title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bihomotypic gap junctions, with scaffolding contributed by zonula occludens-1.
abstract: 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.
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Xinbo
authors.initials: X
authors.lastname: Li
authors.forename: Naomi
authors.initials: N
authors.lastname: Kamasawa
authors.forename: Cristina
authors.initials: C
authors.lastname: Ciolofan
authors.forename: Carl O
authors.initials: CO
authors.lastname: Olson
authors.forename: Shijun
authors.initials: S
authors.lastname: Lu
authors.forename: Kimberly G V
authors.initials: KG
authors.lastname: Davidson
authors.forename: Thomas
authors.initials: T
authors.lastname: Yasumura
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Shigemoto
authors.forename: John E
authors.initials: JE
authors.lastname: Rash
authors.forename: James I
authors.initials: JI
authors.lastname: Nagy
mesh_terms: D000818:Animals; D017630:Connexins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D054351:Electrical Synapses / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D005614:Freeze Fracturing / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008855:Microscopy, Electron, Scanning / Q000379:methods; D010750:Phosphoproteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000166:cytology*; D019108:Tight Junctions / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D062826:Zonula Occludens-1 Protein; D000097004:Gap Junction delta-2 Protein
chemical_list: D017630:Connexins; D049452:Green Fluorescent Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; D000097004:Gap Junction delta-2 Protein; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; C569044:Tjp1 protein, rat; C082920:connexin 45