Calmodulin Colocalizes with Connexins and Plays a Direct Role in Gap Junction Channel Gating

Abstract

The direct calmodulin (CaM) role in chemical gating was tested with CaM mutants, expressed in oocytes, and CaM-connexin labeling methods. CaMCC, a CaM mutant with greater Ca-sensitivity obtained by replacing the N-terminal EF hand pair with a duplication of the C-terminal pair, drastically increased the chemical gating sensitivity of Cx32 channels and decreased their Vj sensitivity. This only occurred when CaMCC was expressed before Cx32, suggesting that CaMCC, and by extension CaM, interacts with Cx32 before junction formation. Direct CaM-Cx interaction at junctional and cytoplasmic spots was demonstrated by confocal immunofluorescence microscopy in HeLa cells transfected with Cx32 and in cryosectioned mouse liver. This was confirmed in HeLa cells coexpressing Cx32-GFP (green) and CaM-RFP (red) or Cx32-CFP (cyan) and CaM-YFP (yellow) fusion proteins. Significantly, these cells did not form gap junctions. In contrast, HeLa cells expressing only one of the two fusion proteins (Cx32-GFP, Cx32-CFP, CaM-RFP or CaM-YFP) revealed both junctional and non-junctional fluorescent spots. In these cells, CaM-Cx32 colocalization was demonstrated by secondary immunofluorescent labeling of Cx32 in cells expressing CaM-YFP or CaM in cells expressing Cx32-GFP. CaM-Cx colocalization was further demonstrated at rat liver gap junctions by Freeze-fracture Replica Immunogold Labeling (FRIL).

Characterization of the Structure and Intermolecular Interactions between the Connexin 32 Carboxyl-terminal Domain and the Protein Partners Synapse-associated Protein 97 and Calmodulin

“…This suggests that alterations in the phosphorylation state of the Cx32CT could also play a role in regulating myelin homeostasis. Consistent with this possibility are studies showing that tyrosine phosphorylation of Cx32 is inhibited in the presence of CaM and CaM binding can be disrupted when its targets are phosphorylated (65,67,69). Therefore, any increased tyrosine kinase activity leading to Cx32CT phosphorylation could also decrease CaM binding leading to the same CMTX phenotype as the Cx32CT mutations.…”

Complex formation between calmodulin and a peptide from the intracellular loop of the gap junction protein connexin43: Molecular conformation and energetics of binding

“…Cx43 has been shown to interact with CaM, and a specific binding site was recently [12] localised to the cytoplasmic loop, between residues 136-158. Similar sites have been characterized or suggested to be present in other connexins [13][14][15][16][17], indicating a putative general mechanism of gap junction regulation by CaM. Currently, no highresolution data on the CaM-cx43 complex exist, and the sequence homology between cx43 and structurally characterized CaM target proteins is low.…”

Cataract-associated connexin 46 mutation alters its interaction with calmodulin and function of hemichannels

“…There are two possible models with regards to how Ca 2+ -CaM and connexin interaction regulates connexin channel function; one is that the interaction with CaM directly regulates Cx32 gap junction channel gating (11); and the other suggests that Ca 2+ -CaM-connexin complex formation may act on other signal pathways, such as PKC or IP3 signaling mechanisms (30,31). Based on our studies with G143R mutant, our data support the first model implicating a direct regulation of connexin channels by CaM.…”

“…CaM undergoes a large conformational change after binding to Ca 2+ , which exposes hydrophobic patches, allowing binding to target proteins and initiating diverse downstream biological reactions. Connexins are also shown to associate with Ca 2+ /CaM as a part of gap junction complex (11). Elevated intracellular Ca 2+ concentration is long known to inhibit gap junction function in cardiac cells (12).…”