An electrodiffusion model for effects of surface glycocalyx layer on microvessel permeability

To investigate the charge effect of the endothelial surface glycocalyx on microvessel permeability, we extended the three-dimensional model developed by Fu et al. (J Biomech Eng 116: 502-513, 1994) for the interendothelial cleft to include a negatively charged glycocalyx layer at the entrance of the cleft. Both electrostatic and steric exclusions on charged solutes were considered within the glycocalyx layer and at the interfaces. Four charge-density profiles were assumed for the glycocalyx layer. Our model indicates that the overall solute permeability across the microvessel wall including the surface glycocalyx layer and the cleft region is independent of the charge-density profiles as long as they have the same maximum value and the same total charge. On the basis of experimental data, this model predicts that the charge density would be 25-35 meq/l in the glycolcalyx of frog mesenteric capillaries. An intriguing prediction of this model is that when the concentrations of cations and anions are unequal in the lumen due to the presence of negatively charged proteins, the negatively charged glycocalyx would provide more resistance to positively charged solutes than to negatively charged ones.     

Structural mechanisms of acute VEGF effect on microvessel permeability

To investigate the ultrastructural mechanisms of acute microvessel hyperpermeability by vascular endothelial growth factor (VEGF), we combined a mathematical model (J Biomech Eng 116: 502-513, 1994) with experimental data of the effect of VEGF on microvessel hydraulic conductivity (L(p)) and permeability of various-sized solutes. We examined the effect of VEGF on microvessel permeability to a small solute (sodium fluorescein, Stokes radius 0.45 nm), an intermediate solute (alpha-lactalbumin, Stokes radius 2.01 nm), and a large solute [albumin (BSA), Stokes radius 3.5 nm]. Exposure to 1 nM VEGF transiently increased apparent permeability to 2.3, 3.3, and 6.2 times their baseline values for sodium fluorescein, alpha-lactalbumin, and BSA, respectively, within 30 s, and all returned to control within 2 min. On the basis of L(p) (DO Bates and FE Curry. Am J Physiol Heart Circ Physiol 271: H2520-H2528, 1996) and permeability data, the prediction from the model suggested that the most likely structural changes in the interendothelial cleft induced by VEGF would be a approximately 2.5-fold increase in its opening width and partial degradation of the surface glycocalyx.     

An electrodiffusion model for the blood-brain barrier permeability to charged molecules

The endothelial surface glycocalyx layer (SGL) and the basement membrane (BM) are two important components of the blood-brain barrier (BBB). They provide large resistance to solute transport across the BBB in addition to the tight junctions in the cleft between adjacent endothelial cells. Due to their glycosaminoglycan compositions, they carry negative charge under physiological conditions. To investigate the charge effect of the SGL and BM on the BBB permeability to charged solutes, we developed an electrodiffusion model for the transport of charged molecules across the BBB. In this model, constant charge densities were assumed in the SGL and in the BM. Both electrostatic and steric interaction and exclusion to charged molecules were considered within the SGL and the BM and at their interfaces with noncharged regions of the BBB. On the basis of permeability data for the positively charged ribonuclease (+4,radius=2.01?nm) and negatively charged α-lactalbumin (-10,radius=2.08?nm) measured in intact rat mesenteric and pial microvessels, our model predicted that the charge density in both SGL and BM would be ～30?mEq/L, which is comparable to that in the SGL of mesenteric microvessels. Interestingly, our model also revealed that due to the largest concentration drop in the BM, there is a region with a higher concentration of negatively charged α-lactalbumin in the uncharged inter-endothelial cleft, although the concentration of α-lactalbumin is always lower than that of positively charged ribonuclease and that of a neutral solute in the charged SGL and BM.     

A model for the modulation of microvessel permeability by junction strands

To investigate the effect of junction strands on microvessel permeability, we extend the previous analytical model developed by Fu et al. (1994, J. Biomech. Eng., 116, pp. 502-513), for the interendothelial cleft to include multiple junction strands in the cleft and an interface between the surface glycocalyx layer and the cleft entrance. Based on the electron microscopic observations by Adamson et al. (1998, Am. J. Physiol., 274(43), pp. H1885-H1894), that elevation of intracellular cAMP levels would increase number of tight junction strands, this two-junction-strand and two-pore model can successfully account for the experimental data for the decreased permeability to water, small and intermediate-sized solutes by cAMP.     

Starling forces that oppose filtration after tissue oncotic pressure is increased

We tested the hypothesis that the effective oncotic force that opposes fluid filtration across the microvessel wall is the local oncotic pressure difference across the endothelial surface glycocalyx and not the global difference between the plasma and tissue. In single frog mesenteric microvessels perfused and superfused with solutions containing 50 mg/ml albumin, the effective oncotic pressure exerted across the microvessel wall was not significantly different from that measured when the perfusate alone contained albumin at 50 mg/ml. Measurements were made during transient and steady-state filtration at capillary pressures between 10 and 35 cmH(2)O. A cellular-level model of coupled water and solute flows in the interendothelial cleft showed water flux through small breaks in the junctional strand limited back diffusion of albumin into the protected space on the tissue side of the glycocalyx. Thus oncotic forces opposing filtration are larger than those estimated from blood-to-tissue protein concentration differences, and transcapillary fluid flux is smaller than estimated from global differences in oncotic and hydrostatic pressures.     

Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells

The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.     

A mechano-electrochemical model of radial deformation of the capillary glycocalyx

A mechano-electrochemical theory of the surface glycocalyx on capillary endothelial cells is presented that models the structure as a mixture of electrostatically charged macromolecules hydrated in an electrolytic fluid. Disturbances arising from mechanical deformation are introduced as perturbations away from a nearly electroneutral equilibrium environment. Under mechanical compression of the layer, such as might occur on the passing of stiff leukocytes through capillaries, the model predicts that gradients in the electrochemical potential of the compressed layer cause a redistribution of mobile ions within the glycocalyx and a rehydration and restoration of the layer to its equilibrium dimensions. Because of the large deformations of the glycocalyx arising from passing leukocytes, nonlinear kinematics associated with finite deformations of the layer are accounted for in the theory. A pseudo-equilibrium approximation is invoked for the transport of the mobile ions that reduces the system of coupled nonlinear integro-differential equations to a single nonlinear partial differential equation that is solved numerically for the compression and recovery of the glycocalyx using a finite difference method on a fixed grid. A linearized model for small strains is also obtained as verification of the finite difference solution. Results of the asymptotic analysis agree well with the nonlinear solution in the limit of small deformations of the layer. Using existing experimental and theoretical estimates of glycocalyx properties, the glycocalyx fixed-charge density is estimated from the analysis to be approximately 1 mEq/l, i.e., we estimate that there exists approximately one fixed charge on the glycocalyx for every 100 ions in blood. Such a charge density would result in a voltage differential between the undeformed glycocalyx and the capillary lumen of approximately 0.1 mV. In addition to providing insight into the mechano-electrochemical dynamics of the layer under deformation, the model suggests several methods for obtaining improved estimates of the glycocalyx fixed-charge density and permeability in vivo.     

Fluorescence correlation spectroscopy can probe albumin dynamics inside lung endothelial glycocalyx

The endothelial glycocalyx is believed to play a major role in capillary permeability by functioning as a macromolecular barrier overlying the intercellular junction. Little is known about the functional attributes of the glycocalyx (i.e., porosity and permeability) or which constituents contribute to its overall structure-function relationship. In this report, we demonstrate the utility of fluorescence correlation spectroscopy (FCS) to measure albumin diffusion rates and concentration profiles above the cell surface and overlying the intercellular junctions of lung capillary endothelial cells. Albumin diffusion rates and concentration profiles were obtained before and after enzymatic digestion of the glycocalyx with pronase, heparanase, or hyaluronidase. The results suggest a structure interacting with albumin located from 1.0 to 2.0 microm above the cell membrane capable of reducing albumin diffusion by 30% while simultaneously increasing albumin concentration fivefold. Digestion of the glycocalyx with pronase or heparanase resulted in only modest changes in albumin diffusion and concentration profiles. Hyaluronidase digestion completely eliminated albumin-glycocalyx interactions. These data also suggest that hyaluronan is a major determinant for albumin interactions with the lung endothelial glycocalyx. Confocal images of heparan sulfate and hyaluronan confirm a cell-surface layer 2-3 mum in thickness, thus supporting FCS measurements. In summary, we report the first use of FCS to probe extracellular structures and further our understanding of the structure-function relationship of the lung microvascular endothelial glycocalyx.     

On the mechanism of oleate transport across human brain microvessel endothelial cells

The blood–brain barrier formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the CNS. As most fatty acids in the brain enter from the blood, we examined the mechanism of oleate (C18:1) transport across primary human brain microvessel endothelial cells (HBMEC). The permeability of [1-¹⁴C]oleate was determined using confluent cells grown on Transwell® inserts in both the absence or presence of bovine serum albumin in the basolateral media, and following inhibition of various fatty acid transporters. The passage of [1-¹⁴C]oleate across confluent HBMEC monolayers was significantly enhanced when fatty acid free albumin was present in the basolateral media. The presence of the non-specific fatty acid uptake inhibitor phloretin significantly decreased [1-¹⁴C]oleate uptake by HBMEC and the subsequent release of [1-¹⁴C]oleate into the basolateral medium. Knockdown of fatty acid transport protein-1 or fatty acid translocase/CD36 significantly decreased [1-¹⁴C]oleate transport across the HBMEC monolayer from either apical as well as basolateral sides. The findings indicate that a fatty acid acceptor is a requirement for oleate transport across HBMEC monolayers. In addition, transport of oleate across HBMEC is, in part, a transcellular process mediated by fatty acid transport proteins.     

A 1-D model to explore the effects of tissue loading and tissue concentration gradients in the revised Starling principle

The recent experiments in Hu et al. (Am J Physiol Heart Circ Physiol 279: H1724-H1736, 2000) and Adamson et al. (J Physiol 557: 889-907, 2004) in frog and rat mesentery microvessels have provided strong evidence supporting the Michel-Weinbaum hypothesis for a revised asymmetric Starling principle in which the Starling force balance is applied locally across the endothelial glycocalyx layer rather than between lumen and tissue. These experiments were interpreted by a three-dimensional (3-D) mathematical model (Hu et al.; Microvasc Res 58: 281-304, 1999) to describe the coupled water and albumin fluxes in the glycocalyx layer, the cleft with its tight junction strand, and the surrounding tissue. This numerical 3-D model converges if the tissue is at uniform concentration or has significant tissue gradients due to tissue loading. However, for most physiological conditions, tissue gradients are two to three orders of magnitude smaller than the albumin gradients in the cleft, and the numerical model does not converge. A simpler multilayer one-dimensional (1-D) analytical model has been developed to describe these conditions. This model is used to extend Michel and Phillips's original 1-D analysis of the matrix layer (J Physiol 388: 421-435, 1987) to include a cleft with a tight junction strand, to explain the observation of Levick (Exp Physiol 76: 825-857, 1991) that most tissues have an equilibrium tissue concentration that is close to 0.4 lumen concentration, and to explore the role of vesicular transport in achieving this equilibrium. The model predicts the surprising finding that one can have steady-state reabsorption at low pressures, in contrast to the experiments in Michel and Phillips, if a backward-standing gradient is established in the cleft that prevents the concentration from rising behind the glycocalyx.     

Segmental barrier properties of the pulmonary microvascular bed.

We determined liquid flux across single pulmonary microvessels of dog, ferret, and rat by our split-drop technique (J. Appl. Physiol. 64: 2562-2567, 1988). Data are reported from 58 lungs excised under halothane or pentobarbital sodium anesthesia and then blood perfused. We stopped blood flow at known vascular pressures and then micropunctured microvessels to inject oil, which we split with albumin solution. From measurements of vessel diameter and split oil drop length, we calculated Jv, the liquid transport rate per unit surface area [x 10(-6) ml/(cm2.s)]. At constant vascular pressure, Jv was not significantly different after different periods of oil-endothelium contact and at different sites within a single vessel. From measurements of Jv at different vascular pressures, we determined Lp, the hydraulic conductivity [x 10(-7) ml/(cm2.s.cmH2O)], and Pzf, the zero filtration pressure. From determinations of Pzf at different albumin concentrations, we quantified sigma alb, the albumin reflection coefficient. Lp and Pzf did not differ among venules of the same lung. However, in venules, Lp was 40% higher and sigma alb 25% lower than in arterioles (P less than 0.01). We conclude that 1) micropuncture procedures incidental to our split-drop technique do not progressively deteriorate the experimental microvessel and 2) in lung, permeability is higher in venules than in arterioles.     

Studies of a serum albumin-liposome complex as a model lipoprotein membrane.

Electron microscopy shows that the lipoprotein dispersions formed from the interaction of negatively charged liposomes with bovine serum albumin contain closed, vesicu lar, multilamellar structures. Discontinuous density gradient studies indicate that the lipoprotein suspensions are vesicles in which bovine serum albumin homogenously associates with lipid. Low angle X-ray diffraction results show that all the systems, positively and negatively charged, with and without protein, have the characteristic lamellar structure observed in biological membranes. The lamellar spacing (bilayer plus water layer) of negatively charged liposomes without bovine serum albumin is 55 A. The same lamellar separation in the positively charged system is 108 A. The lamellar spacing corresponding to bilayer, water, and protein for the negatively charged lipoprotein system is 93 A while that for the positively charged lipoprotein system is 91 A. These dimensions suggest that a layer of protein one molecule thick is incorporated between the lamellae bound to the surface of the bilayer. Wide angle X-ray diffraction results indicate no major effect of the protein on the 4.1 A spacing, characteristic of hexagonal packing of the hydrocarbon chains. A classical light scattering technique is used to show that the lipoprotein systems are osmotically active. The solute permeability exhibited by these lipoprotein systems follows the sequence (glucose smaller than arabinose smaller than malonamide smaller than glycerol). K+ diffusion from negatively charged lipoprotein systems is greater than that found for positively charged lipoprotein systems.     

A model for the blood–brain barrier permeability to water and small solutes

The blood–brain barrier (BBB) has unique structures in order to protect the central nervous system. In addition to the tight junction of the microvessel endothelium, there is a uniform and narrow matrix-like basement membrane (BM) sandwiched between the vessel wall and the astrocyte foot processes ensheathing the cerebral microvessel. To understand the mechanism by which these structural components modulate permeability of the BBB, we developed a mathematical model for water and solute transport across the BBB. The fluid flow in the cleft regions of the BBB were approximated by the Poiseuille flow while those in the endothelial surface glycocalyx layer (SGL) and BM were approximated by the Darcy and Brinkman flows, respectively. Diffusion equations in each region were solved for the solute transport. The anatomical parameters were obtained from electron microscopy studies in the literature. Our model predicts that compared to the peripheral microvessels with endothelium only, the BM and the wrapping astrocytes can reduce hydraulic conductivity (L_p) of the BBB and the permeability to sodium fluorescein (P^NaF) by up to 6-fold when the fiber density in the BM is the same as that in the SGL. Even when the SGL and the tight junctions of the endothelium are compromised, the BM and astrocyte foot processes can still maintain the low L_p and P^NaF of the BBB. Our model predictions indicate that the BM and astrocytes of the BBB provide a great protection to the CNS under both physiological and pathological conditions.     

Studies of a serum albumin-liposome complex as a model lipoprotein membrane

Electron microscopy shows that the lipoprotein dispersions formed from the interaction of negatively charged liposomes with bovine serum albumin contain closed, vesicular, multilamellar structures. Discontinuous density gradient studies indicate that the lipoprotein suspensions are vesicles in which bovine serum albumin homogenously associate with lipid.Low angle X-ray diffraction results show that all the systems, positively and negatively charged, with and without protein, have the characteristic lamellar structure observed in biological membranes. The lamellar spacing (bilayer plus water layer) of negatively charged liposomes without bovine serum albumin is 55 Å. The same lamellar separation in the positively charged system is 108 Å. The lamellar spacing corresponding to bilayer, water, and protein for the negatively charged lipoprotein system is 93 Å while that for the positively charged lipoprotein system is 91 Å. These dimensions suggest that a layer of protein one molecule thick is incorporated between the lamellae bound to the surface of the bilayer.Wide angle X-ray diffraction results indicate no major effect of the protein on the 4.1 Å spacing, characteristic of hexagonal packing of the hydrocarbon chains.A classical light scattering technique is to used to show that the lipoprotein systems are osmotically active. The solute permeability exhibited by these lipoprotein systems follows the sequence (glucose < arabinose < malonamide < glycerol). K⁺ diffusion from negatively charged lipoprotein systems is greater than that found for positively charged lipoprotein systems.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Capillary endothelial surface layer selectively reduces plasma solute distribution volume

We previously reported that a 0.4- to 0.5-microm-thick endothelial surface layer confines Dextran 70 (70 kDa) to the central core of hamster cremaster muscle capillaries. In the present study we used a variety of plasma tracers to probe the barrier properties of the endothelial surface layer using combined fluorescence and brightfield intravital microscopy. No permeation of the endothelial surface layer was observed for either neutral or anionic dextrans >/=70 kDa, but a neutral Dextran 40 (40 kDa) and neutral free dye (rhodamine, 0.4 kDa) equilibrated with the endothelial surface layer within 1 min. In contrast, small anionic tracers of similar size (0. 4-40 kDa) permeated the endothelial surface layer relatively slowly with half-times (tau(50)) between 11 and 60 min, depending on tracer size. Furthermore, two plasma proteins, fibrinogen (340 kDa) and albumin (67 kDa), moved slowly into the endothelial surface layer at the same rates, despite greatly differing sizes (tau(50) approximately 40 min). Dextran 70, which did not enter the glycocalyx over the course of these experiments, entered at the same rate as free albumin when it was conjugated to albumin. These findings demonstrate that for anionic molecules size and charge have a profound effect on the penetration rate into the glycocalyx. The equal rates of penetration of the glycocalyx demonstrated by the different protein molecules suggests that multiple factors may influence the penetration of the barrier, including molecular size, charge, and structure.     

Nanogels for oligonucleotide delivery to the brain

Systemic delivery of oligonucleotides (ODN) to the central nervous system is needed for development of therapeutic and diagnostic modalities for treatment of neurodegenerative disorders. Macromolecules injected in blood are poorly transported across the blood-brain barrier (BBB) and rapidly cleared from circulation. In this work we propose a novel system for ODN delivery to the brain based on nanoscale network of cross-linked poly(ethylene glycol) and polyethylenimine ("nanogel"). The methods of synthesis of nanogel and its modification with specific targeting molecules are described. Nanogels can bind and encapsulate spontaneously negatively charged ODN, resulting in formation of stable aqueous dispersion of polyelectrolyte complex with particle sizes less than 100 nm. Using polarized monolayers of bovine brain microvessel endothelial cells as an in vitro model this study demonstrates that ODN incorporated in nanogel formulations can be effectively transported across the BBB. The transport efficacy is further increased when the surface of the nanogel is modified with transferrin or insulin. Importantly the ODN is transported across the brain microvessel cells through the transcellular pathway; after transport, ODN remains mostly incorporated in the nanogel and ODN displays little degradation compared to the free ODN. Using mouse model for biodistribution studies in vivo, this work demonstrated that as a result of incorporation into nanogel 1 h after intravenous injection the accumulation of a phosphorothioate ODN in the brain increases by over 15 fold while in liver and spleen decreases by 2-fold compared to the free ODN. Overall, this study suggests that nanogel is a promising system for delivery of ODN to the brain.     

Interaction of albumin with the endothelial cell surface

Endothelial cells (EC) are covered with cell-borne proteoglycans and glycoproteins. Blood plasma proteins (e.g., albumin) adsorb to this glycocalyx forming a complex endothelial surface layer (ESL). We determined the molecular mobility of albumin by electron spin resonance (ESR) in the presence and absence of ECs to analyze interactions with the ESL. Albumin was spin labeled with 5- or 12-4,4-dimethyloxazolidine-N-oxyl (DOXYL)-stearic acid yielding information on the mobility of the molecular surface (5-DOXYL) or the entire protein (12-DOXYL). EC cultures grown on glass coverslips were immersed in labeled albumin and placed in the temperature-regulated cavity of an ESR spectrometer. Alternatively, ECs were labeled and then exposed to native albumin. At 37 degrees C, rotational correlation times determined by modified saturation transfer ESR (ST-ESR) were 26 and 48 ns for 5-DOXYL- and 12-DOXYL-labeled albumin, respectively. Presence of ECs increased rotational correlation time values for 5-DOXYL-stearic acid to 37 ns but not for 12-DOXYL-stearic acid. Albumin was able to completely take up the label from labeled EC within 2 min. The present study shows that modified ST-ESR can be used to determine the mobility of biological macromolecules interacting with cellular surfaces. Reduction in albumin surface mobility in the presence of EC at unchanged mobility of protein proper and fast removal of labeled fatty acids from EC membranes indicate rapid transient interactions between albumin surface and ESL but no rigid incorporation of albumin into a macromolecular network that would interfere with its transport function for poorly water-soluble substances.     

Gap junction permeability: selectivity for anionic and cationic probes

Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the permeability of homotypic connexin26 (Cx26), Cx40, Cx43, and Cx45 gap junction channels stably expressed in HeLa cells to solutes with different size and net charge. Channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe. All four connexins allowed passage of both cationic and anionic probes, but the transfer rates were connexin dependent. The negatively charged probes [Lucifer yellow (LY; median axial diameter 9.9 ?, charge -2), carboxyfluorescein (CF; 8.2 ?; -2), and Alexa Fluor350 (AF350, 5.4 ?; -1)] exhibited the following permeability order: Cx43 > Cx45 > Cx26 > Cx40. In contrast, for the positively charged species permeability, the orders were as follows: Cx26 ≈ Cx43 ≈ Cx40 ≈ Cx45 for N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl) amino] ethanaminium (NBD-m-TMA; 5.5 ?, +1) and Cx26 ≥ Cx43 ≈ Cx40 > Cx45 for ethidium bromide (10.3 ?, +1). Comparison of probe permeability relative to K(+) revealed that Cx43 and Cx45 exhibited similar permeability for NBD-m-TMA and AF350, indicating weak charge selectivity. However, lesser transfer of CF and LY through Cx45 relative to Cx43 channels suggests stronger size-dependent discrimination of solute. The permeability of NBD-m-TMA for Cx40 and Cx26 channels was approximately three times higher than to anionic AF350 despite the fact that both have similar minor diameters, suggesting charge selectivity. In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling.     

The first extracellular domain of claudin-7 affects paracellular Cl- permeability

Tight junctions (TJ) constitute paracellular diffusion channels regulating the passage of ions and solutes across epithelia. We recently demonstrated that overexpression of the TJ membrane protein claudin-7 in LLC-PK1 cells decreases paracellular permeability to Cl(-) and increases paracellular permeability to Na(+). To investigate the effect of charged amino acid residues in extracellular domains (ED) of claudin-7 on paracellular charge selectivity, we created claudin-7 mutants by replacing negatively charged amino acids on ED with positively charged amino acids. Immunofluorescence light microscopy showed that these mutant proteins were correctly targeted to the cell junction. Ultrastructure examination of TJ morphology did not reveal any difference between cells expressing wildtype (WT) and mutant claudin-7. However, electrophysiological studies showed increased Cl(-) permeability in cells expressing first extracellular domain (ED1) mutants, but not second extracellular domain (ED2) mutants, compared to that of WT claudin-7. Our results demonstrate that negatively charged amino acids in ED1 of claudin-7 are involved in modulating paracellular Cl(-) permeability.