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 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.
Kasri
Nael Nadif
NN
Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, O/N Herestraat 49/802, B-3000 Leuven, Belgium.
Török
Katalin
K
Galione
Antony
A
Garnham
Clive
C
Callewaert
Geert
G
Missiaen
Ludwig
L
Parys
Jan B
JB
De Smedt
Humbert
H
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
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