Calmodulin dependent protein kinase increases conductance at gap junctions formed by the neuronal gap junction protein connexin36

Abstract

The major neuronal gap junction protein Connexin 36 (Cx36) exhibits the remarkable property of “run-up”, in which junctional conductance typically increases by ten-fold or more within 5–10 min following cell break-in with patch pipettes. Such conductance “run-up” is a unique property of Cx36, as it has not been seen in cell pairs expressing other connexins. Because of the recent observation describing CaMKII binding and phosphorylation sites in Cx36 and evidence that calmodulin dependent protein kinase II (CaMKII) may potentiate electrical coupling in neurons of teleosts, we have explored whether CaMKII activates mammalian Cx36. Consistent with this hypothesis, certain Cx36 mutants lacking the CaMKII binding and phosphorylation sites or wild type Cx36 treated with certain cognate peptides corresponding to binding or phosphorylation sites blocked or strongly attenuated run-up of junctional conductance. Likewise, KN-93, an inhibitor of CaMKII, blocked run-up, as did a membrane permeable peptide corresponding to the CaMKII autoinhibitory domain. Furthermore, run-up was blocked by phosphatase delivered within the pipette and not affected by treatment with the phosphatase inhibitor okadaic acid. These results imply that phosphorylation by CaMKII strengthens junctional currents of Cx36 channels, thereby conferring functional plasticity on electrical synapses formed of this protein.

NMDA Receptor Activation Strengthens Weak Electrical Coupling in Mammalian Brain

“…Our findings were consistent with previous reports demonstrating the interaction of CaMKII with Cx36 in the central synapse of the teleost VIIIth nerve (Pereda et al, 1998), rabbit retina (Kothmann et al, 2012) and in a mouse neuroblastoma expression system (Del Corsso et al, 2012). Interestingly, CaMKII stimulation enables not only its enzymatic kinase activity, but also its direct binding to the NMDAR subunit GluN2B and to the GJ protein Cx36 (Alev et al, 2008; Coultrap and Bayer, 2012).…”

Electrical synapses and their functional interactions with chemical synapses

“…4b). CaMKII activation was shown to lead to an increase in gap junction conductance not only at goldfish Club endings, but also in Cx36-containing gap junctions and cell expression systems 125 . Consistent with these findings, CaMKII was found to phosphorylate Cx36 at retinal electrical synapses at sites that enhance coupling between these cells 126 .…”

Two-color fluorescent analysis of connexin 36 turnover: relationship to functional plasticity

“…We used membrane-permeant fluorescent ligands for this study. The C-terminus of Cx36 is the site of a number of known protein-protein interactions and regulatory phosphorylation events (Li et al, 2004;Ouyang et al, 2005;Kothmann et al, 2007;Alev et al, 2008;del Corsso et al, 2012;Li et al, 2012). We chose to insert the HaloTag open reading frame between the two regulatory phosphorylation sites (S293 and S315) (Fig.…”