Connexin30.2: In Vitro Interaction with Connexin36 in HeLa Cells and Expression in AII Amacrine Cells and Intrinsically Photosensitive Ganglion Cells in the Mouse Retina

Abstract

Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36—both expressed in AII amacrine cells—are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners.

Electrotonic signal processing in AII amacrine cells: compartmental models and passive membrane properties for a gap junction-coupled retinal neuron

“…First, it was essentially impossible to obtain a good fit of the current responses of the models to the physiological recordings obtained before and during onset of the action of MFA, suggesting that the physiologically recorded responses are markedly influenced by the electrical coupling. Second, whereas an alternative strategy could be to record from AII amacrine cells in the retina of genetically modified mice that lack Cx36 (the connexin involved in gap junction coupling of both AII amacrines and ON-cone bipolar cells), there is evidence that tracer coupling may not be completely abolished in Cx36 knockout mice (Deans et al 2002), potentially consistent with the suggestion that coupling between AII amacrines could involve additional connexins (Meyer et al 2016), or that compensatory mechanisms in knock-out animals could be triggered and influence expression of other connexins.…”

Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14–3–3 proteins in vitro

“…This confirmed that binding of PDZ 10 of MUPP1 to mmCx36 is mediated by the PDZ binding domain at the very C-terminal end of the protein. Interestingly, while several studies 19 , 25 earlier reported that the truncated protein (mmCx36 S318 ter) fails to form gap junctions in transfected HeLa cells, the same construct assembled into gap junctions in HEK293 cells, suggesting that the assembly machinery differs between cell lines.…”

“…Therefore, we tested in cultured cells whether the phosphorylation of S315 has an influence on the size of gap junctions. As we have successfully used mmCx36 in a mammalian expression system to measure gap junction volume 25 and sequences for mouse and perch connexins are conserved with respect to the phosphorylated serine and PDZ binding domain (Fig. 1 b), we cloned an mmCx36 variant, in which S315 was mutated to aspartate (S315D) to mimic the phosphorylation of the serine residue.…”

“…HEK293 cells were transfected with either wild-type mmCx36 or the phosphomimetic construct and gap junctions were 3D reconstructed and measured in volume (Fig. 3 ) as described 25 . The volume of gap junctions between adjacent cells was similar between wild-type mmCx36 and the S315D mutant (Fig.…”

“…After several washes, adsorbed proteins were eluted with pre-heated (95 °C) elution buffer, containing 50 mM Tris HCl (pH 6.8), 50 mM dithiothreitol, 1% SDS, 1 mM EDTA, 0.005% bromophenol blue, 10% glycerol. SDS-PAGE (10% gels) and western blot analysis were performed as previously described 25 .…”