Connexin 32 of gap junctions contains two cytoplasmic calmodulin-binding domains.
A fluorescent calmodulin derivative, 2-chloro-[4-(epsilon-amino-Lys75)]-[6-(4- diethylaminophenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-calmodulin) [Török and Trentham (1994) Biochemistry 33, 12807-12820], and equilibrium fluorescence methods were used to identify calmodulin-binding domains of connexin subunits of gap junctions. Synthetic peptides corresponding to six extramembrane regions of connexin 32, a major component of rat liver gap junctions, and peptides derived from connexin 43 and 26, were tested. Two cytoplasmically oriented peptides that correspond to an N-terminal 21-amino-acid sequence and a 15-amino-acid sequence at the C-terminal tail of connexin 32 bound TA-calmodulin in a Ca2+-dependent manner. The dissociation constants (Kd) of TA-calmodulin binding to GAP 10 (MNWTGLYTLLSGVNRHSTAIG, residues 1-21) and GAP 8M (ACARRAQRRSNPPSR, residues 216-230) were 27 nM and 1.2 microM respectively at 150 mM ionic strength, 2 mM MgCl2, 100 microM CaCl2, pH 7.0 and 21 degrees C. Both halves of each peptide were required for calmodulin binding. Substitution of Trp3 present in all connexins by Tyr increased Kd for TA-calmodulin by 40-fold. Liver gap junctions (whose connexons contain mainly connexin 32) and recombinant connexons constructed of connexin 26 expressed by baculovirus-infected insect cells exhibited weaker binding of TA-calmodulin with variable Ca2+-dependence. These studies identify two calmodulin-binding amino-acid sequences in connexin 32, and provide independent evidence that calmodulin may function as an intracellular ligand, regulating Ca2+-dependent intercellular communication across gap junctions.
Török
K
K
Department of Physiological Sciences, University of Newcastle upon Tyne Medical School, U.K.
Stauffer
K
K
Evans
W H
WH
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000031:analogs & derivatives / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017630:Connexins / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D017629:Gap Junctions / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D000097002:Gap Junction beta-1 Protein
D002118:Calcium; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D017630:Connexins; D010446:Peptide Fragments; D000097002:Gap Junction beta-1 Protein; C090205:calmodulin, 2-chloro-(epsilon-amino-lysine(75))-(6-(4-(N,N-diethylamino)phenyl)-1,3,5-triazin-4-yl)-