The Water Permeability and Pore Entrance Structure of Aquaporin-4 Depend on Lipid Bilayer Thickness.

Aquaporin-4 (AQP4), the primary water channel in glial cells of the mammalian brain, plays a critical role in water transport in the central nervous system. Previous experiments have shown that the water permeability of AQP4 depends on the cholesterol content in the lipid bilayer, but it was not clear whether changes in permeability were due to direct cholesterol-AQP4 interactions or to indirect effects caused by cholesterol-induced changes in bilayer elasticity or bilayer thickness. To determine the effects resulting only from bilayer thickness, here we use a combination of experiments and simulations to analyze AQP4 in cholesterol-free phospholipid bilayers with similar elastic properties but different hydrocarbon core thicknesses previously determined by x-ray diffraction.

Lipid-mediated DNA and siRNA Transfection Efficiency Depends on Peptide Headgroup.

A series of amphiphiles with differing cationic tri- and di- peptide headgroups, designed and synthesized based on lysine (K), ornithine (O), arginine (R), and glycine (G), have been characterized and evaluated for DNA and siRNA delivery. DNA-lipoplexes formed from the tri- and di- lipopeptides possessed lipid:nucleic acid charge ratios of 7:1 to 10:1, diameters of ~200 nm to 375 nm, zeta potentials of 23 mV to 41 mV, melting temperatures of 12 °C to 46 °C, and lamellar repeat periods of 6 nm to 8 nm. These lipid-DNA complexes formed supramolecular structures in which DNA is entrapped at the surface between multilamellar liposomal vesicles.

The water permeability of lens aquaporin-0 depends on its lipid bilayer environment.

Aquaporin-0 (AQP0), the primary water channel in lens fiber cells, is critical to lens development, organization, and function. In the avascular lens there is thought to be an internal microcirculation associated with fluid movement. Although AQP0 is known to be important in fluid fluxes across membranes, the water permeability of this channel has only been measured in Xenopus oocytes and in outer lens cortical membranes, but not in inner nuclear membranes, which have an increased cholesterol/phospholipid ratio.

Water permeability of aquaporin-4 channel depends on bilayer composition, thickness, and elasticity.

Aquaporin-4 (AQP4) is the primary water channel in the mammalian brain, particularly abundant in astrocytes, whose plasma membranes normally contain high concentrations of cholesterol. Here we test the hypothesis that the water permeabilities of two naturally occurring isoforms (AQP4-M1 and AQP4-M23) depend on bilayer mechanical/structural properties modulated by cholesterol and phospholipid composition. Osmotic stress measurements were performed with proteoliposomes containing AQP4 and three different lipid mixtures: 1), phosphatidylcholine (PC) and phosphatidylglycerol (PG); 2), PC, PG, with 40 mol % cholesterol; and 3), sphingomyelin (SM), PG, with 40 mol % cholesterol.

Charge-reversal lipids, peptide-based lipids, and nucleoside-based lipids for gene delivery.

Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapy's appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to myriad diseases through treatments tailored to a specific individual's genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when non-viral vectors are used have lead to an increased focus on engineering new gene delivery vectors.

From Brittle to Pliant Viscoelastic Materials with Solid State Linear Polyphosphonium - Carboxylate Assemblies.

A polystyrenylphosphonium polymer was synthesized and complexed with various carboxylic acid derivatives to form new solid-state polyelectrolyte-surfactant assemblies. The properties of these ionic materials were highly dependent on the nature of the anion and included a brittle material, a rubbery ball that bounces, or a sticky fiber. The values for the equilibrium modulus, storage modulus, and loss modulus were dependent on the composition of the carboxylic acid and the number of electrostatic interactions.

Functional lipids and lipoplexes for improved gene delivery.

Cationic lipids are the most common non-viral vectors used in gene delivery with a few currently being investigated in clinical trials. However, like most other synthetic vectors, these vectors suffer from low transfection efficiencies. Among the various approaches to address this challenge, functional lipids (i.

Identification of a binding motif in the S5 helix that confers cholesterol sensitivity to the TRPV1 ion channel.

The TRPV1 ion channel serves as an integrator of noxious stimuli with its activation linked to pain and neurogenic inflammation. Cholesterol, a major component of cell membranes, modifies the function of several types of ion channels. Here, using measurements of capsaicin-activated currents in excised patches from TRPV1-expressing HEK cells, we show that enrichment with cholesterol, but not its diastereoisomer epicholesterol, markedly decreased wild-type rat TRPV1 currents.

Synthesis, characterization, and in vitro transfection activity of charge-reversal amphiphiles for DNA delivery.

A series of charge-reversal lipids were synthesized that possess varying chain lengths and end functionalities. These lipids were designed to bind and then release DNA based on a change in electrostatic interaction with DNA. Specifically, a cleavable ester linkage is located at the ends of the hydrocarbon chains.

The effect of charge-reversal amphiphile spacer composition on DNA and siRNA delivery.

A series of charge-reversal amphiphiles with different spacers separating the headgroup from the hydrophobic chains are described for delivery of DNA and siRNA. Among them, the amphiphiles possessing a glycine spacer (e.g.

Sorting of lens aquaporins and connexins into raft and nonraft bilayers: role of protein homo-oligomerization.

Two classes of channel-forming proteins in the eye lens, the water channel aquaporin-0 (AQP-0) and the connexins Cx46 and Cx50, are preferentially located in different regions of lens plasma membranes (1,2). Because these membranes contain high concentrations of cholesterol and sphingomyelin, as well as phospholipids such as phosphatidylcholine with unsaturated hydrocarbon chains, microdomains (rafts) form in these membranes. Here we test the hypothesis that sorting into lipid microdomains can play a role in the disposition of AQP-0 and the connexins in the plane of the membrane.

A new helper phospholipid for gene delivery.

Enhanced gene transfection activity is observed when using a new helper lipid with DOTAP, compared to DOPE.

X-ray diffraction to determine the thickness of raft and nonraft bilayers.

Low-angle X-ray diffraction is a powerful method to analyze the structure of membrane bilayers. Specifically, the technique can be used to determine accurately the thickness of fully hydrated bilayers. Herein details are presented showing how this technique can measure the difference in thickness of bilayers in detergent-resistant membranes and detergent-soluble membranes extracted from model systems known to contain both raft and nonraft domains.

Overview of membrane rafts.

Transient lateral microdomains (rafts) in cell membranes have been postulated to perform a number of important functions in normal cells, and are also thought to be critically involved in several pathological conditions. However, there are still a number of fundamental unanswered questions concerning the composition, size, dynamics, and stability of membrane rafts. These questions are currently being addressed by a number of sophisticated biophysical, biochemical, and computational methodologies.

Lipophilic peptides for gene delivery.

DNA transfections are widely performed in research laboratories and in vivo gene delivery holds the promise for curing many diseases. The synthetic carriers or vectors for DNA are typically cationic lipids. However, in biology, the recognition of nucleic acids by proteins involves both electrostatic and stacking contributions.

Non-viral charge reversal vectors for pDNA delivery.

A synthetic vector that transform from a cationic to an anionic lipid intracellularly is described. This charge-reversal lipid was synthesized and characterized, including the supramolecular complex it forms with DNA. Enhanced gene transfection was observed using this synthetic vector compared to current cationic lipids.

Role of GAP-43 in sequestering phosphatidylinositol 4,5-bisphosphate to Raft bilayers.

The lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) is critical for a number of physiological functions, and its presence in membrane microdomains (rafts) appears to be important for several of these spatially localized events. However, lipids like PIP(2) that contain polyunsaturated hydrocarbon chains are usually excluded from rafts, which are enriched in phospholipids (such as sphingomyelin) containing saturated or monounsaturated chains. Here we tested a mechanism by which multivalent PIP(2) molecules could be transferred into rafts through electrostatic interactions with polybasic cytoplasmic proteins, such as GAP-43, which bind to rafts via their acylated N-termini.

Roles of bilayer material properties in function and distribution of membrane proteins.

Structural, compositional, and material (elastic) properties of lipid bilayers exert strong influences on the interactions of water-soluble proteins and peptides with membranes, the distribution of transmembrane proteins in the plane of the membrane, and the function of specific membrane channels. Theoretical and experimental studies show that the binding of either cytoplasmic proteins or extracellular peptides to membranes is regulated by the presence of charged lipids and that the sorting of transmembrane proteins into or out of membrane microdomains (rafts) depends on several factors, including bilayer material properties governed by the presence of cholesterol. Recent studies have also shown that bilayer material properties modify the permeability of membrane pores, formed either by protein channels or by cell-lytic peptides.

Transbilayer peptide sorting between raft and nonraft bilayers: comparisons of detergent extraction and confocal microscopy.

Membrane microdomains ("rafts") that sequester specific proteins and lipids are often characterized by their resistance to detergent extraction. Because rafts are enriched in sphingomyelin and cholesterol, raft bilayers are thicker and have larger area compressibility moduli than nonraft bilayers. It has been postulated that rafts concentrate proteins with long transmembrane domains (TMDs) because of "hydrophobic matching" between the TMDs and the thick raft bilayers.

Packing and viscoelasticity of polyunsaturated omega-3 and omega-6 lipid bilayers as seen by (2)H NMR and X-ray diffraction.

Polyunsaturated phospholipids of the omega-3 and omega-6 classes play key roles in cellular functions, yet their mechanisms of biological action are still a matter of debate. Using deuterium ((2)H) NMR spectroscopy and small-angle X-ray diffraction, we show how membrane properties are modified by docosahexaenoic (DHA; 22:6) and arachidonic (AA; 20:4) acyl chains of the omega-3 and the omega-6 families, respectively. Structural and dynamical differences due to polyunsaturation are evident in both the ordered and disordered phases of mixed-chain (16:0)(22:6)PC and (16:0)(20:4)PC bilayers.

Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores.

The membrane-lytic peptide melittin has previously been shown to form pores in lipid bilayers that have been described in terms of two different structural models. In the "barrel stave" model the bilayer remains more or less flat, with the peptides penetrating across the bilayer hydrocarbon region and aggregating to form a pore, whereas in the "toroidal pore" melittin induces defects in the bilayer such that the bilayer bends sharply inward to form a pore lined by both peptides and lipid headgroups. Here we test these models by measuring both the free energy of melittin transfer (DeltaG degrees ) and melittin-induced leakage as a function of bilayer elastic (material) properties that determine the energetics of bilayer bending, including the area compressibility modulus (K(a)), bilayer bending modulus (k(c)), and monolayer spontaneous curvature (R(o)).

Charge-reversal amphiphiles for gene delivery.

Delivering a missing gene or a functional substitute of a defective gene has the potential to revolutionize current medical care. Of the two gene delivery approaches, viral and synthetic vectors, synthetic cationic vectors possess several practical advantages but suffer from poor transfection efficiency. A new approach to gene delivery using charge-reversal amphiphiles is described.

Jumping to rafts: gatekeeper role of bilayer elasticity.

Two of the physiologically important processes that take place in biological membranes are the partitioning of water-soluble proteins into the membrane and the sequestering of specific transmembrane proteins into membrane microdomains or 'rafts'. Although these two processes often involve different classes of protein, recent biophysical studies indicate that they both strongly depend on the structural and elastic properties of the membrane bilayer. That is, both the partitioning of peptides into membranes and the distribution of transmembrane peptides in the plane of the membrane are modulated by physical properties of the lipid bilayer that are controlled by cholesterol content and the composition of the phospholipid hydrocarbon chain.