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Calmodulin mediates the Ca2+-dependent regulation of Cx44 gap junctions.

ABSTRACT: We have shown previously that the Ca2+-dependent inhibition of lens epithelial cell-to-cell communication is mediated in part by the direct association of calmodulin (CaM) with connexin43 (Cx43), the major connexin in these cells. We now show that elevation of [Ca2+](i) in HeLa cells transfected with the lens fiber cell gap junction protein sheep Cx44 also results in the inhibition of cell-to-cell dye transfer. A peptide comprising the putative CaM binding domain (aa 129-150) of the intracellular loop region of this connexin exhibited a high affinity, stoichiometric interaction with Ca2+-CaM. NMR studies indicate that the binding of Cx44 peptide to CaM reflects a classical embracing mode of interaction. The interaction is an exothermic event that is both enthalpically and entropically driven in which electrostatic interactions play an important role. The binding of the Cx44 peptide to CaM increases the CaM intradomain cooperativity and enhances the Ca2+-binding affinities of the C-domain of CaM more than twofold by slowing the rate of Ca2+ release from the complex. Our data suggest a common mechanism by which the Ca2+-dependent inhibition of the alpha-class of gap junction proteins is mediated by the direct association of an intracellular loop region of these proteins with Ca2+-CaM.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC2711270 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Calmodulin mediates the Ca2+-dependent regulation of Cx44 gap junctions.

Zhou Yubin Y Yang Wei W Lurtz Monica M MM Chen Yanyi Y Jiang Jie J Huang Yun Y Louis Charles F CF Yang Jenny J JJ

Biophysical journal 20090401 7

We have shown previously that the Ca2+-dependent inhibition of lens epithelial cell-to-cell communication is mediated in part by the direct association of calmodulin (CaM) with connexin43 (Cx43), the major connexin in these cells. We now show that elevation of [Ca2+](i) in HeLa cells transfected with the lens fiber cell gap junction protein sheep Cx44 also results in the inhibition of cell-to-cell dye transfer. A peptide comprising the putative CaM binding domain (aa 129-150) of the intracellula ...[more]

PMID: 19348766

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Project description:Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation at chemical synapses, has recently been described to potentiate electrical coupling in the retina and several other brain areas via phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown. We recently identified CaMKII-? at electrical synapses in the mouse retina. Now, we set out to identify cell types containing Cx36 gap junctions that also express CaMKII-?. To ensure precise description, we first tested the specificity of two commercially available antibodies on CaMKII-?-deficient retinas. We found that a polyclonal antibody was highly specific for CaMKII-?. However, a monoclonal antibody (CB-?-1) recognized CaMKII-? but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII-? expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII-? also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII-? influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII-?-deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII-? is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII-? with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII-? may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs.

Dataset Information

Calmodulin mediates the Ca2+-dependent regulation of Cx44 gap junctions.

Publications

Calmodulin mediates the Ca2+-dependent regulation of Cx44 gap junctions.

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