Endogenously bound calmodulin is essential for the function of the inositol 1,4,5-trisphosphate receptor

Calmodulin ( CaM) is a ubiquitous Ca2+ sensor protein that plays an important role in regulating a large number of Ca2+ channels, including the inositol 1,4,5- trisphosphate receptor ( IP3R). Despite many efforts, the exact mechanism by which CaM regulates the IP3R still remains elusive. Here we show, using unidirectional Ca-45(2+) flux experiments on permeabilized L15 fibroblasts and COS- 1 cells, that endogenously bound CaM is essential for the proper activation of the IP3R. Removing endogenously bound CaM by titration with a high affinity ( pM) CaM- binding peptide derived from smooth muscle myosin light- chain kinase ( MLCK peptide) strongly inhibited IP3- induced Ca2+ release. This inhibition was concentration- and time- dependent. Removing endogenously bound CaM affected the maximum release capacity but not its sensitivity to IP3. A mutant peptide with a strongly reduced affinity for CaM did not affect inhibited IP3- induced Ca2+ release. Furthermore, the inhibition by the MLCK peptide was fully reversible. Re- adding exogenous CaM, but not CaM1234, reactivated the IP3R. These data suggest that, by using a specific CaM- binding peptide, we removed endogenously bound CaM from a high affinity CaM- binding site on the IP3R, and this resulted in a complete loss of the IP3R activity. Our data support a new model whereby CaM is constitutively associated with the IP3R and functions as an essential subunit for proper functioning of the IP3R.