Identification of the calmodulin binding domain of connexin 43.
Calmodulin (CaM) has been implicated in mediating the Ca(2+)-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136-158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca(2+)-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its alpha-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K(+) is in the range of 0.7-1 microM. Upon binding of the peptide to CaM, the apparent K(d) of Ca(2+) for CaM decreased from 2.9 +/- 0.1 to 1.6 +/- 0.1 microM, and the Hill coefficient n(H) increased from 2.1 +/- 0.1 to 3.3 +/- 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca(2+)-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136-158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca(2+)-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca(2+)-dependent manner, providing a molecular basis for the well characterized Ca(2+)-dependent inhibition of Cx43-containing gap junctions.
Zhou
Yubin
Y
Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, USA.
Yang
Wei
W
Lurtz
Monica M
MM
Ye
Yiming
Y
Huang
Yun
Y
Lee
Hsiau-Wei
HW
Chen
Yanyi
Y
Louis
Charles F
CF
Yang
Jenny J
JJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry*; D018031:Connexin 43 / Q000737:chemistry*; D017629:Gap Junctions / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011188:Potassium / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D020349:Surface Plasmon Resonance
D002147:Calmodulin; D018031:Connexin 43; D011188:Potassium