Abstract
Ca(2+)/calmodulin (Ca(2+)/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca(2+)/CaM is not fully understood. Ca(2+)/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(2+)/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(2+)/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(2+)/CaM with dissociation constants (K(d(+Ca))) 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(2+)-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(2+)-dependent association at resting [Ca(2+)] of 50-100 nM is indicated in these complexes as one of the CaM Ca(2+) binding sites displays high affinity with K(d) of 70 and 30 nM for Ca(2+), 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(2+)/CaM in a dose dependent manner, further cementing Ca(2+)/CaM as a regulator of gap junction function. The motion of a stretched CaM-CL2 complex compacting upon Ca(2+) binding may bring about the Ca(2+)/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.