Ca-Dependent and -Independent Calmodulin Binding to the Cytoplasmic Loop of Gap Junction Connexins.

Ca/calmodulin (Ca/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca/CaM is not fully understood. Ca/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca/CaM and apo-CaM binding to selected representatives of each of the α, β and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca/CaM and apo-CaM interactions of CL2 peptides of β-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca/CaM with dissociation constants () from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca-dependent association at resting [Ca] of 50-100 nM is indicated in these complexes as one of the CaM Ca binding sites displays high affinity with of 70 and 30 nM for Ca, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca/CaM in a dose dependent manner, further cementing Ca/CaM as a regulator of gap junction function. The motion of a stretched CaM-CL2 complex compacting upon Ca binding may bring about the Ca/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane.