Cisplatin and oxaliplatin inhibit gap junctional communication by direct action and by reduction of connexin expression, thereby counteracting cytotoxic efficacy.

Cisplatin [cis-diamminedichloroplatinum(II)]/oxaliplatin [1,2-diamminocyclohexane(trans-1)oxolatoplatinum(II)] toxicity is enhanced by functional gap junctions between treated cells, implying that inhibition of gap junctions may decrease cytotoxic activity of these platinum-based agents. This study investigates the effect of gap junction modulation by cisplatin/oxaliplatin on cytotoxicity in a transformed cell line. The effects were explored using junctional channels expressed in transfected HeLa cells and purified hemichannels. Junctional channels showed a rapid, dose-dependent decrease in dye coupling with exposure to cisplatin/oxaliplatin. With longer exposure, both compounds also decreased connexin expression. Both compounds inhibit the activity of purified connexin hemichannels, over the same concentration range that they inhibit junctional dye permeability, demonstrating that inhibition occurs by direct interaction of the drugs with connexin protein. Cisplatin/oxaliplatin reduced the clonogenic survival of HeLa cells at low density and high density in a dose-dependent manner, but to a greater degree at high density, consistent with a positive effect of gap junctional intercellular communication (GJIC) on cytotoxicity. Reduction of GJIC by genetic or pharmacological means decreased cisplatin/oxaliplatin toxicity. At low cisplatin/oxaliplatin concentrations, where effects on connexin channels are minimal, the toxicity increased with increased cell density. However, higher concentrations strongly inhibited GJIC, and this counteracted the enhancing effect of greater cell density on toxicity. The present results indicate that inhibition of GJIC by cisplatin/oxaliplatin decreases their cytotoxicity. Direct inhibition of GJIC and reduction of connexin expression by cisplatin/oxaliplatin may thereby compromise the effectiveness of these compounds and be a factor in the development of resistance to this class of chemotherapeutic agents.

Connexin channels and phospholipids: association and modulation.

BACKGROUND: For 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. RESULTS: Endogenous 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. CONCLUSION: This 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.

2-aminoethoxydiphenyl borate directly inhibits channels composed of connexin26 and/or connexin32.

2-aminoethoxydiphenyl borate (2-APB), a commonly used blocker of IP3-induced calcium ion release and of store-operated channels, inhibits gap junction conductance when applied to cultured cells. The character and pharmacology of this inhibition was explored using 1) hemichannels composed of connexin32 (Cx32) and/or connexin26 (Cx26) purified from native sources and from transfected HeLa cells in which the connexin had a cleavable C-terminal epitope tag and 2) the corresponding junctional channels. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay (TSF), 2-APB reversibly inhibited homomeric Cx32 and heteromeric Cx26/Cx32 channels from native tissue and their tagged forms from HeLa cells. The IC50-TSF value of the inhibition was approximately 47 microM at pH 6.5. 2-APB did not inhibit tagged homomeric Cx26 channels even after tag cleavage (leaving several amino acids at the carboxyl terminus). Protonated 2-APB is the inhibitory agent, but channel sensitivity to 2-APB also increases as pH is lowered. To help define the chemical requirements for inhibition, the effects of four structural analogs of 2-APB were determined. The inhibitory action of 2-APB was shown to be distinct from that of aminosulfonates. 2-APB and its analogs, except phenytoin, inhibited dye-coupling through junctional channels formed by all the tagged channel forms except Cx26, consistent with the TSF studies. However 2-APB significantly inhibited dye coupling between cells expressing untagged Cx26, suggesting that an unmodified C terminus is required for action on Cx26 channels. These results show that protonated 2-APB directly and reversibly inhibits connexin channels composed of Cx26 and/or Cx32 and suggest involvement of the carboxyl-terminal domain.

Heteromeric, but not homomeric, connexin channels are selectively permeable to inositol phosphates.

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.

Biochemical requirements for inhibition of Connexin26-containing channels by natural and synthetic taurine analogs.

Previous work has shown that protonated taurine and aminosulfonate pH buffers, including HEPES, can directly and reversibly inhibit connexin channels that contain connexin26 (Cx26) (Bevans, C. G., and Harris, A. L. (1999) J. Biol. Chem. 274, 3711-3719). The structural requirements for this inhibition were explored by studies of the effects of structural analogs of taurine on the activity of Cx26-containing reconstituted hemichannels from native tissue. Several analogs inhibited the channels, with a range of relative affinities and efficacies. Each active compound contains a protonated amine separated from an ionized sulfonate or sulfinate moiety by several methylene groups. The inhibition is eliminated if the sulfonate/sulfinate moiety or the amine is not present. Compounds that contain a protonated amine but lack a sulfonate/sulfinate moiety do not inhibit but do competitively block the effect of the active compounds. Compounds that lack the protonated amine do not significantly inhibit or antagonize inhibition. The results suggest involvement of the protonated amine in binding and of the ionized sulfur-containing moiety in effecting the inhibition. The maximal effect of the inhibitory compounds is enhanced when a carboxyl group is linked to the alpha-carbon. Inhibition but not binding is stereospecific, with l-isomers being inhibitory and the corresponding d-isomers being inactive but able to antagonize inhibition by the l-isomers. Whereas not all connexins are sensitive to aminosulfonates, the well defined structural requirements described here argue strongly for a highly specific regulatory interaction with some connexins. The finding that cytoplasmic aminosulfonates inhibit connexin channels whereas other cytoplasmic compounds antagonize the inhibition suggests that gap junction channels are regulated by a complex interplay of cytoplasmic ligands.

Reversible pore block of connexin channels by cyclodextrins.

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.