Mammalian retinas contain abundant neuronal gap junctions, particularly in the inner plexiform layer (IPL), where the two principal neuronal connexin proteins are Cx36 and Cx45. Currently undetermined are coupling relationships between these connexins and whether both are expressed together or separately in a neuronal subtype-specific manner. Although Cx45-expressing neurons strongly couple with Cx36-expressing neurons, possibly via heterotypic gap junctions, Cx45 and Cx36 failed to form functional heterotypic channels in vitro. We now show that Cx36 and Cx45 coexpressed in HeLa cells were colocalized in immunofluorescent puncta between contacting cells, demonstrating targeting/scaffolding competence for both connexins in vitro. However, Cx36 and Cx45 expressed separately did not form immunofluorescent puncta containing both connexins, supporting lack of heterotypic coupling competence. In IPL, 87% of Cx45-immunofluorescent puncta were colocalized with Cx36, supporting either widespread heterotypic coupling or bihomotypic coupling. Ultrastructurally, Cx45 was detected in 9% of IPL gap junction hemiplaques, 90 -100% of which also contained Cx36, demonstrating connexin coexpression and cotargeting in virtually all IPL neurons that express Cx45. Moreover, double replicas revealed both connexins in separate domains mirrored on both sides of matched hemiplaques. With previous evidence that Cx36 interacts with PDZ1 domain of zonula occludens-1 (ZO-1), we show that Cx45 interacts with PDZ2 domain of ZO-1, and that Cx36, Cx45, and ZO-1 coimmunoprecipitate, suggesting that ZO-1 provides for coscaffolding of Cx45 with Cx36. These data document that in Cx45-expressing neurons of IPL, Cx45 is almost always accompanied by Cx36, forming "bihomotypic" gap junctions, with Cx45 structurally coupling to Cx45 and Cx36 coupling to Cx36.

Most gap junctions between neurons in mammalian retina contain abundant connexin36, often in association with the scaffolding protein zonula occludens-1. We now investigate co-association of connexin36, zonula occludens-1, zonula occludens-2 and Y-box transcription factor 3 (zonula occludens-1-associated nucleic acid-binding protein) in mouse and rat retina. By immunoblotting, zonula occludens-1-associated nucleic acid-binding protein and zonula occludens-2 were both detected in retina, and zonula occludens-2 in retina was found to co-immunoprecipitate with connexin36. By immunofluorescence, the four proteins appeared as puncta distributed in the plexiform layers. In the inner plexiform layer, most connexin36-puncta were co-localized with zonula occludens-1, and many were co-localized with zonula occludens-1-associated nucleic acid-binding protein. Moreover, zonula occludens-1-associated nucleic acid-binding protein was often co-localized with zonula occludens-1. Nearly all zonula occludens-2-puncta were positive for connexin36, zonula occludens-1 and zonula occludens-1-associated nucleic acid-binding protein. In the outer plexiform layer, connexin36 was also often co-localized with zonula occludens-1-associated nucleic acid-binding protein. In connexin36 knockout mice, labeling of zonula occludens-1 was slightly reduced in the inner plexiform layer, zonula occludens-1-associated nucleic acid-binding protein was decreased in the outer plexiform layer, and both zonula occludens-1-associated nucleic acid-binding protein and zonula occludens-2 were markedly decreased in the inner sublamina of the inner plexiform layer, whereas zonula occludens-1, zonula occludens-2 and zonula occludens-1-associated nucleic acid-binding protein puncta persisted and remained co-localized in the outer sublamina of the inner plexiform layer. By freeze-fracture replica immunogold labeling, connexin36 was found to be co-localized with zonula occludens-2 within individual neuronal gap junctions. In addition, zonula occludens-1-associated nucleic acid-binding protein was abundant in a portion of ultrastructurally-defined gap junctions throughout the inner plexiform layer, and some of these junctions contained both connexin36 and zonula occludens-1-associated nucleic acid-binding protein. These distinct patterns of connexin36 association with zonula occludens-1, zonula occludens-2 and zonula occludens-1-associated nucleic acid-binding protein in different sublaminae of retina, and differential responses of these proteins to connexin36 gene deletion suggest differential regulatory and scaffolding roles of these gap junction accessory proteins. Further, the persistence of a subpopulation of zonula occludens-1/zonula occludens-2/zonula occludens-1-associated nucleic acid-binding protein co-localized puncta in the outer part of the inner plexiform layer of connexin36 knockout mice suggests close association of these proteins with other structures in retina, possibly including gap junctions composed of an as-yet-unidentified connexin.

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