Abstract
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.
Highlights
Gap junction channels form by end-to-end interaction of hemichannels, each consisting of six connexin monomers
The essential findings of this study are as follows: (a) that connexin channels can discriminate among highly homologous inositol phosphates, most strikingly among inositol triphosphates, and (b) that only heteromeric channels were capable of this discrimination, with the corresponding homomeric channels being nonselective
Our findings suggest a unique functional role of heteromeric channels, with implications for connexin channel cell biology
Summary
Gap junction channels form by end-to-end interaction of hemichannels, each consisting of six connexin monomers. We found that in populations of native heteromeric channels, composed of channels with a range of isoform stoichiometries and arrangements, some of the channels were permeable to cAMP, and a much larger fraction of the channels was permeable to cGMP This selectivity between highly similar biological signaling molecules was unanticipated from dye permeability studies of these channels. The lifetime of inositol 1,4,5triphosphate ((1,4,5)-IP3) in cytoplasm ranges from 9 to 60 s, depending on cell type (26, 27) For this reason, we utilized a well characterized liposome-based technique for assessing permeability of functional connexin channels (20, 28 –34). We report here the surprising ability of heteromeric, but not homomeric, connexin channels to distinguish among biologically active IPs, including those with the same molecular
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