title: 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.
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Yanyi
authors.initials: Y
authors.lastname: Chen
authors.forename: Yubin
authors.initials: Y
authors.lastname: Zhou
authors.forename: Xianming
authors.initials: X
authors.lastname: Lin
authors.forename: Hing-Cheung
authors.initials: HC
authors.lastname: Wong
authors.forename: Qin
authors.initials: Q
authors.lastname: Xu
authors.forename: Jie
authors.initials: J
authors.lastname: Jiang
authors.forename: Siming
authors.initials: S
authors.lastname: Wang
authors.forename: Monica M
authors.initials: MM
authors.lastname: Lurtz
authors.forename: Charles F
authors.initials: CF
authors.lastname: Louis
authors.forename: Richard D
authors.initials: RD
authors.lastname: Veenstra
authors.forename: Jenny J
authors.initials: JJ
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D017630:Connexins / Q000737:chemistry / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism*; D017629:Gap Junctions / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D011487:Protein Conformation; D000099264:Connexin 50
chemical_list: D002118:Calcium; D002147:Calmodulin; D017630:Connexins; D005136:Eye Proteins; D000099264:Connexin 50