Lens extraction is a strong risk factor for the development of late-onset OAG after uncomplicated PPV. While the overall incidence of OAG development after PPV is substantial, it is more so among eyes that have had CE. The absence of substantial OAG incidence in phakic patients points toward a combined mechanism for late-onset post-PPV OAG involving PPV and CE at any time. Preoperative PPV counseling should include the risk of glaucoma development in addition to cataract development and the connection between the two. Patients who have undergone PPV, and especially those who also had CE in the same eye, should be carefully monitored for glaucoma.

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10-20 microg of pure connexin protein from 2.5x10(8) HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)-agarose make this affinity tagging and purification procedure easily applicable to other proteins.

The isoelectric points of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) were determined by isoelectric focusing in free fluids. The isoelectric points were significantly more acidic than predicted from amino acid sequences and different from each other, allowing homomeric channels to be resolved separately. The isoelectric points of the homomeric channels bracketed the isoelectric points of heteromeric Cx26/Cx32 channels. For heteromeric channels, Cx26 and Cx32 were found in overlapping, pH-focused fractions, indicating quaternary structure was retained. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify post-translational modifications of Cx26 and Cx32 cytoplasmic domains, including the first reported post-translational modifications of Cx26. Suspected modifications were hydroxylation and/or phosphorylation near the amino terminus of both connexins, gamma-carboxyglutamate residues in the cytoplasmic loop of both connexins, phosphorylation in the carboxyl-terminal domain of Cx32, and palmitoylation at the carboxyl-terminus of Cx32. These modifications contribute to the measured acidic isoelectric points of Cx26 and Cx32, whereas their low molecular masses would not appreciably change connexin SDS-PAGE mobility. Most of these modifications have not previously been identified for connexins and may be instrumental in guiding and understanding novel aspects of channel trafficking and molecular mechanisms of channel regulation.

Previous work has shown that channels formed by both connexin (Cx)26 and Cx32 (heteromeric Cx26/Cx32 hemichannels) are selectively permeable to cAMP and cGMP. To further investigate differential connexin channel permeability among second messengers, and the influence of connexin channel composition on the selectivity, the permeability of inositol phosphates with one to four phosphate groups through homomeric Cx26, homomeric Cx32, and heteromeric Cx26/Cx32 channels was examined. Connexin channels were purified from transfected HeLa cells and from rat, mouse, and guinea pig livers, resulting in channels with a broad range of Cx26/ Cx32 aggregate ratios. Permeability to inositol phosphates was assessed by flux through reconstituted channels. Surprisingly, myoinositol and all inositol phosphates tested were permeable through homomeric Cx32 and homomeric Cx26 channels. Even more surprising, heteromeric Cx26/Cx32 channels showed striking differences in permeability among inositol phosphates with three or four phosphate groups and among isomers of inositol triphosphate. Thus, heteromeric channels are selectively permeable among inositol phosphates, whereas the corresponding homomeric channels are not. There was no discernible difference in the permeability of channels with similar Cx26/Cx32 ratios purified from native and heterologous sources. The molecular selectivity of heteromeric channels among three inositol triphosphates could not be accounted for by simple connexin isoform stoichiometry distributions and therefore may depend on specific isoform radial arrangements within the hexameric channels. Dynamic regulation of channel composition in vivo may effectively and efficiently modulate intercellular signaling by inositol phosphates.Connexin channels, which compose most vertebrate gap junctions, mediate direct intercellular movement of ions and molecules. There are ϳ20 isoforms of connexin protein, each forming channels with distinct functional properties. Every known functional deletion of a connexin isoform produces a distinct pathology, and genetic replacement of one connexin (Cx) 2 by another ("knock in") fails to fully compensate (1-3). Pathologies that arise from altered connexin channel function must arise from abnormal molecular movement through connexin channels, whether in magnitude, regulation, or molecular specificity (reviewed in Ref. 4).Gap junction channels form by end-to-end interaction of hemichannels, each consisting of six connexin monomers. Hemichannels are either homomeric (composed of a single connexin isoform) or heteromeric (more than one isoform). Dramatic and surprising degrees of ionic and molecular permselectivity have been observed for homomeric channels (5-13). However, most cells express more than one connexin, and heteromeric connexin channels are common in vivo (14 -18). Heteromeric mixing of different connexin isoforms, producing variation in channel stoichiometry and/or arrangements of isoforms within the hemichannels, may allow cells to dynamically regulate their intercellular co...

Cyclodextrins (CDs), a series of hollow cyclic glucosaccharides, can reversibly block molecular permeation through channels formed by connexin-32 and/or connexin-26 reconstituted into liposomes. The character of the block changes as a function of the size of the CD relative to the connexin pore diameter, suggesting that the block occurs via entry of the CD into the pore lumen and occlusion of the permeability pathway. The block occurs only when the CD is applied to the side of the pore that is normally cytoplasmic and not from the side that is normally extracellular. The block is potentiated when organic analytes are sequestered in the hydrophobic interior of the CDs. CDs may be useful as molecular tools with which to explore the structure of the connexin pore and to alter molecular movement through connexin channels.