Size selectivity between gap junction channels composed of different connexins

Gap junction channels are traditionally viewed as large, nonspecific pores connecting cells. Recently the diversity in the connexin family has drawn more attention to their permeability characteristics. Several studies have shown that both size and charge contribute to the permeability of gap junctional channels. We have used a graded series of neutral polyethylene glycol probes (PEGs), which eliminate charge contribution completely, to specifically assess the physical exclusion limits of gap junction channels formed by different connexins. Cx 26, 32 and 37 were expressed in paired Xenopus oocytes to form homotypic gap junctional channels. PEG probes were perfused intracellularly into one side of the oocyte pair. A reversible drop in conductance of the gap juctional channels indicated that the probe was small enough to enter the pore and hinder ion flux. Our data suggest that Cx32 channels have a size cut-off between PEG 400 (11.2 A) and PEG 300 (9.6 A) despite their relatively small single channel conductance (approximately 55 pS). Cx26 channels (approximately 130 pS single channel conductance) have a size exclusion limit around PEG 200 (8.0 A), while Cx37 channels show the most restricted size cut-off between PEG 200 (8.0 A) and TriEG (6.8 A), despite having the largest unitary conductance (approximately 300 pS).     

Wide nanoscopic pore of maxi-anion channel suits its function as an ATP-conductive pathway

The newly proposed function of the maxi-anion channel as a conductive pathway for ATP release requires that its pore is sufficiently large to permit passage of a bulky ATP(4-) anion. We found a linear relationship between relative permeability of organic anions of different size and their relative ionic mobility (measured as the ratio of ionic conductance) with a slope close to 1, suggesting that organic anions tested with radii up to 0.49 nm (lactobionate) move inside the channel by free diffusion. In the second approach, we, for the first time, succeeded in pore sizing by the nonelectrolyte exclusion method in single-channel patch-clamp experiments. The cutoff radii of PEG molecules that could access the channel from intracellular (1.16 nm) and extracellular (1.42 nm) sides indicated an asymmetry of the two entrances to the channel pore. Measurements by symmetrical two-sided application of PEG molecules yielded an average functional pore radius of approximately 1.3 nm. These three estimates are considerably larger than the radius of ATP(4-) (0.57-0.65 nm) and MgATP(2-) (approximately 0.60 nm). We therefore conclude that the nanoscopic maxi-anion channel pore provides sufficient room to accommodate ATP and is well suited to its function as a conductive pathway for ATP release in cell-to-cell communication.     

Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion

The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200-1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100-300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300-1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600-1500, whereas 100 mOsm solutions of PEG300-400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300-400, but not to PEG600-1500. The swelling-activated channels conducting PEG300-400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R_h of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of ∼ 0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300-1500, the smallest PEG (PEG200, R_h = 0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues.     

Use of Nonelectrolytes Reveals the Channel Size and Oligomeric Constitution of the Borrelia burgdorferi P66 Porin

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than ‘normal’ Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be ≤1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80–90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.     

Inducible or constitutive polyethylene glycol dehydrogenase involved in the aerobic metabolism of polyethylene glycol

2, 6-Dichlorophenolindophenol (DCIP)-dependent polyethylene glycol (PEG) dehydrogenase activity was found in the particulate fractions of cell-free extracts prepared from PEG-utilizing bacteria (Pseudomonas and Flavobacterium species). This result suggested that PEG dehydrogenase is linked to the respiratory chain of each bacterium and that the enzyme plays a major role in the aerobic metabolism of PEG. Enzyme activities were strongly inhibited by 1, 4-benzoquinone. No metal ion was indispensable for the enzyme activities. Enzyme activities of PEG-utilizing bacteria were induced by PEG except for the activity of PEG 4000-utilizing Flavobacterium sp. no. 203 which had a constitutive enzyme. Although PEG-utilizing bacteria had different growth substrate specificities toward PEGs 200–20,000, their PEG dehydrogenases oxidized the same molecular wt. range of PEGs (dimer-20,000). Cell-free extracts of PEG 400-, 1000- or 4000-utilizing bacteria oxidized PEG 6000 and 20,000 though these bigger PEGs could not be utilized as the sole carbon and energy sources by the bacteria. Methanol, ethylene glycol and glycerol were not or only barely dehydrogenated by all the enzyme preparations.     

Analysis of PEG 400 and 4000 in urine for gut permeability assessment using solid phase extraction and gel permeation chromatography with refractometric detection

We developed a treatment of urine samples allowing the analysis of two intestinal permeability markers: polyethylene glycol (PEG) 400 (highly diffusible; basal permeability indicator) and PEG 4000 (poorly diffusible; indicator of an abnormal increase of permeability) by a unique gel permeation chromatography (GPC) with refractometric detection. Urinary PEG were extracted using a mixed-bed resin composed of C2 and C18 layers. Permeability mean values determined in 11 human healthy subjects were 24.20 +/- 9.30% and 0.12 +/- 0.08% for, respectively, PEG 400 and 4000. The percentage of the PEG 4000 permeability value to the one of PEG 400 corresponded to an intestinal permeability index (IPI) of 0.52 +/- 0.35 expressing a low diffusion of this poorly permeability marker.     

Integrated effects of fractional laser microablation and sonophoresis on skin immersion optical clearing in vivo

This study is aimed to find an approach for effective skin optical clearing in vivo using polyethylene glycol 300 (PEG‐300) as an optical clearing agent in combination with physical enhancers: fractional laser microablation (FLMA) and/or low‐frequency sonophoresis. In this study albino outbred rats were used. Light attenuation coefficient and optical clearing potential (OCP) of these approaches were evaluated in upper (from ~70 to ~200 μm) and middle (from ~200 to ~400 μm) dermis separately using optical coherence tomography. In 30 minutes, OCP of sonophoresis in combination with FLMA and PEG‐300 in the upper dermis was the maximal (2.3 ± 0.4) in comparison with other treatments in this time point. The most effective approach for optical clearing of middle dermis was PEG‐300 and sonophoresis; but the maximal value of OCP (1.6 ± 0.1) was achieved only in 90 minutes.     

Sizing the Bacillus anthracis PA63 channel with nonelectrolyte poly(ethylene glycols)

Nonelectrolyte polymers of poly(ethylene glycol) (PEG) were used to estimate the diameter of the ion channel formed by the Bacillus anthracis protective antigen 63 (PA₆₃). Based on the ability of different molecular weight PEGs to partition into the pore and reduce channel conductance, the pore appears to be narrower than the one formed by Staphylococcus aureus α-hemolysin. Numerical integration of the PEG sample mass spectra and the channel conductance data were used to refine the estimate of the pore's PEG molecular mass cutoff (∼1400 g/mol). The results suggest that the limiting diameter of the PA₆₃ pore is <2 nm, which is consistent with an all-atom model of the PA₆₃ channel and previous experiments using large ions.     

Poroelastic bulk properties of the tectorial membrane measured with osmotic stress

The equilibrium stress-strain relation and the pore radius of the isolated tectorial membrane (TM) of the mouse were determined. Polyethylene glycol (PEG), with molecular mass (MM) in the range 20-511 kDa, added to the TM bathing solution was used to exert an osmotic pressure. Strain on the TM induced by isosmotic PEG solutions of different molecular masses was approximately the same for MM > or = 200 kDa. However, for MM < or = 100 kDa, the TM strain was appreciably smaller. We infer that for the smaller molecular mass, PEG entered the TM and exerted a smaller effective osmotic pressure. The pore radius of the TM was estimated as 22 nm. The equilibrium stress-strain relation of the TM was measured using PEG with a molecular mass of 511 kDa. This relation was nonlinear and was fit with a power function. In the radial cochlear direction, the transverse stiffness of the TM was 20% stiffer in the inner than in the outer region. TM segments from the basal region had a larger transverse stiffness on average compared to sections from the apical-middle region. These measurements provide a quantitative basis for a poroelastic model of the TM.     

Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol).

The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.     

Polymeric nonelectrolytes to probe pore geometry: application to the alpha-toxin transmembrane channel

Asymmetrical (one-sided) application of penetrating water-soluble polymers, polyethylene glycols (PEGs), to a well-defined channel formed by Staphylococcus aureus alpha-toxin is shown to probe channel pore geometry in more detail than their symmetrical (two-sided) application. Polymers added to the cis side of the planar lipid membrane (the side of protein addition) affect channel conductance differently than polymers added to the trans side. Because a satisfactory theory quantitatively describing PEG partitioning into a channel pore does not exist, we apply the simple empirical rules proposed previously (, J. Membr. Biol. 161:83-92) to gauge the size of pore openings as well as the size and position of constrictions along the pore axis. We estimate the radii of the two openings of the channel to be practically identical and equal to 1. 2-1.3 nm. Two apparent constrictions with radii of approximately 0. 9 nm and approximately 0.6-0.7 nm are inferred to be present in the channel lumen, the larger one being closer to the cis side. These structural findings agree well with crystallographic data on the channel structure (, Science. 274:1859-1866) and verify the practicality of polymer probing. The general features of PEG partitioning are examined using available theoretical considerations, assuming there is no attraction between PEG and the channel lumen. It is shown that the sharp dependence of the partition coefficient on polymer molecular weight found under both symmetrical and asymmetrical polymer application can be rationalized within a "hard sphere nonideal solution model." This finding is rather surprising because PEG forms highly flexible coils in water with a Kuhn length of only several Angstroms.     

Polyethylene glycol behaves like weak organic solvent

Effect of polyethylene glycol (PEG) on protein solubility has been primarily ascribed to its large hydrodynamic size and thereby molecular crowding effect. However, PEG also shows characteristics of organic solvents. Here, we have examined the solubility of glycine and aliphatic and aromatic amino acids in PEG solutions. PEG400, PEG4000, and PEG20000 decreased the solubility of glycine, though to a much smaller magnitude than the level achieved by typical organic solvents, including ethanol and dimethyl sulfoxide. PEG4000 showed varying degree of interactions with amino acid side chains. The free energy of aliphatic side chains marginally increased by the addition of PEG4000, indicating their weak unfavorable interactions. However, it significantly decreased the free energy of the aromatic side chains and hence stabilized them. Thus, it was concluded that PEG behaves like weak organic solvents; namely PEG destabilized (interacted unfavorably with) polar and charged groups and stabilized (interacted favorably with) aromatic groups. Interestingly, the interaction of PEG20000, but neither PEG400 nor PEG4000, with glycine resulted in phase separation under the saturated concentration of glycine.     

Poly(Ethylene Glycol) as a Scaffold for High-Affinity Open-Channel Blockers of the Mouse Nicotinic Acetylcholine Receptor

High-affinity blockers for an ion channel often have complex molecular structures that are synthetically challenging and/or laborious. Here we show that high-affinity blockers for the mouse nicotinic acetylcholine receptor (AChR) can be prepared from a structurally simple material, poly(ethylene glycol) (PEG). The PEG-based blockers (PQ1–5), comprised of a flexible octa(ethylene glycol) scaffold and two terminal quaternary ammonium groups, exert low- to sub-micromolar affinities for the open AChR pore (measured via single-channel analysis of AChRs expressed in human embryonic kidney cells). PQ1–5 are comparable in pore-binding affinity to the strongest AChR open-channel blockers previously reported, which have complex molecular structures. These results suggest a general approach for designing potent open-channel blockers from a structurally flexible polymer. This design strategy involves simple synthetic procedures and does not require detailed information about the structure of an ion-channel pore.     

Lipase immobilization on magnetic microspheres via spacer arms: Effect of steric hindrance on the activity

Poly(styrene-acrylic acid) magnetic microspheres with an average diameter of 2 μm were successfully prepared and used as carriers to immobilize lipase. Lipase immobilized on microspheres with no spacer arm exhibited low activities, which were attributed to steric hindrance on the lipase conformation. To avoid steric effects, ethylenediamine and poly(ethylene glycol) (PEG) 400/800/4000 were utilized as spacer arms to bind the lipase to the microspheres. The immobilized lipase activities were improved using PEG 800/4000 as a spacer arm. Furthermore, the influence of enzyme loading on lipase activity was investigated, and the results indicated that enzyme overloading could exert steric effect on lipase activity. The degree of PEG modification was demonstrated to affect lipase activity because excess PEG on the surface of microspheres could interact with lipase due to its mobility, consequently reducing lipase activity.     

Preparation of self-assembled silk sericin nanoparticles

Silk sericin (SS) possessing moisture-retaining property was reacted with activated poly(ethylene glycol) (PEG) to obtain self-assembled SS nanoparticles. The aliphatic and aromatic hydroxyl groups of serine and tyrosine residues as the reaction sites in SS were clarified by amino acid analysis and 1H NMR spectroscopy, respectively. From IR and circular dichroism (CD) measurements, introduction of PEG into SS induced the conformational change from random coil to beta-sheet. DSC thermogram of sericin-PEG conjugate suggests that mutual miscibility between PEG and SS chains was poor. Nanoparticles of sericin-PEG conjugate with sizes measured by dynamic light scattering ranging about 200-400 nm in diameter, were prepared by the diafiltration method. Shape of sericin-PEG conjugate nanoparticles observed by scanning and transmission electron microscopes was spherical. The results suggest that sericin-PEG conjugates are self-associated to form spherical nanoparticles through hydrophobic interaction.     

Asymmetry of syringomycin E channel studied by polymer partitioning

To probe the size of the ion channel formed by Pseudomonas syringae lipodepsipeptide syringomycin E, we use the partial blockage of ion current by penetrating poly(ethylene glycol)s. Earlier experiments with symmetric application of these polymers yielded a radius estimate of approximately 1 nm. Now, motivated by the asymmetric non-ohmic current-voltage curves reported for this channel, we explore its structural asymmetry. We gauge this asymmetry by studying the channel conductance after one-sided addition of differently sized poly(ethylene glycol)s. We find that small polymers added to the cis-side of the membrane (the side of lipodepsipeptide addition) reduce channel conductance much less than do the same polymers added to the trans-side. We interpret our results to suggest that the water-filled pore of the channel is conical with cis- and trans-radii differing by a factor of 2-3 and that the smaller cis-radius is in the 0.25-0.35 nm range. In symmetric, two-sided addition, polymers entering the pore from the larger opening dominate blockage.     

Separation and purification of glucoamylase in aqueous two-phase systems by a two-step extraction

Extraction in two steps of glucoamylase was studied in poly(ethylene glycol) (PEG) and potassium phosphate systems at pH values of 6, 7 and 9. Ten different conditions using PEG 300, 600, 1500, 4000 and 6000 were studied. The bottom phase of the first extraction step, with the enzyme, was reused in an appropriate concentration of PEG to form the second extraction step. The optimal partitioning conditions for glucoamylase separation were obtained in PEG 4000 (first step), PEG 1500 (second step) at pH 7 and resulted in a three-fold increase in glucoamylase purification.     

Development of an improved medium for germination of Cajanus cajan (L.) Millsp. pollen in vitro.

A simple, reliable medium for pollen germination of Cajanus cajan was developed by modifying Brewbaker and Kwack (BK) medium. Past attempts of C. cajan pollen germination in artificial media were not successful. A medium containing polyethylene glycol 4000 (PEG) showed more than 90% germination for C. cajan var. Pusa 33 only when the young buds (36 h before anthesis) were kept in pollen germination medium (PGM) for 36 h before pollen extraction. Supplementation of PGM with epsilon-amino caproic acid (EACA), an amino acid, showed improved pollen germination in Pusa 33 and also helped to avoid preconditioning of young buds before pollen extraction. It was also observed that there is a genotypic difference in the level of EACA required for in vitro pollen germination. Thus a complete medium for C. cajan genotypes consists of 37.5% sucrose+ 15% PEG 4000+250 mg l(-1) boric acid+300 mg l(-1) calcium nitrate+100 mg l(-1) potassium nitrate+ 200 mg l(-1) magnesium sulphate+1% agar+EACA (0, 100, 250, 500, 750 or 1000 mg l(-1)).     

Cluster organization of ion channels formed by the antibiotic syringomycin E in bilayer lipid membranes.

The cyclic lipodepsipeptide, syringomycin E, when incorporated into planar lipid bilayer membranes, forms two types of channels (small and large) that are different in conductance by a factor of sixfold. To discriminate between a cluster organization-type channel structure and other possible different structures for the two channel types, their ionic selectivity and pore size were determined. Pore size was assessed using water-soluble polymers. Ion selectivity was found to be essentially the same for both the small and large channels. Their reversal (zero current) potentials with the sign corresponding to anionic selectivity did not differ by more than 3 mV at a twofold electrolyte gradient across the bilayer. Reduction in the single-channel conductance induced by poly(ethylene glycol)s of different molecular weights demonstrated that the aqueous pore sizes of the small and large channels did not differ by more than 2% and were close to 1 nm. Based on their virtually identical selectivity and size, we conclude that large syringomycin E channels are clusters of small ones exhibiting synchronous opening and closing.     

Low molecular weight linear polyethylenimine-b-poly(ethylene glycol)-b-polyethylenimine triblock copolymers: synthesis, characterization, and in vitro gene transfer properties

Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.