Structural and Functional Consequences of Connexin 36 (Cx36) Interaction with Calmodulin

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.


INTRODUCTION
Electrical and chemical synapses can form mixed synapses conferring synchronous activity among coupled neurons in the vertebrate nervous system (Hamzei-Sichani et al., 2012;Nagy et al., 2013;Rubio and Nagy, 2015). This enables synchronous oscillations that temporally correlate firing patterns in otherwise heterogeneous networks (Galarreta and Hestrin, 1999;Deans et al., 2001;Bennett and Zukin, 2004;Vervaeke et al., 2010;Haas et al., 2011;Postma et al., 2011). In many regions of the nervous system, synchronous rhythmic oscillations serve as a mechanism to entrain the activity of distributed neuronal networks, and promote processes related to memory formation and retrieval, sensory perception, motor control, attention and plasticity (Deans et al., 2001;Hormuzdi et al., 2004;Van Der Giessen et al., 2008;Pereda, 2014).
Neurons express connexin36 (Cx36) and form gap junctions, which are the molecular substrate of electrical synapses. Functional plasticity of neuronal gap junctions in goldfish Mauthner cells (Pereda et al., 1998(Pereda et al., , 2003(Pereda et al., , 2004Flores et al., 2010), and in both teleost and rodent retina (Li et al., 2009;Cachope and Pereda, 2012;Kothmann et al., 2012) involves interaction of Cx36 with the calcium/calmodulin-dependent kinase II (CaMKII). In this process, CaMKII binds Cx36 in a manner analogous to binding of this kinase to the NR2B subunit of the NMDA receptor and phosphorylates the connexin protein both in situ and in vitro (Alev et al., 2008). Blocking of Cx36-CaMKII binding and phosphorylation by decoy inhibition, and deletion of CaMKII binding sites, as well as inhibition of phosphorylation sites through site-directed mutagenesis and peptide inhibition, leads to a significant loss of the "run up" activity, the term coined by us to describe the plasticity of the process (Zoidl et al., 2002;Del Corsso et al., 2012).
Interaction of Cx36 with CaMKII endows electrical synapses with functional plasticity but is in competition with binding of calmodulin (CaM), another multifunctional Ca 2+ signaling protein (Burgoyne, 2007), to a region overlapping with the CaMKII binding motif (Burr et al., 2005). At chemical synapses both CaM and the NMDA receptor subunit NR2B synergize to lock CaMKII in an active conformation conferring calciumdependent plasticity (Bayer et al., 2001;Lisman et al., 2002;Bhattacharyya et al., 2016). A similar mechanism involving Cx36, CaM and CaMKII appears unlikely due to the overlapping binding site. Addressing this conflict, we have proposed a model predicting sequential, competitive processing of interaction between CaM, CaMKII, and Cx36 (Alev et al., 2008). To resolve this conflict, the neuroblastoma cell line Neuro2a was used to interrogate how calcium signaling converges on Cx36 through CaM binding. Using Förster resonance energy transfer analysis (FRET), we demonstrate that formation of the Cx36-CaM binding complex is dependent on the capacity of CaM to bind calcium and strengthened when intracellular calcium concentrations rise. The formation of the Cx36-CaM complex precedes arrival of the Cx36 protein at the gap junction plaque (GJP) or is established after removal of Cx36 from the GJP. Functional dye transfer/dye recovery assays in cell pairs under physiological conditions showed that Cx36-CaM binding endows GJPs with the capacity to exchange a fluorescent dye between adjacent cells. Pharmacological blocking of CaM or mutation of the critical amino acid W277 within the core CaM binding motif of Cx36 significantly diminished this property. This observation led us to propose a revised model of calcium signaling where the localization of Cx36 in the cell affects the participants (CaM and/or CaMKII) and consequently, the outcomes arising from these interactions. This model is additionally supported by detailed structural and kinetic view of the Cx36-CaM interaction using a combination of the NMR and calorimetric approaches. In summary, this study demonstrates that priming of Cx36 by CaM represents a critical stage in the creation of plasticity at electrical synapses.

Confocal Microscopy
Transfected cells were fixed with 4% paraformaldehyde for 20 min at RT, washed with PBS, and mounted with ProLong R Antifade Mountant (Thermo Fisher Inc., Mississauga, ON, Canada) for imaging. Samples were visualized using a Zeiss LSM 700 confocal microscope with a Plan-Apochromat 63x/1.4 Oil DIC M27 objective and the ZEN 2010 program to control all hardware parameters. Images were collected by line averaging (4×) at high resolution (2048 × 2048 pixels) using single planes or z-stacks. The GJ frequency was quantified as described previously (Qu et al., 2009), by counting the number of EGFP-expressing cell pairs against the number of cell pairs having GJPs in images from randomly selected, non-overlapping visual fields collected from experimental replicates (n > 3). The GJ area was quantified using ImageJ. Here, the ImageJ free hand tool was used to draw the contours of GJs between cell pairs, followed by quantification of the GJ area using the measure tool. Mander's overlap coefficients were calculated using the co-localization analysis if the ZEN 2010 program (Manders et al., 1993). Images were exported and further processed using ImageJ or the Imaris Scientific 3D/4D Image Processing & Analysis Software (Bitplane USA, Concord, MA, USA). Images were combined using Adobe Photoshop CS6 for presentation.

FRET Analysis
The acceptor bleach protocol was established by testing different combinations of fluorescently tagged Cx36 and CaM proteins using a Zeiss LSM 700 confocal microscope (data not shown).
In the experiments shown, Neuro2a cells were transiently transfected with DsRed-monomer and CFP tagged expression vectors and processed as described above. Baseline readings were measured prior to the acceptor bleach protocol. Here, DsRed-tagged proteins were bleached using the 555 nm laser line (set to 100% intensity) and the intensity change of CFP tagged proteins was recorded using the 405 nm laser line. The experiment was completed when the bleaching of the acceptor channel reached 10% of the initial intensity. FRET efficiency was then calculated by using the FRET efficiency formula: FRET eff = (D post − D pre )/D post , where D post is the average intensity after the bleach, and D pre is the average intensity before the bleach (Okamoto et al., 2004) after subtracting the values of the background (noise). The FRET distance (FRET d ) between the interacting proteins was calculated using the formula: R = R o ((1/E) − 1) 1/6 , where R o is the reference distance between the two fluorescent tags (DsRed and ECFP: 5.09) (Erickson et al., 2003), and E is the FRET efficiency. The mean values of the intensity change and distance were calculated and formulated into graphical representations. Error bars denote the standard error of the mean (SEM) of each protein-protein interaction.

Ethidium Bromide Uptake and Recovery after Photobleaching Assay
This assay is a variation of the ethidium bromide uptake assay previously reported (Prochnow et al., 2009;Kurtenbach et al., 2013). Transfected Neuro2a cells grown for 48 h in 3.5 cm MatTek cell culture dishes were incubated with 10 µM of ethidium bromide in complete growth medium for 10 min at 37 • C and 5% CO 2. MatTek chambers were placed in a livecell imaging chamber and imaged at 37 • C using a Zeiss 700 confocal microscope. Cell pairs expressing Cx36-EGFP were selected and a time-lapse baseline image was recorded (defined as 100%). Next, one cell was bleached using the 555 nm laser line with the intensity set to 100% laser power. The number of iterations in the bleaching protocol was terminated when 40-50% reduction in fluorescence was achieved. In this process, a volume of 36.2 µm × 36.2 µm × 1.6 µm was bleached using a non-confocal imaging mode. The recovery of ethidium bromide fluorescence in the center plane of this volume after bleaching was measured over time in confocal imaging mode.
In all experiments, three regions of interest (ROI: R0, R1, R2) were selected for analysis. R0 was placed outside the cell pair to determine background the fluorescence. ROIs were placed inside the bleached cell close (R1) and distant (R2) to the GJP. The fluorescence recovery in percent was calculated using the formula:

NMR Spectroscopy
Spectra were acquired at 298 K with a 700 MHz Bruker Avance spectrometer equipped with a TXI cold probe. Backbone and side chain chemical shift assignments were obtained using a suite of standard heteronuclear, triple resonance experiments (HNCACB, CBCACONH, HNCO, HNCACO, HNCA, CCH-TOCSY, HCCH-TOCSY) with non-uniform Poisson gap sparse sampling at 10-15%. Distance restraint data were obtained from 15 N-edited and 13 C-edited 3D-NOESY spectra (120 ms mixing time). All datasets were processed with NMRPipe (Delaglio et al., 1995) and analyzed with CcpNmr Analysis (Skinner et al., 2015).

Structure Determination
CcpNmr Analysis was used to integrate NOEs and calculate distance restraints based on an r −6 function spanning 1.8-5.5 Å with a reference distance set at 3.2 Å. These restrains were used as input to CYANA (Buchner and Güntert, 2015) along with Ca 2+ coordination distance restraints, hydrogen bonding distance restraints and torsion angle restraints predicted by the DANGLE method embedded in CCPN Analysis. Allowed backbone torsion angles and side chain rotamers were enforced by the ramaaco and rotameraco methods embedded in the CYANA suite. The final representative ensemble of 20 structures based on lowest CYANA target function was selected from an initial calculation of 400 structures. As this ensemble demonstrated good geometry and bonding, no additional refinement was pursued.

Databases
The structural ensemble was deposited in the Protein Data Bank under the accession number 2N6A.

Isothermal Titration Calorimetry (ITC)
Isothermal titration calorimetry (ITC) measurements were performed on MicroCal VP-ITC calorimeter (MicroCal Inc., Northampton, MA, USA). The recombinant H 6 -CaM (affinity purified as described above) was titrated with Cx36 derived peptides reflecting the wild type (GSGWRKIK LAVRGAQAKRKSVYEIR; CanPeptide Inc., Montréal, QC, Canada) or the W227A mutant (GSGARKIKLAVRGAQ AKRKSVY; BioBasic, Markham, ON, Canada). An optimal titration was achieved with 25 µM CaM in the reaction cell and 250 µM Cx36 peptide in the syringe, each in a buffer containing 50 mM NaCl, 5 mM BisTris pH 7.0, 5 mM CaCl 2 . After an initial injection of 2 µL, the bulk of the titration consisted of 34 successive 8 µL injections with an equilibration delay of 300 s. Heats of dilution were determined by titrating the same peptide solutions into buffer alone. Titration profiles corrected for the heat of dilution were fitted into one-binding-site model using MicroCal Origin v5.0. All values reported were calculated based on three individual experiments.

Statistics
Statistical analysis and data presentation was performed using the IBM-SPSS and R-software packages. Results shown derive from experimental replicates with n ≥ 3. All data were analyzed for data distribution and subjected to Mann-Whitney U tests for independent samples or a paired t-test, when appropriate.

Cx36-CaM at Gap Junctions in Neuro2A Cells
Previous work (Burr et al., 2005) and the sequence alignment of Cx36 orthologs representing major vertebrate phyla ranging from primitive fish to humans suggested the existence of a highly conserved potential CaM binding motif in the carboxy-terminus of Cx36 resembling a variation of the 1-8-14 subclass. This motif overlapped with a binding site for the calcium-dependent calmodulin kinase II ( Figure 1A) (Alev et al., 2008).
The role of W277 in position 1 of the putative CaMbinding site was investigated after sequential alanine-scanning mutagenesis of positions 276 to 278 of the rat Cx36 protein.

Mutation W277A Alters Subcellular Distribution and Exchange between Cell Pairs
Two types of experiments were performed to clarify the distribution of Cx36 and W277A in Neuro2A cells. When single transfected Neuro2A cells expressing Cx36-EGFP or Cx36-DsRed monomer were paired and analyzed 48 h after transfection, 3D imaging of cell pairs showed mixed GJPs (see inset in Figure 2A; Supplementary Figure S1), and limited presentation of Cx36 in mixed vesicles. Cell pairs expressing the W277A mutation showed mixed GJPs (see Supplementary Figure S2), and an extensive display of vesicular structures exchanged between cell pairs, suggesting that the mutation altered GJP stability (Figure 2A).
Next, the subcellular localization of Cx36 was visualized using Neuro2a cells double transfected with EGFP tagged Cx36 and genetically encoded cell organelle markers for Frontiers in Molecular Neuroscience | www.frontiersin.org Quantification of gap junction size and frequency demonstrating that gap junction size is not affected by mutations of the core CaM binding region, but mutation W277A affects GJP frequency. Error bars box plot: maximum and minimum range with median; Mann-Whitney U test, * * * p < 0.001. endoplasmic reticulum (ER), Golgi, and mitochondria ( Figure 2B, Supplementary Figure S3). Wild-type Cx36 was predominantly localized in vesicles and at GJPs. Outside of GJPs, both 2D images (data not shown) and 3D reconstructions of images from double transfected cells showed Cx36 in vesicles of variable size embedded in, or attached to ER and Golgi (Supplementary Figure S4). Co-localization analysis revealed a significant overlap of Cx36 with ER (0.695 ± 0.0413, n = 30, p = 9.837 × 10 −6 ) and Golgi organelles (0.651 ± 0.0355, n = 30, p = 4.209 × 10 −5 ). The Mander's overlap coefficient for Cx36 and mitochondria was significantly lower (0.356 ± 0.0413, n = 30) demonstrating that exogenously expressed, tagged wild-type Cx36 is not redistributed into this organelle type ( Figure 2C; Supplementary Figure S5). The distribution of Cx36 in cell pairs, together with the localization of tagged Cx36 in ER, Golgi and GJPs is consistent with the subcellular distribution of other connexin proteins. When Cx36-W277A was expressed, the Mander's overlap coefficient was significantly higher than wild-type Cx36 in all organelles (ER: 0.814 ± 0.012, p = 0.009; Golgi: 0.811 ± 0.017, p = 1.6 × 10 −5 ; mitochondria: 0.812 ± 0.010, p = 2.22 × 10 −9 ). The distinct subcellular distribution of the W277A mutant suggests a mechanism that interferes with the transport, formation and/or stabilization of GJPs in transfected Neuro2a cells.
Next, FRET-Distance (FRET dis ) for Cx36-CFP and CaM-DsRed was compared at GJPs and the vesicles. Typically, if two interacting proteins are within 10 nm distance, FRET can be achieved (threshold indicated by the dashed line). No significant difference at the GJPs (non-stimulated: 9.11 ± 0.24, stimulated: 9.61 ± 0.33, p = 0.33), but a significant reduction in FRET dis was observed at cytoplasmic vesicles when [Ca 2+ ] I was increased, (non-stimulated: 9.27 ± 0.22, stimulated: 8.09 ± 0.19, p = 0.002). When CaM E1234Q was used to test the interaction with Cx36 WT-CFP, neither at the GJP (non-stimulated: 10.93 ± 0.63, stimulated: 10.13 ± 0.53, p = 0.21) nor the vesicles (nonstimulated: 9.96 ± 0.58, stimulated: 10.81 ± 0.46, p = 0.23) was significantly different ( Figure 3E). All values were below 10 nm except for CaM E1234Q , which indicated FRET was not achieved between wild-type Cx36 and CaM E1234A . We concluded that changes in [Ca 2+ ] I are relayed to Cx36 before reaching GJPs, and that the interaction between Cx36 and CaM is critically dependent on the capacity of CaM to bind calcium.

Reduced Dye-Redistribution in Neuro2a Cells Expressing W277A
The W277A mutation decreased the GJP frequency, but not the GJP size (Figures 1D,E), and caused a reduced response to [Ca 2+ ] I elevation (Figures 4A,B). To investigate impaired gap junction communication prompted by the W277A mutation, the non-invasive uptake and redistribution of ethidium bromide across GJPs under normal cell growth conditions was quantified using a method based on gap-FRAP (Abbaci et al., 2007). Pairs of Neuro2a cells expressing Cx36-ECFP or W277A-ECFP, and forming GJPs were selected for imaging (Figures 5A-D, left  column). Sample traces for each experimental condition show the fluorescence in regions of interest (R1, red trace; R2, black trace) over the entire time course (Figures 5A-D, right column). Regions of interest were placed close to the GJP (R1) and at a more distant location (R2) in the same cell to capture recovery of ethidium bromide after photobleaching. Quantifications shown below ( Figure 5E) demonstrated that treatment with 2 mM [Ca 2+ ] E and 2 µM ionomycin caused a significant ethidium bromide fluorescence recovery in Neuro2a cells expressing the Cx36-ECFP protein (values in %: non-stimulated: 5.82 ± 4.22, stimulated: 47.54 ± 13.56, p = 0.0045). Blocking of CaM with W-7 led to a significant reduction of fluorescence recovery when intracellular calcium levels were increased suggesting that CaM binding is critical for this response (non-stimulated: 16.60 ± 8.01, stimulated: 3.77 ± 3.41, p = 0.1427). Treatment with the gap junction blocker carbenoxolone (CBX) modulated fluorescence recovery (non-stimulated: −8.66 ± 4.65, stimulated: 8.91 ± 1.89, p = 0.0032). When Neuro2a cells expressed Cx36-W277A, no significant recovery was seen after the same treatment (nonstimulated: 8.12 ± 4.21, stimulated: −0.55 ± 8.35, p = 0.425). It was concluded that W277A expression impairs dye redistribution between cell pairs in a way similar to a gap junction blocker. Control experiments using non-transfected Neuro2a cells with and without ionomycin treatment showed limited recovery (Neuro2a without ionomycin treatment: −15.72 ± 17.52, Neuro2a with ionomycin treatment: 3.40 ± 1.23, p = 0.001). The recovery observed after CBX or ionomycin treatment likely reflects alterations to the biophysical properties of the cell membranes caused by the compounds applied.

A High Resolution Solution Structure of Cx36 and CaM
Following a strategy used successfully in earlier structure determination (Ogura et al., 2012), a single-chain protein hybrid consisting of human CaM, a short glycine-rich linker,  (Wallace et al., 1995) of key interactions observed in the Cx36-CaM complex. Green lines denote hydrogen bonds and ionic interactions. and the CaM binding region of rat Cx36 (Figure 6A) was expressed and uniformly 13 C, 15 N labeled for NMR spectroscopy. Amide 1 H, 15 N HSQC spectra of this CaM-Cx36 hybrid protein (Supplementary Figure S6) and a sample of 15 N-labeled CaM in a 1:1 complex with a wild-type 25 amino acid long Cx36 carboxyterminal tail peptide were similar suggesting that the structure of the CaM-Cx36 hybrid was an accurate representation of a binary Cx36-CaM interaction.
The Cx36-CaM hybrid protein structure was determined using 1100 distance restraints from 15 N-and 13 C-edited 3D-NOESY spectra, supplemented with a 12 C-filtered, 13 C-edited 2D-NOESY of a 13 C-CaM/ 12 C-Cx36 peptide complex ( Table 2). The ensemble of 20 lowest energies structures is shown in Supplementary Figure S7. From the representative structure of the Cx36-CaM complex, CaM binds Cx36 in a wellknown compact form ( Figure 6B) with both Ca 2+ saturated lobes enveloping a helical ligand with amphipathic features (Figure 6C). Based on the spacing of hydrophobic amino acids with W277 at anchor position 1, Cx36 resembles a 1-8-14 class ligand except that position 14 is occupied by K280 where a hydrophobic acid would typically be found. Accordingly, the Cx36-CaM protein structure compares favorably to other crystal structures of compact CaM complexes bearing 1-8-(14) ligands from smooth muscle myosin light chain kinase (Meador et al., 1992) (smMLCK; PDB:1CDL, 1.63 Å) and NMDA receptor NR1 C1 region (Ataman et al., 2007) (NMDAR NR1, PDB:2HQW, 1.81 Å) (Figure 6D). A contact map of Cx36-CaM from position 8 onward is shown in Figure 6E. The guanidino group of arginine at position 9 (R285 in Cx36) is balanced by ionic contacts from E84 in CaM. Likewise, the hydrophobic amino acid position 11 (A287 in Cx36) is supported by F19, L32, and V91 in CaM. At the penultimate contact position 14, the helix of Cx36 terminates earlier than smMLCK and NMDAR NR1 ligands. Despite this difference, three methionines in CaM (M36, M51, and M72) still contribute important hydrophobic contacts to Cx36. A lysine at position 14 in Cx36 is protruding from the CaM binding cleft toward a series of acidic amino acids in CaM (E47, D50, and E54). Taken together, a continuous network of contacts is observed between CaM and Cx36 thereby establishing Cx36 as bona fide ligand of CaM.

DISCUSSION
The interaction of the neuronal gap junction protein Cx36 with CaM was investigated in Neuro2a cells and the amino acid W277 identified as a critical residue in the CaM binding motif. The binding of Cx36 to CaM was demonstrated in intracellular vesicles and at GJPs, but only the interaction at vesicles showed a calcium-dependent increase, revealed by pharmacological blocking of CaM, manipulation of intracellular calcium or overexpression of a calcium insensitive CaM protein.
Mutation W277A, as well as pharmacological blocking of CaM caused a significant reduction in dye transfer between cell pairs, implicating CaM binding to Cx36 in gap junction communication. In vitro, CaM binds Cx36 with a comparable affinity to other connexins with contacts that follow a 1-8-14 motif found in other CaM-binding proteins such as smMLCK, NMDAR-NR1, and MARCKS.

The Cx36-CaM Binding Motif Is Distinct from Other Connexins
The novel details of CaM binding to rat Cx36 build on the original findings using mouse Cx36 and two perch Cx35 variants (Burr et al., 2005). The Cx36 binding motif is highly conserved across species from humans to lower vertebrates, like lamprey and zebrafish, and overlapping with a CaMKII binding site (Alev et al., 2008). In contrast, the CaM binding sites of Cx35/Cx36 are distinct from other connexins including Cx32, Cx43, Cx44, Cx46, and Cx50 in terms of ligand class (1-8 versus 1-5-10), location of the ligand binding sites, and resemble to date the only connexins activated by CaM (Zhou et al., 2007(Zhou et al., , 2009Dodd et al., 2008;Myllykoski et al., 2009;Chen et al., 2011;Xu et al., 2012;Mruk et al., 2014). The similarity of the Cx36 binding motifs across different species is reflected in comparable dissociation kinetics and thermodynamic characteristics found in mouse, the two  perch Cx35 variants (Burr et al., 2005) and the rat Cx36 protein sequence investigated in this study. In more general terms, connexins bind CaM with dissociation kinetics and thermodynamic characteristics covering more than a 100-fold range (Dodd et al., 2008;Myllykoski et al., 2009;Zhou et al., 2009;Chen et al., 2011). Together, the specific localization of binding motifs, distinct subclasses and different dissociation kinetics suggest functional adaptations specific for each connexins protein.
The low affinity of Cx36 to CaM, at ∼500 nM, may have arisen due to the substitution of K290 at position 14 of typical 1-8-14 ligands like smMLCK, NMDAR-NR1, or MARCKS, which CaM encounters in the compact state. Furthermore, the less extensive array of hydrophobic contacts arising from the shorter side chains throughout Cx36, namely A283 and A287, cannot be compensated by larger hydrophobic amino at position 14 (K290 in Cx36). Regardless of the precise structural basis, a lower affinity may help Cx36 quickly sample CaM, CaMKII and possibly other proteins, depending upon the numerous signaling contexts it participates in throughout the life cycle of this protein.
Calmodulin Confers Calcium Signaling to Cx36 Outside the GJP Generally, CaM binding to connexins has been investigated in the context of gap junction communication, mainly focusing on implications for channel functions in the cell membrane. On the basis of the subcellular distribution of Cx36 and mutants thereof, we have expanded this focus, showing that binding already occurs before Cx36 reaches the cell membrane forming gap junctions, or after removal from the GJP. Interestingly, the W277A mutation caused retention in vesicles and showed a reduced gap junction coupling, reflecting in part similar observations made for Cx43 (Zhou et al., 2007). Further, gap junctions made by W277A are less frequent and more prone to removal as annular plaques found distributed in cell pairs, suggesting that decorating Cx36 with CaM is critical and most likely serving multiple purposes during the life cycle of this connexin.

Ménage à Trios: Competition or Cooperation?
We have previously proposed a model predicting the sequential, cooperative processing of interaction between CaM, CaMKII, and Cx36 at the GJP (Alev et al., 2008). The main results of this study are more consistent with a sequential and competitive process, sequestering calcium-dependent actions between vesicles and GJPs. To emphasize the new findings, we propose a revised model for plasticity at electrical synapses. As highlighted in Figure 8, both CaM and CaMKII have the capacity to bind Cx36 at vesicles, with CaMKII binding at physiological calcium levels, and CaM binding only when intracellular calcium is elevated. At GJPs, CaMKII is calcium-responsive, with CaM showing no further strengthening of binding in response to calcium increase. This sequence of actions differs significantly from the activation of NMDA receptors (Bayer et al., 2001), and lead us to conclude that electrical synapses share a portfolio of interacting proteins, but mechanisms of achieving plasticity do not appear to fully recapitulate those found at chemical synapses. Instead, CaMKII binding at Cx36 at GJP seems to be the critical interaction conferring calcium-dependent plasticity at electrical synapses, while calcium-dependent sensitization of CaM appears restricted to spatially separate compartments. This could endow electrical synapses with a tunable molecular machinery using calcium as the main regulator for activation through CaMKII, while interaction of Cx36 with CaM open new avenues for linking this channel to intracellular calcium signaling pathways.
What could be the function(s) of CaM binding to Cx36? The work by Burr et al. (2005) and our data suggest that CaM binding to Cx36 will occur when [Ca 2+ ] in levels are elevated. Such conditions typically are found during synaptic activity in neurons (Gamble and Koch, 1987). Since elevation of [Ca 2+ ] in increases Cx36-CaMKII, but not Cx36-CaM interaction at GJP, we conclude that calcium signals converge through CaM onto Cx36 before the protein reaches the GJP, ruling out a major role in plasticity.
It is reasonable to speculate that Cx36-CaM binding at vesicles may be sensitive to competition with other proteins with higher CaM affinity, suggesting that CaM binding could be orchestrated and/or sequestered by calcium affinities of CaM relative to CaMKII, or other, yet unidentified calcium binding proteins, or those activated or sensitized by calcium. Prior to its arrival at GJPs, Cx36-CaM could promote vesicular transport to the cell membrane and/or post-translational modifications affecting the oligomerization of connexins in the Golgi complex (Musil and Goodenough, 1993), as well as the assembly or disassembly of Cx36 into GJPs, potentially in a way similar to glutamate receptor trafficking to dendritic spines (Korkotian and Segal, 2007;Loo et al., 2014). Alternatively, calcium-dependent binding of CaM to Cx36 could link functional Cx36 channels in the ER directly to intracellular calcium signaling as suggested for Panx1, Panx3 or the transient receptor potential vanilloid 1 channel (TRPV1) (Vanden Abeele et al., 2006;Gallego-Sandin et al., 2009;Ishikawa et al., 2011), or by limiting calcium leakage from the ER as exemplified by CaM binding to Sec61 alpha (Erdmann et al., 2011). Addressing these consequences in future experiments will close this important knowledge gap.

AUTHOR CONTRIBUTIONS
RS, ES, CB, CZ, and LD performed experiments. All authors analyzed data and were involved in the manuscript preparation. DS and GZ designed the study and finalized the manuscript.

ACKNOWLEDGMENT
We thank Ms. Lesia Szyca for generating the illustrations.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnmol.2016. 00120/full#supplementary-material FIGURE S1 | Representative 3D images of Neuro2A cells expressing wild-type Cx36, tagged with EGFP, or DsRed monomer. The original projection based on the raw Z-stack before processing is shown top left. By rotating the 3D image and zooming into structural details of the Cx36 distribution it is possible to highlight the exchange of Cx36 proteins between adjacent cells connected by a GJP. Note that GJPs share a mixture of EGFP (in turquoise) and DsRed monomer (in red) tagged Cx36 proteins, whereas redistribution between cells as indicated by red and turquoise vesicles is low (Scale bar: 10 µm).
FIGURE S2 | Representative 3D images of Neuro2A cells expressing the Cx36 W277A mutant. The original projection based on the raw Z-stack before processing is shown top left. Note the mixed red and turquoise GJP and the increased redistribution of red and turquoise vesicles between adjacent cells indicating the reduced stability of the GJP and increase in protein turnover (Scale bar: 10 µm).

FIGURE S3 | Western blot detection of ER and Golgi tagged EGFP after transient expression in Neuro2a cells (in red, position indicated by lines).
β-actin expression was used as internal protein standard.
FIGURE S5 | Representative 3D images of Neuro2a cells expressing wild-type Cx36-ECFP (in red) and DsRed monomer tagged mitochondria (in light turquoise). This image is a typical example showing the lack of co-localization between Cx36 and mitochondria (Scale bar: 10 µm).
FIGURE S6 | A 15 N-edited HSQC spectum of 13 C, 15 N-labeled CaM-Cx36 hybrid protein acquired at 700 MHz. Cx36 specific amino acids are labeled red. Amino side chain resonances of asparagine and glutamine are indicated by dashed lines. An asterisk indicates an unassigned resonance. A plus sign indicates the position of a resonance below the contour limit of the plot.
FIGURE S7 | Superposition of the ensemble of 20 CaM-Cx36 NMR solution structures deposited in the Protein Data Bank (2N6A). The protein is presented as a Cα ribbon (CaM lobe 1, green; CaM lobe 2, blue; linker, gray; Cx36, orange) and the calcium ions are presented as spheres.