Cam Interaction and Binding Mode Study with Peptide from Intracellular Loop of Cx50

Abstract

Connexin 50 (Cx50) is a member of the α family of gap junction proteins expressed in the lens of the eye where it has been shown to be essential for normal lens development. We have identified calmodulin (CaM) binding sites in the intracellular loop near to the 3rd transmembrane region of Connexin43 (Cx43) and Connexin44 (Cx44) which belong to the same α connexin family as Cx50. Sequence alignment of the candidate CaM binding regions of Cx43 and Cx44, with Cx50 identified a region encompassing residues 141-166 of Cx50 with a high predicted affinity for CaM. A peptide Cx50141-166 was synthesized to study the interaction of CaM with this domain of Cx50. Biophysical results indicate that in the presence of Ca2+, Cx50141-166 binds with high affinity (0.14 μM) to CaM, as monitored by NMR spectroscopy and measured by the fluorescence intensity change of IAEDANS that was covalently attached to the C-terminal Cys of CaM. Electrophysiological data support the hypothesis that elevated intracellular Ca2+ concentration inhibits Cx50 gap junctions because omission of Ca2+ from the 1 μM ionomycin bath saline prevented the 95% decline in junctional conductance (gj) observed in the presence of 1.8 mM CaCl2. This is likely CaM-mediated, because inclusion of a CaM inhibitor also prevented this Ca2+-dependent inhibition of Cx50 gap junctions. The involvement of the Cx50 CaM binding domain in this Ca2+/CaM-dependent regulation was further demonstrated by inclusion ofthe Cx50141-166 peptide in both whole cell patch pipettes, which effectively prevented the usual decrease in Cx50 gj. 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.