Abstract
The gap junctional protein connexin 36 (Cx36) has been co-purified with the lipid raft protein caveolin-1 (Cav-1). The relevance of an interaction between the two proteins is unknown. In this study, we explored the significance of Cav-1 interaction in the context of intracellular and membrane transport of Cx36. Coimmunoprecipitation assays and Förster resonance energy transfer analysis (FRET) were used to confirm the interaction between the two proteins in the Neuro 2a cell line. We found that the Cx36 and Cav-1 interaction was dependent on the intracellular calcium levels. By employing different microscopy techniques, we demonstrated that Cav-1 enhances the vesicular transport of Cx36. Pharmacological interventions coupled with cell surface biotinylation assays and FRET analysis revealed that Cav-1 regulates membrane localization of Cx36. Our data indicate that the interaction between Cx36 and Cav-1 plays a role in the internalization of Cx36 by a caveolin-dependent pathway.
Highlights
The intracellular transport of connexins, their assembly and channel formation, and removal are governed by complex interactions with regulatory, transport, and structural proteins [1,2]
Förster Resonance Energy Transfer Analysis (FRET) efficiency above the threshold of 1.7% signified that proximity between two proteins is less than 10 nm, meaning that they are close enough to interact with each other (Figure 1C)
Because connexin 36 (Cx36) monomers oligomerize into hexamers, Cx36-Cx36 pairs served as a positive control with a FRET efficiency value of 10.27 ± 0.60 (n = 40)
Summary
The intracellular transport of connexins, their assembly and channel formation, and removal are governed by complex interactions with regulatory, transport, and structural proteins [1,2]. The turnover of connexins from the cell membrane is, in particular, challenging for connexin 36 (Cx36), the major component of electrical synapses. In these gap junctions of the brain, Cx36 has been found in axo-axonal, axo-dendritic, and dendro-dendritic contact sites [3,4,5]. The deletion of CaMKII binding and phosphorylation regions in Cx36 led to the loss of this “run-up” property, signifying that this interaction is essential for the functional plasticity of electrical synapses formed by Cx36. Calmodulin (CaM), another multifunctional calcium signaling protein, has been shown to bind Cx36 [8] Both CaMKII and CaM share a binding motif and interact with Cx36 competitively. A recent study demonstrated that Cx36 interaction with tubulin potentiates the synaptic strength of Cx36 by tubulin-mediated delivery of channels to the gap junction plaques [14]
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