Darren Locke

BIOLOGICAL AND BIOPHYSICAL PROPERTIES OF VASCULAR CONNEXIN CHANNELS

Intercellular channels formed by connexin proteins play a pivotal role in the direct movement of ions and larger cytoplasmic solutes between vascular endothelial cells, between vascular smooth muscle cells, and between endothelial and smooth muscle cells. Multiple genetic and epigenetic factors modulate connexin expression levels and/or channel function, including cell-type-independent and cell-type-specific transcription factors, posttranslational modifications, and localized membrane targeting. Additionally, differences in protein-protein interactions, including those between connexins, significantly contribute to both vascular homeostasis and disease progression. The biophysical properties of the connexin channels identified in the vasculature, those formed by Cx37, Cx40, Cx43 and/or Cx45 proteins, are discussed in this chapter in the physiological and pathophysiological context of vessel function.

Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland.

During the development of the mammary gland, duct‐lining epithelial cells progress through a program of expansive proliferation, followed by a terminal differentiation that allows for the biosynthesis and secretion of milk during lactation. The role of gap junction proteins, connexins, in the development and function of this secretory epithelium was investigated. Connexins, Cx26 and Cx32, were differentially expressed throughout pregnancy and lactation in alveolar cells. Cx26 poly‐(A)+ RNA and protein levels increased from early pregnancy, whereas Cx32 was detectable only during lactation. At this time, immunolocalization of connexins by confocal microscopy and immunogold labeling of high‐pressure frozen freeze‐substituted tissue showed that both connexins colocalized to the same junctional plaque. Analysis of gap junction hemichannels (connexons) isolated from lactating mammary gland plasma membranes by a rate‐density centrifugation procedure, followed by immunoprecipitation and by size‐exclusion chromatography, showed that Cx26 and Cx32 were organized as homomeric and heteromeric connexons. Structural diversity in the assembly of gap junction hemichannels demonstrated between pregnant and lactating mammary gland may account for differences in ionic and molecular signaling that may physiologically influence the onset and/or maintenance of the secretory phenotype of alveolar epithelial cells. J. Cell. Physiol. 183:228–237, 2000.

Post-translational modifications of connexin26 revealed by mass spectrometry

Gap junctions play important roles in auditory function and skin biology; mutations in the Cx26 (connexin26) gene are the predominant cause of inherited non-syndromic deafness and cause disfiguring skin disorders. Mass spectrometry (MS) was used to identify PTMs (post-translational modifications) of Cx26 and to determine whether they occur at sites of disease-causing mutations. Cx26 was isolated from transfected HeLa cells by sequential immunoaffinity and metal chelate chromatography using a tandem C-terminal haemagglutinin epitope and a (His-Asn)6 sequence. In-gel and in-solution enzymatic digestions were carried out in parallel with trypsin, chymotrypsin and endoproteinase GluC. Peptides were fractionated using a reversed-phase matrix by stepwise elution with increasing concentrations of organic solvent. To improve detection of low-abundance peptides and to maximize sequence coverage, MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; MS) and MALDI-TOF/TOF-MS/MS (matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry; MS/MS) spectra were acquired from each elution step using an Applied Biosystems 4800 tandem mass spectrometer. Acquisition, processing and interpretation parameters were optimized to improve ionization and fragmentation of hydrophobic peptides. MS and MS/MS coverage of Cx26 was significantly above that reported for other membrane proteins: 71.3% by MS, with 29.9% by MS/MS. MS coverage was 92.6% if peptides resulting from in-source collisions and/or partial enzymatic cleavages were considered. A variety of putative PTMs of Cx26 were identified, including acetylation, hydroxylation, gamma-carboxyglutamation, methylation and phosphorylation, some of which are at sites of deafness-causing mutations. Knowledge of the PTMs of Cx26 will be instrumental in understanding how alterations in the cellular mechanisms of Cx26 channel biogenesis and function lead to losses in auditory function and disfiguring skin disorders.

Isoelectric points and post-translational modifications of connexin26 and connexin32

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, γ‐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.—Locke, D., Koreen, I. V., Harris, A. L. Isoelectric points and post‐translational modifications of connexin26 and connexin32. FASEB J. 20, E426–E438 (2006)

Connexin channels and phospholipids: association and modulation

BackgroundFor membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood.ResultsEndogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred.ConclusionThis study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.

Nature of Cx30-containing channels in the adult mouse mammary gland

Oligonucleotide microarray analysis uniquely shows that several members of the connexin family of gap junction proteins are expressed by the epithelium during mouse mammary gland development. Connexin 26 (Cx26) is present throughout pregnancy and lactation, is then undetectable shortly after weaning, but reappears during involution. Additionally, Cx30 is abundant in late-pregnant and early lactating gland epithelium. From mid-pregnancy into early lactation, Cx26 and Cx30 co-localize in junctional plaques between epithelial cells, forming hemichannels of mixed connexin content. Microarray analysis also shows Cx32 is developmentally restricted to parturition, suggesting that specific modification of gap junction channel composition and/or intercellular communication pathways occurs at parturition. Specifically, heteromeric channels of all pairwise combinations are formed when these connexins are expressed within the same cells. Of these hemichannels, Cx26/Cx32 pores are increasingly sensitive to closure by taurine (an osmolyte implicated in milk protein synthesis) with increasing Cx26 content. In contrast, physiological taurine concentrations have no effect on Cx26/Cx30 and Cx30/Cx32 channel activity. Such changes in connexin expression and channel composition and their chemical modulation are discussed in relation to the various stages of mammary gland development in the adult mouse.

Mechanism for modulation of gating of connexin26-containing channels by taurine

The mechanisms of action of endogenous modulatory ligands of connexin channels are largely unknown. Previous work showed that protonated aminosulfonates (AS), notably taurine, directly and reversibly inhibit homomeric and heteromeric channels that contain Cx26, a widely distributed connexin, but not homomeric Cx32 channels. The present study investigated the molecular mechanisms of connexin channel modulation by taurine, using hemichannels and junctional channels composed of Cx26 (homomeric) and Cx26/Cx32 (heteromeric). The addition of a 28–amino acid “tag” to the carboxyl-terminal domain (CT) of Cx26 (Cx26T) eliminated taurine sensitivity of homomeric and heteromeric hemichannels in cells and liposomes. Cleavage of all but four residues of the tag (Cx26Tc) resulted in taurine-induced pore narrowing in homomeric hemichannels, and restored taurine inhibition of heteromeric hemichannels (Cx26Tc/Cx32). Taurine actions on junctional channels were fully consistent with those on hemichannels. Taurine-induced inhibition of Cx26/Cx32T and nontagged Cx26 junctional channels was blocked by extracellular HEPES, a blocker of the taurine transporter, confirming that the taurine-sensitive site of Cx26 is cytoplasmic. Nuclear magnetic resonance of peptides corresponding to Cx26 cytoplasmic domains showed that taurine binds to the cytoplasmic loop (CL) and not the CT, and that the CT and CL directly interact. ELISA showed that taurine disrupts a pH-dependent interaction between the CT and the CT-proximal half of the CL. These studies reveal that AS disrupt a pH-driven cytoplasmic interdomain interaction in Cx26-containing channels, causing closure, and that the Cx26CT has a modulatory role in Cx26 function.

Open pore block of connexin26 and connexin32 hemichannels by neutral, acidic and basic glycoconjugates.

The mechanisms of molecular discrimination by connexin channels are of acute biological and medical importance. The availability of affinity or open-pore blocking reagents for reliable and specific study of the connexin permeability pathway, would make possible the rigorous cellular and physiological studies required to inform, in molecular terms, the underlying role of intercellular communication pathways in development and disease. Previous work utilized a series of glucosaccharides labeled with an uncharged fluorescent aminopyridine (PA-) group to establish steric constraints to permeability through connexin hemichannels. In that work, the smallest probe permeable through homomeric Cx26 and heteromeric Cx26–Cx32 channels was the PA-disaccharide, and the smallest probe permeable through homomeric Cx32 channels was the PA-trisaccharide. The larger impermeable probes did not block permeation of the smaller probes. Building on this work, a new set of glucosaccharide probes was developed in which the label was one of a homologous series of novel anthranilic acid derivatives (ABG) that carry negative or positive formal charge or remain neutral at physiological pH. When the PA-label of the smallest impermeant PA-derivatized oligosaccharides was replaced by ABG label, the resulting probes acted as reversible, high-affinity inhibitors of large molecule permeation through connexin pores in a size and connexin-specific manner.