Abstract
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.
Highlights
Ca2+/calmodulin-dependent protein kinase II (CaMKII) binding and phosphorylation of mammalian connexin-36 (Cx36) potentiate electrical coupling
Results emphasize that the ionotropic NMDA receptormediated rise in [Ca2+]I was an essential signal requirement to increase a compact conformation of the Cx36–CaMKII complex and to open Cx36 channels
Mutations in protein domains targeting the interaction between Cx36 and either CaM or CaMKII, and pharmacological interventions disrupting the NMDA receptor or CaMKII activation, abolished [Ca2+]I signaling the opening of Cx36 channels located in gap junction plaque (GJP)
Summary
Ca2+/calmodulin-dependent protein kinase II (CaMKII) binding and phosphorylation of mammalian connexin-36 (Cx36) potentiate electrical coupling. 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 Pannexin[1] open Cx36. Cx36 GJCs show functional plasticity similar to chemical synapses[4,5], in which the interaction of Cx36 with Ca2+-activated calmodulin (CaM) and calmodulin protein kinase II (CaMKII) is considered analogous to the interaction of ionotropic N-methyl-D-aspartate (NMDA) receptors with CaM/CaMKII6. Similar to the NR2B subunit of the NMDA receptor, both Cx36 binding sites exhibit phosphorylation-dependent interaction and autonomous activation of CaMKII, suggesting that the functional efficacy of both modes of interneuronal communication share common molecular features. Panx[1], together with metabotropic and ionotropic NMDA receptors and Src family kinases (SFKs) have been implicated in forming signaling complexes[11,12,13]
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