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 g_j (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 g_j. 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 g_j. 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.