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.

ZO-1 and the Spatial Organization of Gap Junctions and Glutamate Receptors in the Outer Plexiform Layer of the Mammalian Retina

“…In addition, Li et al (2008) presented data from electron microscopic (FRIL) experiments, indicating a coscaffolding of Cx45 and Cx36 by ZO-1 at bihomotypic gap junctions in the IPL. In their model of gap junctional architecture, ZO-1 intracellularly covers the whole area of the gap junction.…”

Gap Junction-Mediated Death of Retinal Neurons Is Connexin and Insult Specific: A Potential Target for Neuroprotection

“…Our results using the nonspecific GJ blockers MFA and 18␤-GA did not address this issue. To test this idea, we therefore examined the extent of excitotoxic and ischemic cell death in mice in which Cx36 and/or Cx45, the two most highly expressed subtypes in the IPL (Li et al, 2008;Bloomfield and Völgyi, 2009;Pan et al, 2010), were genetically deleted.…”

Neuronal connexin-36 can functionally replace connexin-45 in mouse retina but not in the developing heart

“…Whether Cx45 and Cx36 form heterotypic (Maxeiner et al, 2005;Dedek et al, 2006) or bi-homotypic (Li et al, 2008) gap junction channels between mouse AII amacrine and ON cone bipolar cells is still controversial. Although Cx45 is expressed in distinct types of amacrine cells, it could not be detected in AII amacrine cells (Maxeiner et al, 2005;Dedek et al, 2006).…”

“…Cx45 has been suggested to participate in this heterotypic synapse (Maxeiner et al, 2005;Dedek et al, 2006) and data derived from single-cell RT-PCR experiments point to a complex and cellsubtype-specific expression of Cx36 and Cx45 in ON cone bipolar cells (Lin et al, 2005;Han and Massey, 2005). But colocalization of Cx36 and Cx45 could not be detected in vitro in cell culture , and findings from double-replica analysis of gap junctions containing Cx36 and Cx45 in the inner plexiform layer (IPL) of the rat retina revealed the presence of both Cx36 and Cx45 in apposed hemiplaques (Li et al, 2008). Hence, Li and colleagues (Li et al, 2008) proposed that electrical synapses between AII amacrine and ON cone bipolar cells are formed by bi-homotypic gap junctions.…”

“…But colocalization of Cx36 and Cx45 could not be detected in vitro in cell culture , and findings from double-replica analysis of gap junctions containing Cx36 and Cx45 in the inner plexiform layer (IPL) of the rat retina revealed the presence of both Cx36 and Cx45 in apposed hemiplaques (Li et al, 2008). Hence, Li and colleagues (Li et al, 2008) proposed that electrical synapses between AII amacrine and ON cone bipolar cells are formed by bi-homotypic gap junctions. Currently, the composition of these gap junctions between AII amacrine and ON cone bipolar cells is not clear.…”