Functional plasticity of neuronal gap junctions involves the interaction of the neuronal connexin36 with calcium/calmodulin-dependent kinase II (CaMKII). The important relationship between Cx36 and CaMKII must also be considered in the context of another protein partner, Ca2+ loaded calmodulin, binding an overlapping site in the carboxy-terminus of Cx36. We demonstrate that CaM and CaMKII binding to Cx36 is calcium-dependent, with Cx36 able to engage with CaM outside of the gap junction plaque. Furthermore, Ca2+ loaded calmodulin activates Cx36 channels, which is different to other connexins. The NMR solution structure demonstrates that CaM binds Cx36 in its characteristic compact state with major hydrophobic contributions arising from W277 at anchor position 1 and V284 at position 8 of Cx36. Our results establish Cx36 as a hub binding Ca2+ loaded CaM and they identify this interaction as a critical step with implications for functions preceding the initiation of CaMKII mediated plasticity at electrical synapses.

BackgroundCASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions.ResultsWe demonstrate that the crystal structure of the Sterile Alpha Motif (SAM) domain tandem (SAM1-SAM2) oligomer from CASKIN2 is different than CASKIN1, with the minimal repeating unit being a dimer, rather than a monomer. Analytical ultracentrifugation sedimentation velocity methods revealed differences in monomer/dimer equilibria across a range of concentrations and ionic strengths for the wild type CASKIN2 SAM tandem and a structure-directed double mutant that could not oligomerize. Further distinguishing CASKIN2 from CASKIN1, EGFP-tagged SAM tandem proteins expressed in Neuro2a cells produced punctae that were distinct both in shape and size.ConclusionsThis study illustrates a new way in which neuronal SAM domains can assemble into large macromolecular assemblies that might concentrate and amplify synaptic responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0140-3) contains supplementary material, which is available to authorized users.

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Ca2+/calmodulin-dependent protein kinase II (CaMKII) binding and phosphorylation of mammalian connexin-36 (Cx36) potentiate electrical coupling. To explain the molecular mechanism of how Cx36 modifies plasticity at gap junctions, we investigated the roles of ionotropic N-methyl-D-aspartate receptors and pannexin1 (Panx1) channels in regulating Cx36 binding to CaMKII. Pharmacological interference and site-directed mutagenesis of protein interaction sites shows that NMDA receptor activation opens Cx36 channels, causing the Cx36- CaMKII binding complex to adopt a compact conformation. Ectopic Panx1 expression in a Panx1 knock-down cell line is required to restore CaMKII mediated opening of Cx36. Furthermore, blocking of Src-family kinase activation of Panx1 is sufficient to prevent the opening of Cx36 channels. Our research demonstrates that the efficacy of Cx36 channels requires convergent calcium-dependent signaling processes in which activation of ionotropic N-methyl-D-aspartate receptor, Src-family kinase, and Pannexin1 open Cx36. Our results add to the best of our knowledge a new twist to mounting evidence for molecular communication between these core components of electrical and chemical synapses.

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