Abstract
In contrast to chemical transmission, few proteins have been shown associated with gap junction-mediated electrical synapses. Mixed (electrical and glutamatergic) synaptic terminals on the teleost Mauthner cell known as “Club endings” constitute because of their unusual large size and presence of connexin 35 (Cx35), an ortholog of the widespread mammalian Cx36, a valuable model for the study of electrical transmission. Remarkably, both components of their mixed synaptic response undergo activity-dependent potentiation. Changes in electrical transmission result from interactions with colocalized glutamatergic synapses, the activity of which leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII), required for the induction of changes in both forms of transmission. However, the distribution of this kinase and potential localization to electrical synapses remains undetermined. Taking advantage of the unparalleled experimental accessibility of Club endings, we explored the presence and intraterminal distribution of CaMKII within these terminals. Here we show that (1) unlike other proteins, both CaMKII labeling and distribution were highly variable between contiguous contacts, and (2) CaMKII was not restricted to the periphery of the terminals, in which glutamatergic synapses are located, but also was present at the center in which gap junctions predominate. Accordingly, double immunolabeling indicated that Cx35 and CaMKII were colocalized, and biochemical analysis showed that these proteins associate. Because CaMKII characteristically undergoes activity-dependent translocation, the observed variability of labeling likely reflects physiological differences between electrical synapses of contiguous Club endings, which remarkably coexist with differing degrees of conductance. Together, our results indicate that CaMKII should be considered a component of electrical synapses, although its association is nonobligatory and likely driven by activity.
Introduction
As a result of their unusually large size (∼10 μm), a group of contacts on the lateral dendrite of the teleost Mauthner (M) cells has provided since their discovery (Bartelmez, 1915) the opportunity of examining diverse structural features of vertebrate synapses. These “striking synapses” are terminations of auditory afferents originating in the anterior part of the sacculus and are known as “large myelinated Club endings” [Club endings (CEs)] (Bartelmez, 1915). Studies on CEs provided early evidence for the lack of protoplasmatic continuity between presynaptic and postsynaptic elements (Bartelmez and Hoerr, 1933) and, with the advent of electron microscopy, early evidence for the structural basis of electrical transmission: the gap junction (Robertson et al., 1963). Ultrastructural (Tuttle et al., 1986) and confocal microscopy (Pereda et al., 2003) studies show that, although gap junctions (up to ∼200) are distributed throughout the surface of the contact, chemical synapses are predominantly restricted to the periphery (Tuttle et al., 1986). Thus, the large size of these terminals and differential segregation of the structural components for both forms of transmission makes them amenable for exploring the presence and detailed subcellular distribution of various proteins associated with these modalities of synaptic transmission.
The physiological features of CEs can also be easily explored, allowing the uncommon correlation between synaptic structure and function (for review, see Pereda et al., 2004). Consistent with the coexistence of gap junctions and chemical synapses at these terminals (“mixed synapses”), electrical stimulation of saccular afferents evokes a mixed, electrical and chemical, synaptic potential in the lateral dendrite of the M-cell (Furshpan, 1964; Lin and Faber, 1988a). Although chemical transmission is mediated by glutamate (Wolszon et al., 1997), gap junctions contain connexin 35 (Cx35) (Pereda et al., 2003), the fish ortholog of the neuronal connexin 36 (Cx36), which is widely expressed throughout mammalian brain (Condorelli et al., 1998). Strikingly, both components of the mixed synaptic response undergo activity-dependent potentiation of their respective strengths (Yang et al., 1990; Pereda and Faber, 1996). Thus, changes are not restricted to chemical synapses but also involve the regulation of gap junction-mediated electrical synapses. The mechanism underlying modifications in electrical transmission reflects a functional interaction with neighboring colocalized glutamatergic synapses, in which activity leads to the activation of the multifunctional Ca2+/calmodulin-dependent kinase II (CaMKII), necessary for the induction of changes in both forms of transmission (Pereda et al., 1998). However, the distribution and relationship of CaMKII to chemical and electrical synapses within CEs remains undetermined.
CaMKII has been implicated in mechanisms of activity-dependent plasticity in chemical synapses (Fink and Meyer, 2002; Schulman 2004; Merrill et al., 2005; Wayman et al., 2008). This kinase is an essential and abundant component of glutamatergic postsynaptic densities (PSDs) (Kennedy, 2000) in which it associates with other proteins, such as NMDA receptors (Yang and Schulman, 1999; Strack et al., 2000). Recent data indicate that this kinase can also molecularly interact with Cx36 (Alev et al., 2008). Together with the well established functional role of CaMKII in CEs (Pereda et al., 1998), the high homology of Cx36 with its fish ortholog suggests a potential association of this kinase with Cx35 at these terminals. Moreover, the dynamic spatial properties of CaMKII, which are based on its ability to translocate to active synapses (Shen and Meyer, 1999; Merrill et al., 2005; Lee et al., 2009; Rose et al., 2009), including in fish (Gleason et al., 2003), suggest that its intraterminal distribution could reflect its functional role in these terminals.
To investigate the distribution of CaMKII in CEs, we took advantage of the unique experimental accessibility of the M-cell system for imaging single synaptic terminals using confocal microscopy. Here we show that the distribution of CaMKII was highly variable between contiguous CEs. Furthermore, CaMKII was found not only in the periphery of the terminals, in which glutamatergic PSDs are located, but also at the center, in which gap junctions predominate. Double immunolabeling and biochemical approaches showed that Cx35 and CaMKII associate, indicating that these proteins functionally interact at electrical synapses in CEs. We speculate that the observed heterogeneinity of labeling may reflect differences in functional states between adjacent CEs.
Materials and Methods
Immunocytochemistry.
Goldfish (Carassius auratus), 2–5 inches long, were perfused with PBS (1×) at pH 7.4 for 10–15 min, followed by cold 4% paraformaldehyde in 0.1 m phosphate buffer for 10–15 min. Brains were dissected out and kept overnight at 4°C in 4% paraformaldehyde in 0.1 m phosphate buffer. Brains were sectioned (40–50 μm) with a TPI microtome (Vibratome; Technical Products International). The sections were rinsed at room temperature three times for 10 min each with 1× PBS, blocked, and permeabilized for 45 min to 1 h at room temperature in PBStr (1× PBS and 0.3–0.4% Triton X-100, pH 7.4) plus 10% normal goat serum. Sections were incubated overnight at 4°C using a moving platform with rabbit polyclonal G301 anti-αCaMKII (1:1000) and/or mouse monoclonal anti-Cx35/Cx36 (1:250/1:500; Millipore Bioscience Research Reagents). A previously characterized teleost anti-NR1 antibody directed against the NR1 subunit of the electric fish Apteronotus (Berman et al., 2001) (gift from Drs. L. Maler, University of Ottawa, Ottawa, Ontario, Canada, and R. Dunn, McGill University, Montreal, Quebec, Canada) was used as a marker for glutamatergic synapses. Then, sections were rinsed in PBStr four times for 10 min each wash and incubated for 1–2 h at room temperature with Alexa Fluor 594-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated goat anti-mouse (1:500; Invitrogen) or Texas Red goat anti-rabbit (1:500; Jackson ImmunoResearch) or FITC goat anti-mouse (1:250; Millipore Bioscience Research Reagents) secondary antibodies. Finally, sections were rinsed with PBStr three times for 10 min each wash and then 10 min with 50 mm phosphate buffer, pH 7.4, and mounted on slides in a propyl gallate-based antifading solution to reduce photobleaching. Control sections were routinely incubated with secondary antibodies in the absence of primary antibodies.
Confocal microscopy and image processing.
Sections were imaged with an Olympus BX61WI confocal microscope with a mortised fixed stage with 20× air, 40× apo/340 water, and 60× oil-objective lenses. FLUOVIEW FV500 software was used for data acquisition. x–y images were scanned in the z-axis at 0.5–0.8 μm intervals for three-dimensional reconstruction. z-plane sections and z-plane stacks were analyzed using NIH Image J software. For presentation purposes, some images were processed with Adobe Photoshop (Adobe Systems) and Canvas X (ACD Systems). For the analysis of CaMKII labeling, 8 bit images of individual CEs were background subtracted and thresholded using NIH Image J to include signals at least 1.5-fold to 2-fold greater than the background signal. Regions of interest corresponding to the surface area of individual CEs were identified using differential interference contrast (DIC) microscopy and/or Cx35 labeling during double-labeling experiments. To investigate its distribution at CEs, CaMKII labeling at two concentric, peripheral and central, areas of each CE (considered for this purpose as an ellipse) (see Fig. 4D) were quantified using NIH Image J (see Fig. 4D). These areas represented 36 and 49% of the total surface of the terminal, respectively. A transition ring between these two regions (15%) was not taken into account (see Fig. 4D) to better contrast the distribution of labeling between these areas. The rationale behind this analysis was that, although CaMKII is expected to be present in the periphery of the terminals in which the majority of the glutamatergic PSDs are located, its localization in the center would be suggestive of its association to gap junctions, which predominate in this area. To illustrate the variability in the distribution of CaMKII labeling within the population of CEs, we used a ratio between labeling at central and peripheral areas. This index was defined as follows: periphery/center index = labeling center − labeling periphery/labeling center + labeling periphery, where labeling center = Σ (labeling intensity above threshold at center × number of pixels)/center area, and labeling periphery Σ = (labeling intensity above threshold at periphery × number of pixels)/periphery area. Indices approximating −1 indicate prevalence of labeling at the periphery of the CE, whereas those approximating +1 indicate prevalence of labeling at the center. Finally, indices approximating 0 are indicative of a comparable labeling between center and periphery. For colocalization analysis, images of individual CEs were background subtracted and thresholded to include signals that were at least twofold greater than the scatter labeling at the dendrite. Regions of interest corresponding to individual CEs were identified in MetaMorph using transmitted light images and Cx35 labeling. Colocalization was measured as the percentage of the area labeled for one channel that was also labeled for the second channel and the converse.
Coimmunoprecipitation.
Fresh tissue samples of ∼90 mg and containing both M-cells, as well other smaller reticulospinal and vestibulospinal neurons, were obtained from goldfish hindbrain ventral to the cerebellum and between rostral and caudal margins of the cerebellar peduncles. Samples were rapidly stored at −80°C until homogenization. At 4°C, immunoprecipitation (IP) buffer (20 mm Tris-HCl, pH 8.0, 140 mm NaCl, 1% Triton X-100, 10% glycerol, 1 mm EGTA, 1.5 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 5 μg/ml of the protease inhibitors leupeptin, pepstatin A, and aprotinin) were added to the thawed samples for homogenization. Homogenates were sonicated at 4°C three times for 10 s each, 35% duty cycle with a W-225R sonicator (Misonix), and centrifuged for 10 min at 14,000 × g at 4°C, and the supernatant was collected for additional analysis (Western blotting and coimmunoprecipitation). Protein was quantified using BCATM Protein Assay kit (Pierce). Aliquots of supernatants (2 mg of protein) were precleared for 1 h at 4°C using 30 μl of protein A-coated agarose beads (Santa Cruz Biotechnology) and centrifuged at 14,000 × g for 10 min at 4°C. Supernatants were incubated with 2 μg of G301 rabbit anti-αCaMKII antibody overnight at 4°C on a moving platform, followed by 1 h incubation with 30 μl of protein A-coated agarose beads. The mix was then centrifuged as above for 10 min at 4°C. The pellets were washed five times with 500 μl to 1 ml of wash buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 0.5% NP-40) and incubated at 60°C for 2 min in SDS-PAGE loading buffer containing 5–10% β-mercaptoethanol. Samples were resolved on 10 or 12% SDS-PAGE gels. Proteins were transferred at 30 V overnight to nitrocellulose membranes (Schleicher and Schuell) in standard Tris-glycine transfer buffer, pH 8.3, containing 0.5% SDS. To confirm transfer efficiency, membranes were stained with Ponceau-S (Sigma). Blots were blocked for 1–3 h at room temperature with 4–5% nonfat dry milk in TSTw (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.1% Tween 20), rinsed briefly in TSTw, and then incubated overnight at 4°C using a moving platform with mouse anti-Cx35/Cx36 (2 μg/ml) or mouse anti-zona occludens-1 (ZO-1) (2–3 μg/ml) antibodies. Antibodies were diluted in TSTw containing 1% nonfat dry milk. Membranes were washed with TSTw three times for 10 min, incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG (diluted 1:7500; Santa Cruz Biotechnology), washed with TSTw four times for 10 min, and once for 10 min with 20 mm Tris-HCl, pH 8.0. Proteins were then resolved by chemiluminescence ECL (GE Healthcare). Control samples were precipitated in the absence of CaMKII antibody.
Electrophysiology and dye coupling.
Surgical and recording techniques were similar to those described previously (Smith and Pereda, 2003; Curti and Pereda, 2004). Briefly, individual nerve VIII afferents were sequentially penetrated outside the brain (electrodes contained 2.5 m KCl; 35–45 MΩ) while a second electrode (5 m KAc; 4–12 MΩ) was kept inserted in the M-cell lateral dendrite, 350–400 μm from the axon cap of the cell. Afferents with electrical synapses on the M-cell were identified by the presence of electrotonic coupling potentials when the M-cell antidromic spike was evoked by stimulating the spinal cord (Furshpan, 1964; Lin and Faber, 1988a; Smith and Pereda, 2003; Curti and Pereda, 2004). The resting potential of the afferents and the M-cell averaged 71 ± 0.5 mV (mean ± SEM; n = 140) and 78.7 ± 2.5 mV (mean ± SEM; n = 95), respectively. Only afferents with resting potentials of at least −60 mV were used for the analysis. For dye coupling evaluation, the M-cell was recorded intracellularly with electrodes containing a 4% solution of Neurobiotin (molecular weight of 322.8) in 2.5 m KCl, and this solution was iontophoretically injected (400 ms pulses of 50 nA for 20 min), visualized with avidin-conjugated DAB, and examined under transmitted light microscopy. To avoid unintentional extracellular leakage of Neurobiotin around the CEs, the injections were performed in the soma, which is ∼400 μm from the terminal field of the CEs. After injections, waiting periods of 1–2 h were used to allow diffusion of the dye (for details, see Smith and Pereda, 2003). Coupling coefficients for individual CEs were estimated as the ratio between the amplitude of the electrical component (or coupling potential) of the unitary synaptic potential and the amplitude of the presynaptic spike (coupling coefficient = coupling potential/presynaptic spike). This approximation is a convenient experimental advantage of the M-cell because (1) the presynaptic action potential is an active signal of constant amplitude between Club endings afferents, and (2) the fast time constant of the M-cell dendrite, estimated in ∼400 μs (Fukami et al., 1965), does not disproportionably attenuate fast voltage transients, allowing reliable measurements of coupling. An average value of 88 ± 1.09 mV (mean ± SEM; n = 150) was used for the presynaptic spike amplitude, because the amplitude observed at the recording site at each might not be representative of its amplitude at the contact. The spike recorded at the site of depolarization regenerates in subsequent nodes and the presynaptic terminal (Lin and Faber 1988a; Smith and Pereda, 2003; Curti et al., 2008).
In silico alignments and predictions of binding and phosphorylation sites. The amino acid sequence of the murine CaMKIIα (NP_803126.1) regulatory site (Rosenberg et al., 2005) was aligned to its fish counterpart (zebrafish CaMKIIα; UnitProKB Q32PV2), and amino acid sequences of αCaMKII binding and phosphorylation sites of mammalian Cx36 (gi|8928062) (Alev et al., 2008) were aligned to both perch Cx35 (gi|3420235) and Cx34.7 (gi|3420237) using EMBOS (European Molecular Biology Laboratory–European Bioinformatics Institute; http://www.ebi.ac.uk/Tools/emboss/align/). In all cases, amino acid homology was expressed in terms of percentage of similarity, which takes into account identity as well conserved amino acid substitutions. Group-based Prediction System (GPS) version 2.1 was used for prediction of αCaMKII phosphorylation sites in mammalian Cx36 and perch Cx35 and Cx34.7, using a high threshold with a cutoff of 2.917. High-threshold analysis has been validated by large-scale predictions of mammalian phosphorylation sites [GPS 2.0 (Xue et al., 2008)]. The cutoff values used in this analysis offered 98.5% accuracy, 100% sensitivity, 98.48% of specificity, and 0.7117 Mathew correlation coefficient (MCC) and were set on a calculated false positive rate (FPR) [GPS 2.0 (Xue et al., 2008)]. The prediction of serine 293 as phosphorylation target site in Cx36 required a lower threshold (medium), which still offers high accuracy and sensitivity (90.71% accuracy, 100% sensitivity, 90.56% of specificity, and 0.4673 MCC).
Results
Identification of Club endings
The CEs represent the most recognizable input to the M-cells and originate from a population of ∼90 saccular afferents that segregate to the distal portion of the lateral dendrite (Fig. 1A) (Bartelmez, 1915). Because of their unusually large size and high expression of Cx35, CEs can be unequivocally identified using antibodies that recognize Cx35 (Fig. 1B) (Pereda et al., 2003; Flores et al., 2008). As illustrated in Figure 1B, the use of an anti-Cx35 monoclonal antibody (Cx35/Cx36; Millipore Bioscience Research Reagents) characteristically yields intense punctate staining at the contact areas between CEs and M-cells (Fig. 1B–D). The number of anti-Cx35 fluorescent puncta observed at individual CEs is consistent with a previous ultrastructural demonstration of 63–200 closely spaced gap junction plaques at individual CEs (Tuttle et al., 1986), suggesting that each punctum represents an individual plaque (Flores et al., 2008). The correspondence of this immunolabeling to CEs was confirmed in double-labeling experiments in which the saccular afferents were also labeled (Fig. 1B–D), in which Cx35 labeling was found at the contact area of each afferent and the M-cell dendrite. These contacts were also easily identifiable on the surface of the lateral dendrite using DIC microscopy (Fig. 1E, inset). Additionally, this labeling pattern was confirmed using other anti-connexin antibodies (Pereda et al., 2003) and anti-ZO-1 antibodies (see below), a protein that exhibits a high degree of colocalization with Cx35 (Flores et al., 2008).
The Club endings: the striking synapses of the Mauthner cell. A, Diagram of the M-cell showing its axon cap, soma, and lateral dendrite. The large myelinated Club endings, referred to here as “Club endings”, are distributed along the distal portion of the lateral dendrite. These contacts belong to primary auditory afferents that establish mixed (electrical and chemical) synapses on the distal portion of the M-cell lateral dendrite. Right, The diagram summarizes the main structural features of CEs; specializations corresponding to chemical synapses are restricted to the periphery of the contact. The presynaptic and postsynaptic elements of the synapse are illustrated separately, for better appreciation of the PSDs. B, Projection image of the distal portion of the lateral dendrite obtained with laser scanning confocal microscopy, illustrating saccular afferents (green) terminating as CEs. Labeling of these afferents was obtained using a phospho-specific anti-Cx35 antibody (Cx35 phospho-Ser110), which is known to cross react to unknown high-molecular-weight phosphoproteins that appear to be expressed primarily in glial cells (Kothmann et al., 2007). Double labeling with monoclonal anti-Cx35/Cx36 antibody (red; Millipore Bioscience Research Reagents) reveals the area of contact between CEs and the distal portion of the M-cell lateral dendrite. C, Higher magnification of the labeled boxed region in B. The observation of multiple Cx35-labeled puncta is consistent with the presence of up to ∼200 gap junction plaques in individual CEs. D, Higher magnification of the labeled boxed region in B. Cx35 labeling is observed at the region of contact between two CEs and the M-cell dendrite. E, Image of a Club ending on the surface of the lateral dendrite obtained using DIC optics. These unusually large contacts are easily recognizable because of their big size and characteristic ring-like appearance caused by their prominent myelination.
Labeling for CaMKII is highly variable between Club endings
To investigate the presence and distribution of CaMKII at CEs, we performed immunofluorescence labeling using an antibody that exhibits relative selectivity for the α subunit of rat brain CaMKII (G301, directed against a sequence in the autoregulatory domain of the rat brain α subunit) and that we showed previously recognizes goldfish CaMKII in Western blots (Pereda et al., 1998). As reported previously, this antibody showed intense labeling of the M-cell (Figs. 2A,B, 3B,C), which is consistent with the fact that CaMKII interacts with proteins associated with cytoskeleton (Sahyoun et al., 1985; Ohta et al., 1986). This distinct pattern constituted an internal control for the specificity of CaMKII labeling. At the surface of the dendrite, this antibody labeled ovoidal structures that were reminiscent, because of the size and location, of CEs. The observed labeling matched that of CEs in double-labeling experiments using a Cx35 antibody (Fig. 2C,D) and when visualizing these terminals with the help of DIC (data not shown). Thus, our results indicate that CaMKII can be detected at the areas of contacts between CEs and the M-cell.
Double immunolabeling of Cx35 and αCaMKII in the lateral dendrite of the Mauthner cells. A, Laser scanning confocal projection image of the distal portion of the M-cell lateral dendrite (average of 5 sections) using a polyclonal anti-αCaMKII antibody (G301; red). The G301 antibody also labeled neurofilaments (asterisks) in the cytoskeleton of the M-cell. B, Higher magnification of the labeled boxed region in A, illustrating αCaMKII labeling at individual CEs. C, Double labeling using the polyclonal anti-αCaMKII (G301; red) and anti-Cx35/Cx36 (green) antibodies, respectively. D, High magnification of the labeled boxed region in C.
Heterogeneity of αCaMKII labeling between contiguous Club endings. A, Confocal projection of the distal portion of the lateral dendrite showing immunolabeling for Cx35 using monoclonal anti-Cx35/Cx36 antibody. Note the regularity of Cx35 labeling between CEs. B, Confocal projection of a similar portion of the distal lateral dendrite showing immunolabeling for αCaMKII (G301 antibody). C, Higher magnification of the labeled boxed region in B. CaMKII distribution is highly variable between adjacent CEs (arrowheads, predominant in the terminal periphery; arrows, predominant on the entire surface of the contact). D–F, Examples of variability of CaM-KII labeling between CEs.
Interestingly, in contrast to the predictable regularity of the labeling observed using the Cx35 antibody (Figs. 1B, 3A), the amount of labeling with the CaMKII antibody was not constant but highly variable between contiguous CEs (Figs. 3B–D, 4C). Such variability of labeling was observed in all analyses of CaMKII immunoreactivity (n = 5 fish). Furthermore, detailed examination of individual CEs using high-resolution confocal microscopy revealed that the spatial distribution of CaMKII within the contact areas was also highly variable. Although gap junctions are known to be distributed throughout the entire surface of the contact (Fig. 4A), glutamatergic synapses are primarily distributed in the periphery (Fig. 4B), in which PSDs are located (Tuttle et al., 1986). Such differential segregation makes CEs ideal for examining the relative distribution of diverse synaptic proteins to these coexistent modalities of transmission (Fig. 1A, diagram). We found that, although CaMKII labeling was predominant in the periphery, which is consistent with localization to glutamatergic PSDs (Figs. 2B, 3D), it was also unexpectedly found at the center of the contacts and, in some cases, distributed throughout their entire surface (Figs. 3E, F, 4C). To document and quantify the difference in the spatial distribution of CaMKII, we expressed this variability as a ratio between the amount of labeling detected at the periphery and that detected at the center of the terminals (Fig. 4D). As illustrated in the summary graph (Fig. 4D), the estimated “periphery/center index” (for details, see Materials and Methods) was highly variable between CEs, indicating that the intraterminal distribution of CaMKII is not a consistent feature at these contacts. Confirming such variability in its spatial distribution, the amount of CaMKII labeling in both the total area and at the center of the contact were highly variable between CEs (supplemental Fig. 1A,B, respectively, available at www.jneurosci.org as supplemental material). Furthermore, these two measurements were highly correlated (supplemental Fig. 1C, available at www.jneurosci.org as supplemental material), indicating that the amount detected at the center substantially contributes to the total amount of CaMKII in CEs.
Intraterminal distribution of αCaMKII in Club endings. Confocal projections of individual Club endings showing the distribution of Cx35 (A), NR1 (B), and αCaMKII (C). A, B, Cx35 labeling is typically punctate and covers most of the surface contact area, whereas NR1 labeling is mostly restricted to the periphery of the synaptic contact [images in A and B were illustrated previously (Pereda et al., 2003)]. C, In contrast, αCaMKII labeling (G301 antibody) is more diffuse and highly variable between contiguous Club endings. Although αCaMKII labeling was always observed at the periphery, in which PSDs are located, it is also found at the center of the contact area of the CEs in some cases (compare top CEs with the bottom one). D, Quantification of the variability of αCaMKII distribution of CEs using a ratio between the labeling at periphery and center of each CE. Diagram of the regions selected for analysis: a peripheral “ring” area (in which PSDs are included) and a center area (in which gap junctions are predominant). A transition area (white) between periphery and center areas was not included in the analysis. The ratio between these areas was defined as the periphery/center index (for details, see Materials and Methods). Graph illustrates the distribution of this index for 43 Club endings.
Association of CaMKII with connexin 35
The unexpectedly variable spatial distribution of CaMKII within CE terminals suggested that it could be associated with proteins other than those located at PSDs. In particular, the presence of CaMKII labeling in the center of the terminals suggests that CaMKII could also associate with proteins at Cx35-containing gap junction plaques, which are highly predominant in this region of the contact (Tuttle et al., 1986). To investigate this possibility, we looked for colocalization of Cx35 and CaMKII in double-labeling experiments (Fig. 5). We found a clear localization of CaMKII at many Cx35-labeled puncta (Fig. 5A,B). The degree of colocalization of Cx35 with CaMKII was lower compared with previous studies of colocalization of this connexin with the scaffold protein ZO-1 (see below), and it was highly variable between adjacent CEs. Averaged over individual endings, 66.7 ± 32.5% (mean ± SD) of the area of Cx35 immunolabeling also showed CaMKII labeling, and, conversely, 70.1 ± 18.6% (mean ± SD) of the area of CaMKII labeling also showed Cx35 labeling (n = 28). As already mentioned above, the degree of colocalization was highly variable, ranging from 6.4 to 81% and 15.9 to 79% for Cx35 over CaMKII and CaMKII over Cx35, respectively. Strikingly, the variability of colocalization was found in immediately adjacent Cx35-labeled puncta, within the same region of a single contact (Fig. 5B). Because each punctum is thought to represent an individual gap junction plaque, such differential labeling of adjacent plaques indicates that the colocalization of Cx35 and CaMKII might not be obligatory in nature and reflective of their functional interaction. Thus, its distribution to the center of the terminals might represent the extent of its functional association with Cx35 at gap junction plaques in CEs.
Connexin35 and αCaMKII colocalize at Club endings and associate in goldfish brain. A, Confocal z-section of a single terminal showing colocalization (yellow) of Cx35 (green) and αCaMKII (red). Labeling of Cx35 and αCaMKII colocalizes at the periphery and the center of the synaptic contact. B, Higher magnification of the labeled boxed region in B. αCaMKII and Cx35 colocalize at some Cx35 puncta (arrowheads) but is absent at others (asterisks). C, Sequence alignment of mouse Cx36 cytoplasmic loop and C-terminal αCaMKII binding site regions (Alev et al., 2008) reveals a high degree of similarity with perch Cx35 (86 and 92%, respectively). D, Immunoblot detection of Cx35 in two different samples (lanes 2 and 3) with monoclonal Cx35/Cx36 antibody after IP of αCaMKII from goldfish hindbrain with G301 antibody. The Cx35 immunoblot exhibits three identifiable bands.
The colocalization of Cx35 and CaMKII together with the high homology of this connexin with its mammalian ortholog Cx36 (O'Brien et al., 1998), which has been shown recently to interact with CaMKII (Alev et al., 2008), suggested that these two proteins might also associate and interact in goldfish brain. To directly explore the possible association of Cx35 with CaMKII, we first performed in silico analysis of mammalian and teleost αCaMKII sequences, as well as of mouse Cx36 and perch Cx35 (see Materials and Methods). Alignments of the mammalian and teleost sequences of the αCaMKII regulatory region revealed that they are highly homologous (100% similarity in the pseudotarget and pseudosubstrate regions) (supplemental Fig. 2A, available at www.jneurosci.org as supplemental material) and that core amino acids for binding of the αCaMKII autoregulatory region to αCaMKII catalytic domain are conserved [Only one gene for αCaMKII has been identified in teleost (ZFIN ZDB-GENE-051113-72).] Furthermore, sequence alignment of the described Cx36 cytoplasmic loop (CL) binding (pseudotarget) and C-terminus (CT) binding (pseudosubstrate) regions (Alev et al., 2008) showed that these areas were highly conserved in Cx35, with 86 and 92% similarity for the CT and CL regions, respectively (Fig. 5C) (supplemental Fig. 2B, available at www.jneurosci.org as supplemental material). Similar results were obtained for Cx34.7, another perch Cx36 homolog (O'Brien et al., 1998), which showed 76.2 and 84% of similarity for the CT and CL regions, respectively (supplemental Fig. 2B, available at www.jneurosci.org as supplemental material), suggesting that CaMKII association mechanisms are highly conserved and likely represent an important regulatory feature of neuronal connexins.
To investigate whether Cx35 and αCaMKII associate in goldfish brain, we performed coimmunoprecipitation studies using the G301 antibody. This analysis was performed in samples obtained from a small area of the goldfish hindbrain containing the colossal M-cells, along with some other smaller reticulospinal and vestibulospinal neurons (supplemental Fig. 3, available at www.jneurosci.org as supplemental material) (see Materials and Methods). Thus, this sample should be representative of the M-cell contents. αCaMKII was immunoprecipitated from these hindbrain homogenates, and immunoblots were probed with the Cx35 monoclonal antibody (Cx35/Cx36; Millipore Bioscience Research Reagents). Immunoprecipitation of αCaMKII resulted in the detection of a Cx35 band at the predicted molecular weight (Fig. 5D, lanes 2 and 3) that was comparable with migration profiles reported previously (Flores et al., 2008). Although Western blots detected a small presence of Cx35 (Fig. 5D, lane 1) in the input, more prominent bands were seen with the immunoprecipitation from two different samples (Fig. 5D, lanes 2 and 3), indicating that αCaMKII more efficiently pulled down this connexin as a result of their association. Interestingly, multiple bands were identified with the Cx35 antibody, suggesting the existence of multiple phosphorylation states of this connexin (Fig. 5D, lanes 2 and 3).
Recently, four αCaMKII phosphorylation sites were described for Cx36 (Alev et al., 2008). To investigate the presence of putative phosphorylation sites for αCaMKII in Cx35, we performed in silico analysis using GPS 2.1 software (see Materials and Methods) in mouse Cx36 and perch Cx35 and Cx34.7 sequences. This analysis confirmed the presence of residues S110, T111, S277, and S315 in Cx36 as αCaMKII targets. Five sites, including S110 and T111, both in the intracellular loop, and S128, S276, and S298 in the C terminus were identified for Cx35 (supplemental Fig. 4, available at www.jneurosci.org as supplemental material), in which the last two correspond to sites S293 and S315 of Cx36, respectively. Residues S110 and S276 (S110 and S293 of Cx36) (Urschel et al., 2006) were described previously as PKA phosphorylation sites (O'Brien et al., 1998; Kothmann et al., 2007), suggesting that they are targets shared by both kinases. Conversely, S298 (equivalent to S315 in Cx36) seems to be exclusive for αCaMKII. Only three putative sites were identified for Cx34.7: S110, S277, and S300, which correspond to residues S110, S276, and S298 of Cx35 and S110, S293, and S315 of Cx36, respectively. Thus, our in silico analysis is consistent with the possibility of multiple phosphorylation states for Cx35.
Together, our in silico and biochemical results suggest that CaMKII associates with Cx35 in goldfish brain. Several proteins have been also shown to interact with connexins, indicating the existence of protein complexes associated with gap junction channels (Hervé et al., 2004). We have reported recently that ZO-1 extensively colocalizes and associates with Cx35 at CEs (Flores et al., 2008). In contrast to the less abundant and highly variable labeling of CaMKII, ZO-1 was shown to extensively colocalize with Cx35 (mean ± SD, 85.53 ± 14.45% of Cx35 colocalizes with ZO-1 labeling and 86.06 ± 15.45% of ZO-1 colocalizes with Cx35; n = 116) (Flores et al., 2008) (Fig. 6A) and to directly interact with the last four amino acids of its C terminus (Flores et al., 2008), suggesting that this scaffold protein might play a structural role in gap junction plaques at these terminals. Thus, we hypothesized that, if CaMKII directly interacts with Cx35, immunoprecipitation studies with the G301 antibody should be also able to detect ZO-1. For this purpose, we performed coimmunoprecipitation studies in which CaMKII was immunoprecipitated from hindbrain homogenates. In this case, immunoblots were probed with a monoclonal ZO-1 antibody, also used for immunolabeling of ZO-1. Immunoprecipitation of CaMKII resulted in the detection of a ZO-1 band at the predicted molecular weight (Fig. 6B). These results suggest that CaMKII associates with Cx35 and, together with ZO-1, is likely to be part of the same macromolecular complex in which channel-forming, scaffold, and regulatory proteins coexist.
αCaMKII associates with ZO-1 in goldfish brain. A, Extensive colocalization of Cx35 (green) and ZO-1 (red) at CEs in the M-cell lateral dendrite. B, Immunoblot detection of ZO-1 (lane 2) with mouse ZO-1 antibody after IP of αCaMKII from goldfish hindbrain with G301 antibody.
Variability of electrical coupling between contiguous terminals
Although both sequence alignments and immunoprecipitation studies suggest that Cx35 and αCaMKII interact at CEs, colocalization studies suggest that this association might not be obligatory, because variable labeling was found even between plaques within the same terminal. Because CaMKII is known to translocate and aggregate to target sites after activity (Shen and Meyer, 1999; Gleason et al., 2003; Merrill et al., 2005; Rose et al., 2009), its intraterminal distribution could be indicative of the degree of potentiation at individual CEs. Accordingly, the variability of CaMKII labeling between immediately adjacent CEs was reminiscent of the variability of unitary electrical components of the mixed (electrical and chemical) (Fig. 7A) synaptic potential evoked by stimulation of a single CE, which differ dramatically in amplitude (Smith and Pereda, 2003). To further demonstrate the extent and consistency of this phenomenon, we compared the strength of electrical coupling between individual CE afferents and the M-cell lateral dendrite between different fish. For this purpose, we obtained multiple simultaneous presynaptic and postsynaptic recordings sequentially from individual CE afferents and the same M-cell lateral dendrite. As shown in Figure 7A in which seven consecutive single terminal recordings are illustrated, the amplitude of the electrical components differed dramatically. [Characteristically, most CE single terminal recordings lack a chemical component (Lin and Faber, 1988b) and transmitter release is enhanced by coactivation of multiple terminals (Pereda et al., 2004).] This same variability was observed for five different fish in which unitary potentials were recorded in each M-cell dendrite during stimulation of six to eight terminals (Fig. 7C). Given that these single terminals originate from ∼90 large saccular afferents (Bodian, 1937), this sample represents ∼8–10% of the total population of CEs in each lateral dendrite. In contrast to previous studies (Smith and Pereda, 2003), we expressed this variability in terms of coupling coefficients of orthodromic coupling produced by presynaptic action potentials (Fig. 7C), an estimate that is greatly facilitated by the fast time constant of the lateral dendrite of the M-cell (Fukami et al., 1965) (see Materials and Methods). The properties of the presynaptic action potentials are highly constant across the population of CEs (Curti et al., 2008). Furthermore, because they were all recorded in the same postsynaptic cell (which makes corrections for differences in input resistance unnecessary), these coupling coefficients are highly correlated with the gap junctional conductance of individual terminals. The differences in conductance were confirmed by tracer coupling to the afferents after injection of the M-cell with Neurobiotin, in which unlabeled afferents and afferents showing different degrees of staining were observed in the same dendritic area (Fig. 7B). This variability of coupling was also observed for the whole population of recorded afferents. Interestingly, both the spatial distribution of CaMKII (expressed as periphery/center index; mean ± SD, −0.11 ± 0.41; n = 43) (Fig. 7D) and the coupling coefficients of individual CEs (mean ± SD, 0.008 ± 0.003; n = 92) (Fig. 7E) showed wide variability and comparable distributions. The similarity opens the possibility that differences in the spatial distribution of CaMKII could represent differences in the functional state of electrical synapses at these terminals, in which the amount and localization of CaMKII to gap junctions would be indicative of the degree of potentiation. Unfortunately, detecting changes in the distribution of CaMKII after the application of the high-frequency stimulating protocols leading to synaptic potentiation (Yang et al., 1990; Pereda and Faber, 1996; Pereda et al., 1998) was impractical and difficult to interpret resulting from (1) the inability of identifying the contacts in the dendrite that correspond to the subpopulation of afferents stimulated by the extracellular electrode in anatomical sections and (2) the fact that, within the population of stimulated afferents, although most synapses potentiate, others were shown to depress as a result of the same stimulating protocol (Smith and Pereda, 2003) (Fig. 4), indicating that both depression and potentiation coexist in the population of stimulated afferents.
Electrical synapses between neighboring Club endings coexist at different degrees of conductance on the M-cell lateral dendrite. A, Differences in gap junctional conductance between CEs evidenced by multiple simultaneous recordings in the same M-cell dendrite. The recordings of unitary synaptic potentials (red top traces) were obtained sequentially from the same dendritic position, and it cannot be explained by differences in the amplitude of the presynaptic action potentials (black bottom traces), in which variability was much lower. Only two of these nine unitary synaptic potentials exhibit a clear chemical component (e.g., “chemical” in recording a). B, Variability in the amplitude of unitary coupling potentials does not represent variability in the electrotonic distances from the recording site, because a similar diversity in coupling strength can be revealed using Neurobiotin dye coupling (Smith and Pereda, 2003). Transfer of Neurobiotin from the M-cell to neighboring CEs differs dramatically (dark, white arrowheads; unlabeled, black arrowheads), indicating that junctions differ in permeability. View of the bifurcation of the lateral dendrite (dark branches) obtained with DIC optics and revealing differences of labeling between neighboring CEs (only a small number of CEs are generally labeled after injection of Neurobiotin into the M-cell, indicating that gap junction permeability at CEs is generally low). Inset, A single terminal distinguished solely by the use of DIC optics. C, Variability of coupling coefficients among individual CEs in five different fish. Inner circle represents the M-cell lateral dendrite. The length of each line is proportional to the coupling coefficient of that CE. For calibration, the circle represents a coupling potential of 0.020. D, Histogram showing the periphery/center index distribution of the CaMKII distribution at single terminals (n = 43). E, Histogram showing the amplitude distribution of the unitary electrical EPSPs (n = 92).
Discussion
“These endings are large enough to be useful for the elucidation of many details and also for the experimental studies on which we are engaged at present.”
As pointed out by Bartelmez and Hoerr 76 years ago (Bartelmez and Hoerr, 1933), the unusually large size of the CEs makes them a valuable model to test specific questions regarding vertebrate synaptic transmission. The present analysis was only possible at these terminals, which offered an unparalleled access for exploring the distribution of CaMKII within a single contact and the possibility to correlate it with well established pharmacological and physiological properties. CEs have become recently a model for the study of interactions between chemical and electrical synapses (Pereda et al., 1998, 2004; Smith and Pereda, 2003). We showed previously that, within these mixed contacts, the activity of glutamatergic synapses not only leads to their own potentiation but also of nearby electrical synapses (Smith and Pereda, 2003). Our results indicate that a likely major molecular candidate for this interaction is the enzyme CaMKII. Furthermore, our analysis of the intraterminal distribution of this kinase and its association to Cx35 indicates that CaMKII should be considered not only a major component of glutamatergic PSDs but also of gap junction-mediated electrical synapses.
Functional significance of CaMKII variability
A characteristic property of CaMKII is its ability to translocate to active synaptic sites (Shen and Meyer, 1999; Gleason et al., 2003; Merrill et al., 2005; Rose et al., 2009). It has been proposed that the level of CaMKII associated with glutamatergic PSDs at any given time may be a combination of (1) transiently associated kinase and (2) a population of this kinase that is retained after translocation and that might encode the history of correlated neuronal activity (Schulman, 2004). Translocation to contiguous synapses has been shown to occur (Merrill et al., 2005; Rose et al., 2009), including to GABAergic synapses (K. C. Madsen and R. C. Caroll, personal communication), suggesting that gap junctions can also be the target of translocation/redistribution of CaMKII from nearby glutamatergic synapses. In contrast to glutamatergic synapses, in which accumulation of this kinase at PSDs can be difficult sometimes to detect (Dosemeci et al., 2001; Merrill et al., 2005), the large number and intraterminal distribution of gap junctions at CEs (predominant at the center of the contact) made it easier to detect their association with CaMKII. Based on the association to Cx35 that we report here (see below), the distribution of CaMKII might similarly reflect the history of synaptic activation of individual CEs. That is, the distribution of CaMKII labeling within the population of CEs is consistent with the variability in coupling between adjacent contacts, shown previously to result from terminal-specific activity-dependent modifications initiated by glutamatergic synapses at each of these terminals (Smith and Pereda, 2003), and they both have comparable distributions. Although this conclusion will require additional confirmation, there is experimental evidence that supports this possibility: (1) activated CaMKII leads to enhancement of electrical and chemical transmission of CEs (Pereda et al., 1998), (2) the activity of CaMKII is required for activity-dependent potentiation of CEs (Pereda et al., 1998), and finally, (3) changes in glutamatergic transmission parallel changes in electrical coupling in CEs, and, accordingly, the amount of CaMKII at individual spines of hippocampal CA1 neurons was shown to be positively correlated with the strength of its glutamatergic synapses (Asrican et al., 2007).
Furthermore, our in silico analysis and biochemical studies, in combination with our previous data (Pereda et al., 1998), suggest that association of CaMKII with Cx35 leads to its activation, ultimately resulting in potentiation of electrical transmission in CEs. According to current models (Bayer et al., 2001; Schulman, 2004), the binding of CaMKII to Cx36 (Alev et al., 2008) and likely to Cx35 (based on the sequence homology and co-IP we report here), will lock this kinase in a persistently active state, even after dissociation of calmodulin. More specifically, the αCaMKII autoinhibitory domain is considered a gate consisting of a pseudosubstrate segment that blocks binding of substrates to the “S site”, as well as a pseudotarget segment that blocks access to its target proteins (“T site”) (Bayer et al., 2001; Hudmon and Schulman, 2002; Schulman, 2004). Binding of Ca2+/calmodulin opens the gate and allows substrates to bind (kinase activation), thereby enabling interactions with some targets, such as to the NR2B subunit of the NMDA receptor (Bayer et al., 2001), Drosophila Eag (ether-a-go-go) potassium channel (Sun et al., 2004), and Cx36 (Alev et al., 2008), all of which contain similar “pseudosubstrate” and “pseudotarget” binding regions. This mechanism would be equivalent to the classic effect of autophosphorylation of Thr286, positioned like a “wedge” in the inhibitory gate, further displacing it (Singla et al., 2001; Schulman, 2004) and enabling interactions with additional targeting proteins (Bayer et al., 2001; Schulman, 2004). As mentioned above, our previous work showed that intradendritic injections of a constitutively active form of αCaMKII in the M-cell led to a persistent potentiation of the electrical component of the mixed synaptic potential evoked by stimulation of CEs, and CaMKII activation was found to be required for the induction of activity-dependent potentiation of this synaptic potential (Pereda et al., 1998). Together, these evidence suggest that the activity of CaMKII indeed leads to potentiation of electrical transmission in CEs, and differences in the spatial distribution of this kinase could thus represent differences in the functional state of electrical synapses at these terminals, in which the amount and localization of CaMKII to gap junctions would be indicative of the degree of potentiation.
Our current experimental approaches made impractical to detect changes in the distribution of CaMKII after activity-dependent protocols. This question could be more easily addressed in future experiments using zebrafish embryos, in which it is possible to track the translocation and/or redistribution of fluorescently tagged kinase to synaptic sites (Gleason et al., 2003). Nonetheless, these results provide, for the first time, evidence of the distribution to and association with CaMKII to identifiable electrical synapses in vivo, in which it is possible to correlate the presence of this kinase with its known functional properties.
CaMKII regulatory mechanisms are conserved
The in silico analysis revealed the existence of multiple phosphorylation sites for αCaMKII in Cx35. These predictions were consistent with those reported recently for Cx36, (Alev et al., 2008), which were confirmed by biochemical approaches, providing a high degree of confidence on this analysis. By extending our analysis to an additional fish homolog of Cx36, Cx34.7, we found that Cx36, Cx35, and Cx34.7 shared most of the identified residues. The analysis also showed that residues S315/S298/S300 in Cx36, Cx35, and Cx34.7, respectively, constitute exclusive phosphorylation sites for CaMKII that are not shared with other kinases. Conversely, residues S110 and S293 in Cx36, S110 and S276 in Cx35, and S110 and S277 in Cx34.7 are phosphorylation sites shared with cAMP-dependent protein kinase A (O'Brien et al., 1998; Mitropoulou and Bruzzone, 2003; Ouyang et al., 2005; Urschel et al., 2006), a kinase also shown to promote enhancement of electrical transmission at these terminals (Pereda et al., 1994; Cachope et al., 2007). Interestingly, our IP experiments (restricted to a small portion of the hindbrain that mainly contains the M-cells) showed multiple bands for Cx35 pulled down by CaMKII. Although final proof would require dephosphorylation of samples with protein phosphatase, this observation is consistent with the possibility that Cx35 coexists at multiple states of phosphorylation. We did not investigate this possibility further, which will be the focus of future research efforts. In summary, our sequence alignment shows that CaMKII binding regions and phosphorylation sites are highly similar between Cx36 and its fish homologs, indicating that essential regulatory mechanisms are highly conserved and likely to pertain to all Cx35- and Cx36-mediated electrical synapses. These mechanisms could include, as shown for glutamate receptors, direct phosphorylation of the channels themselves (Derkach et al., 1999) or the promotion of their insertion in the plasma membrane (Shi et al., 1999, 2001; Liao et al., 2001).
CaMKII is a nonobligatory component of electrical synapses
In contrast to the extensive information on proteins associated with chemical synapses (Kim and Sheng, 2004) involved in channel insertion, anchoring, or removal from the plasma membrane (Migaud et al., 1998; Mori et al., 1998; Sprengel et al., 1998; Shi et al., 1999; Malinow et al., 2000; Steigerwald et al., 2000; Lin et al., 2004), very few proteins have been shown to associate with the neuronal gap junctions. It is currently accepted that gap junctions not only comprise the defining intercellular channel proteins but also associated scaffold and regulatory proteins (Hervé et al., 2004). Neuronal gap junctions in particular characteristically exhibit a PSD-like structure in the electron microscopy sections, described as a “semi-dense cytoplasmatic matrix” (Sotelo and Korn, 1978) and that likely represents (similar to glutamatergic PSDs) proteins associated with these intercellular channels. Cx36 (Condorelli et al., 1998), the most prevalent neuronal connexin that is widely distributed throughout the mammalian brain (Condorelli et al., 2000), was shown to interact with two relevant proteins: CaMKII (Alev et al., 2008) and ZO-1 (Li et al., 2004a, b). ZO-1 is a member of the membrane-associated guanylate kinase family of proteins that was reported to interact with several other connexins (Itoh et al., 1999). Our recent work indicates that ZO-1 also interacts with Cx35 in CEs (Flores et al., 2008). Because its colocalization with Cx35 is extensive (Flores et al., 2008), ZO-1 is likely to play a structural role, and the properties of the ZO-1/Cx35 association suggest the existence of a dynamic relationship between these two proteins, possibly including a role of ZO-1 in regulating gap junctional conductance at these highly modifiable electrical synapses (Flores et al., 2008). Our present results show that CaMKII also associates with Cx35 at CEs. This study extends the observations of Alev et al. (2008), showing that CaMKII associates with neuronal connexins in anatomically identifiable, native, electrical synapses. More importantly, our data also indicate that CaMKII is likely a nonobligatory component of electrical synapses. That is, its presence is highly variable, even between gap junction plaques within the same contact, and likely is linked to differences in the degree of potentiation between individual terminals (see above). Compared with ZO-1, which would play a more permanent or structural role, the presence of CaMKII seems to be nonobligatory and possibly regulated by neural activity (Fig. 8). This conclusion is consistent with the dynamic translocation properties of CaMKII and whose presence is thought to represent the history of synaptic activation. Thus, our results indicate that ZO-1 and CaMKII probably belong to the same macromolecular complex, and, together with other identified proteins [association with zona occludens proteins ZO-2 (Ciolofan et al., 2006) and ZO-3 (Li et al., 2009) and calmodulin (Burr et al., 2005) were also reported] as well as those that might be identified in the future, are likely components of the semi-dense cytoplasmatic matrix associated with intercellular channels in neuronal gap junctions.
Potential mechanism for differences in CaMKII labeling between contiguous Club endings. Electrical synapses at adjacent CEs coexist at different degrees of conductance (indicated by different colors). The extensive colocalization of Cx35 with ZO-1 suggests that this scaffold protein could constitute a structural component of gap junctions at these terminals. Activity of neighboring chemically transmitting regions within the terminal trigger changes in junctional conductance, via a PSD-mediated mechanism (arrows) (Pereda and Faber, 1996; Pereda et al., 1998) promoting the association of CaMKII to Cx35 and ZO-1. The association of CaMKII to electrical synapses would be thus nonobligatory and driven by synaptic activity. For convenience, a simplified gap junction is illustrated; the diagram does not indicate whether the association is exclusively presynaptic or postsynaptic.
In summary, CaMKII is not only a major component of glutamatergic postsynaptic densities but is also an important and dynamic component of gap junction-mediated electrical synapses in CEs. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses, our data suggest that the properties observed at these identifiable gap junctions might also apply to electrical synapses elsewhere, including those in mammalian brain.
Footnotes
This research was supported by National Institutes of Health Grants DC03186, NS0552827 (A.E.P.), and DA10044 (A.C.N.). We thank Carl Castillo for help with immunochemistry and Joseph Zavilovitz for help with the confocal microscope during initial experiments. We also thank Costa Dobrenis and the Morphology Core of the Rose F. Kennedy Center for outstanding support. Finally, we thank David Spray, Rolf Dermietzel, and Theresa Szabo for useful discussions and comments on this manuscript.
- Correspondence should be addressed to Alberto E. Pereda, Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. alberto.pereda{at}einstein.yu.edu
