined the characteristics of LOX ‐1 and the role of L5 in aspirated coronary thrombi of AMI patients. Methods and Results Intracoronary thrombi were aspirated by performing interventional thrombosuction in patients with ST ‐segment–elevation myocardial infarction ( STEMI ; n=32) or non–ST‐segment–elevation myocardial infarction (n=12). LOX ‐1 level and the ratio of sLOX ‐1 to membrane‐bound LOX ‐1 were higher in thrombi of STEMI patients than in those of non–ST‐segment–elevation myocardial infarction patients. In all aspirated thrombi, LOX ‐1 colocalized with apoB100. When we explored the role of L5 in AMI , deconvolution microscopy showed that particles of L5 but not L1 (the least electronegative low‐density lipoprotein) quickly formed aggregates prone to retention in thrombi. Treating human monocytic THP ‐1 cells with L5 or L1 showed that L5 induced cellular adhesion and promoted the differentiation of monocytes into macrophages in a dose‐dependent manner. In a second cohort of AMI patients, the L5 percentage and plasma concentration of sLOX ‐1 were higher in STEMI patients (n=33) than in non–ST‐segment–elevation myocardial infarction patients (n=25), and sLOX ‐1 level positively correlated with L5 level in AMI patients. Conclusions The level of LOX ‐1 and the ratio of sLOX ‐1 to membrane‐bound LOX ‐1 in aspirated thrombi, as well as the circulating level of sLOX ‐1 were higher in STEMI patients than in non–ST‐segment–elevation myocardial infarction patients. L5 may play a role in releasing a high level of sLOX ‐1 into the circulation of STEMI patients.

The structural-functional hallmark of the liver sinusoidal endothelium is the presence of fenestrae grouped in sieve plates. Fenestrae are open membrane bound pores supported by a (sub)membranous cytoskeletal lattice. Changes in number and diameter of fenestrae alter bidirectional transport between the sinusoidal blood and the hepatocytes. Their physiological relevance has been shown in different liver disease models. Although the structural organization of fenestrae has been well documented using different electron microscopy approaches, the dynamic nature of those pores remained an enigma until the recent developments in the research field of four dimensional (4-D) AFM. In this contribution we highlight how AFM as a biophysical nanocharacterization tool enhanced our understanding in the dynamic behaviour of liver sinusoidal endothelial fenestrae. Different AFM probing approaches, including spectroscopy, enabled mapping of topography and nanomechanical properties at unprecedented resolution under live cell imaging conditions. This dynamic biophysical characterization approach provided us with novel information on the ‘short’ life-span, formation, disappearance and closure of hepatic fenestrae. These observations are briefly reviewed against the existing literature. Electronic supplementary material The online version of this article (10.1007/s12551-020-00699-0) contains supplementary material, which is available to authorized users.

Solid-state NMR (SSNMR) is one of the most useful methods to investigate membrane-bound peptides and proteins, in order to elucidate the structural basis of their diverse biological functions. In the case of short peptides, SSNMR is now performed on a routine basis in several labs. By employing side-chain 19F- or 2H-isotope-labelled peptides in static oriented samples, SSNMR can provide information about conformation, alignment, dynamics, oligomerization, and aggregation behaviour. Changes in and transitions between these structural properties tend to be directly related to the functional mechanism of the peptides. Here, we present an easy-to-follow description of the methodological SSNMR approach in the form of flow charts. The successive steps needed, from peptide synthesis and functional tests, on to NMR sample preparation and NMR experiments, and further on to data analysis, are described. At each stage, questions to be answered and tasks to be performed are explained, and some representative results are illustrated. Relevant challenges and pitfalls are discussed, and possible reasons for problems and their possible solutions are considered.

Currently, no data are available regarding the expression levels of CD40L on CD4+ T cells in patients with neuromyelitis optica spectrum disorders (NMOSD). The percentage of circulating CD40L+CD4+ T cells was measured by flow cytometry in 23 NMOSD patients and 10 healthy controls. The ratio of CD40L+CD4+ to CD4+ T cells in patients at acute phase (18.28 ± 15.56%) was significantly higher than that in healthy controls (7.23 ± 5.94%, P = .032) and was positively correlated with disease severity (r = 0.532, P = .041). Thus, our results suggest an important role of this molecule in acute attacks of NMOSD.

The brain is an immune privileged organ, uniquely placed in the body. Two systems involved in maintaining brain homeostasis and in protecting the brain are the blood-brain barrier (BBB) and the complement system. The BBB is present in the vasculature of the brain and is the dynamic interface between brain and body that regulates what enters and leaves the brain, thereby maintaining the brain microenvironment optimal for brain function. The complement system is ubiquitous, being present systemically and in the brain, both membrane bound and in circulation. It is an important arm of the body's defense that helps maintain homeostasis by eliminating debris and damaged cells, participating in destroying pathogens, promoting inflammation and conveying 'danger signals'. Recent studies reveal that the complement system plays an important role in normal brain development. However, when the complement system is overwhelmed, complement activation could contribute to loss of BBB integrity resulting in brain pathology. Studies support an association between complement proteins and BBB dysfunction, with the mechanisms being slowly unraveled. This review will provide an overview of both these systems, how they intersect and interact with each other.

The amphipathic a-helical peptide KIA14 [(KIAGKIA)(2)-NH2] was studied in membranes using circular dichroism and solid-state NMR spectroscopy to obtain global as well as local structural information. By analyzing H-2 NMR data from 10 analogues of KIA14 that were selectively labeled with Ala-d(3), those positions that are properly folded into a helix could be determined within the membrane-bound peptide. The N-terminus was found to be unraveled, whereas positions 4-14 formed an ideal helix all the way to the C-terminus. The helicity did not change when Gly residues were replaced by Ala-d3 but was reduced when Ile was replaced, indicating that large hydrophobic residues are required for membrane binding and helix formation. The reduced helicity was strongly correlated with a decrease in peptide-induced leakage from lipid vesicles. The orientation of the short KIA14 peptide was assessed in several lipid systems and compared with that of the longer KIA21 sequence [(KIAGKIA)(3)-NH2]. In 1,2-dioleoylsn-glycero-3-phosphatidylcholine, both peptides are aligned flat on the membrane surface, whereas in 1,2-dimyristoyl-sn-glycero3-phosphatidylcholine (DMPC)/1-myristoy1-2-hydroxy-sn-glycero-3-phosphatidylcholine (lyso-MPC) both are inserted into the membrane in an upright orientation. These two types of lipid systems had been selected for their strongly negative and positive spontaneous curvature, respectively. We propose that in these cases, the peptide orientation is largely determined by the lipid properties. On the other hand, in plain DMPC and 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine, which have only a slight positive curvature, a marked difference in orientation is evident: the short KIA14 lies almost flat on the membrane surface, whereas the longer KIA21 is more tilted. We thus propose that out of the lipid systems tested here, DMPC (with hardly any curvature) is the least biased,lipid system in which peptide orientation and realignment can be studied, allowing to compare and discriminate the intrinsic effects of the properties of the peptides as such.

Rationale: In cardiomyocytes, Na V 1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. Objective: The objective of this study was to test whether Na V 1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. Methods and Results: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1 Δ314-315 or Kir2.1 R44A/R46A with wild-type (WT) Na V 1.5 WT in heterologous cells reduced inward sodium current compared with Na V 1.5 WT alone or coexpressed with Kir2.1 WT . In cell surface biotinylation experiments, expression of Kir2.1 Δ314-315 reduced Na V 1.5 channel surface expression. Glycosylation analysis suggested that Na V 1.5 WT and Kir2.1 WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of Na V 1.5 WT , and vice versa, whereas coexpression with Kir2.1 Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1 Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with Na V 1.5 WT and Kir2.1 WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1 WT , Na V 1.5 WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that Na V 1.5 WT channels interact with AP1 through the Na V 1.5 Y1810 residue, suggesting that, like for Kir2.1 WT , AP1 can mark Na V 1.5 channels for incorporation into clathrin-coated vesicles at the trans -Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. Conclusions: The Na V 1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of Na V 1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

A series of novel aromatic primary sulfonamides decorated with diversely substituted 1,2,4-oxadiazole periphery groups has been prepared using a parallel chemistry approach. The compounds displayed a potent inhibition of cytosolic hCA II and membrane-bound hCA IX isoforms. Due to a different cellular localization of the two target enzymes, the compounds can be viewed as selective inhibition tools for either isoform, depending on the cellular permeability profile. The SAR findings revealed in this study has been well rationalized by docking simulation of the key compounds against the crystal structures of the relevant hCA isoforms.