Molecular interaction and functional regulation of connexin50 gap junctions by calmodulin

Abstract

Cx50 (connexin50), a member of the α-family of gap junction proteins expressed in the lens of the eye, has been shown to be essential for normal lens development. In the present study, we identified a CaMBD [CaM (calmodulin)-binding domain] (residues 141–166) in the intracellular loop of Cx50. Elevations in intracellular Ca2+ concentration effected a 95 % decline in gj (junctional conductance) of Cx50 in N2a cells that is likely to be mediated by CaM, because inclusion of the CaM inhibitor calmidazolium prevented this Ca2+-dependent decrease in gj. The direct involvement of the Cx50 CaMBD in this Ca2+/CaM-dependent regulation was demonstrated further by the inclusion of a synthetic peptide encompassing the CaMBD in both whole-cell patch pipettes, which effectively prevented the intracellular Ca2+-dependent decline in gj. Biophysical studies using NMR and fluorescence spectroscopy reveal further that the peptide stoichiometrically binds to Ca2+/CaM with an affinity of ~ 5 nM. The binding of the peptide expanded the Ca2+-sensing range of CaM by increasing the Ca2+ affinity of the C-lobe of CaM, while decreasing the Ca2+ affinity of the N-lobe of CaM. Overall, these results demonstrate that the binding of Ca2+/CaM to the intracellular loop of Cx50 is critical for mediating the Ca2+-dependent inhibition of Cx50 gap junctions in the lens of the eye.

Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes

“…Moreover, injection of the proteo-liposome further showed that the Cx26 channel can be regulated by intracellular Ca 2+ [42], a result consistent with a previous findings [43]. The channel activities we reported are a net of outward minus inward activities.…”

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

“…This mutant can even interact with CaM without Ca 2+ -activation of CaM possibly due to an increase in overall positive charges of Cx46 IL domain. This result also agrees with the study by Chen et al (2011) (29), which shows that several positively charged residues on Cx50 are important for Cx50-CaM interaction.…”

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

“…CaM has been implicated in mediating the Ca 2ϩ -dependent regulation of gap junctions via interacting directly with the intracellular domains of connexins (13,18,19 (Fig. 5A).…”

“…CaM is a Ca 2ϩ -binding protein that acts by transducing the effect of a rise in intracellular Ca 2ϩ to create physiological responses; specifically it has been implicated in mediating the Ca 2ϩ -dependent down-regulation of Cx32 gap junction channels (18,19). Cx32 has been shown to interact with CaM directly via the Cx32CT domain in a Ca 2ϩ -dependent manner (13).…”