Regulation of Intercellular Regulation of Signaling via Gap Junction

Abstract

Gap junction, allowing the intercellular transmission of molecules through its specialized cell membrane channels, plays a major role in intercellular calcium signaling between adjacent cells. Connexin 43, 44 and 50 (Cx43, Cx44 and Cx50) are members of the α family of gap junction proteins expressed in heart and the lens of the eye where are essential for normal heart and lens development. We have identified calmodulin (CaM) binding sites in Cx43, Cx44 and Cx50. Biophysical results indicate that in the presence of calcium, synthesized Cx peptide fragments encompassing predicted CaM binding regions are able to bind with high affinity to CaM using NMR spectroscopy and fluorescence method with dansylated CaM. In addition, electrophysiological studies demonstrate that elevated intracellular Ca2+ concentration inhibits Cx50 gap junctions with the 95% decline in junctional conductance (gj). Inclusion of either CaM inhibitor or the designed CaM binding Cx peptide is able to prevent this calcium-dependent inhibition of Cx50 gap junctions. Further, we have predicted several calcium binding pockets using our developed MUG algorithm. We have further revealed the site-specific calcium binding capability of the predicted calcium binding site in gap junctions by grafting approach. Our results elucidate the molecular mechanism of regulation of gap junction by both calcium and calcium dependent CaM actions and provide insight into molecular basis of human diseases.