A fluorescent calmodulin derivative, 2-chloro-[4-(ϵ-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 μM respectively at 150 mM ionic strength, 2 mM MgCl2, 100 μM CaCl2, pH 7.0 and 21 °C. Both halves of each peptide were required for calmodulin binding. Substitution of Trp3 present in all connexins by Tyr increasedKd 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.
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