CONNEXIN-32 OF GAP-JUNCTIONS CONTAINS 2 CYTOPLASMIC CALMODULIN-BINDING DOMAINS

A fluorescent calmodulin derivative, psilon-amino-Lys(75))]-[6-(4-diet hylaminophenyl)-1 ,3,5-triazin-4-yl]-calmodulin (TA-calmodulin) [Torok and Trentham (1994) Biochemistry 33, 12807-12820], and equilibrium fl uorescence 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, wer e 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 (K-d) of TA-calmodulin binding to GAP 10 (MNWTGLYTLLSGVNRHSTAIG, residues 1-21) and GAP 8M (ACARRAQRRSNP PSR, residues 216-230) were 27 nM and 1.2 mu M respectively at 150 mM ionic strength, 2 mM MgCl2, 100 mu M CaCl2, pH 7.0 and 21 degrees C. B oth halves of each peptide were required for calmodulin binding. Subst itution of Trp(3) present in all connexins by Tyr increased K-d for TA -calmodulin by 40-fold. Liver gap junctions (whose connexons contain m ainly connexin 32) and recombinant connexons constructed of connexin 2 6 expressed by baculovirus-infected insect cells exhibited weaker bind ing of TA-calmodulin with variable Ca2+-dependence. These studies iden tify two calmodulin-binding amino-acid sequences in connexin 32, and p rovide independent evidence that calmodulin may function as an intrace llular ligand, regulating Ca2+-dependent intercellular communication a cross gap junctions.