title: Endogenously bound calmodulin is essential for the function of the inositol 1,4,5-trisphosphate receptor.
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.
authors.affiliation: Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, O/N Herestraat 49/802, B-3000 Leuven, Belgium.
authors.forename: Nael Nadif
authors.initials: NN
authors.lastname: Kasri
authors.forename: Katalin
authors.initials: K
authors.lastname: Török
authors.forename: Antony
authors.initials: A
authors.lastname: Galione
authors.forename: Clive
authors.initials: C
authors.lastname: Garnham
authors.forename: Geert
authors.initials: G
authors.lastname: Callewaert
authors.forename: Ludwig
authors.initials: L
authors.lastname: Missiaen
authors.forename: Jan B
authors.initials: JB
authors.lastname: Parys
authors.forename: Humbert
authors.initials: H
authors.lastname: De Smedt
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism* / Q000502:physiology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008168:Lung / Q000166:cytology; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012617:Sea Urchins / Q000166:cytology; D018411:Spodoptera / Q000166:cytology / Q000235:genetics
chemical_list: D015220:Calcium Channels; D002147:Calmodulin; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D009219:Myosin-Light-Chain Kinase; D002118:Calcium