Author:

Keywords:

Animals, Binding Sites, COS Cells, Calcium, Calcium Channels, Calmodulin, Cell Line, Cercopithecus aethiops, Comparative Study, Dose-Response Relationship, Drug, Female, Fibroblasts, Humans, Kinetics, Lung, Myosin-Light-Chain Kinase, Oocytes, Protein Binding, Protein Isoforms, Receptors, Cytoplasmic and Nuclear, Research Support, Non-U.S. Gov't, Sea Urchins, Spodoptera, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, SPONTANEOUS CA2+ SPARKS, FROG SKELETAL-MUSCLE, BINDING DOMAIN, TRISPHOSPHATE RECEPTORS, CALCIUM-RELEASE, CA2+-INDEPENDENT INHIBITION, ENDOPLASMIC-RETICULUM, LIGAND-BINDING, CHANNELS, CELLS, Chlorocebus aethiops, Inositol 1,4,5-Trisphosphate Receptors, 03 Chemical Sciences, 06 Biological Sciences, 11 Medical and Health Sciences, 31 Biological sciences, 32 Biomedical and clinical sciences, 34 Chemical sciences

Abstract:

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 45Ca2+ 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.