Effect of mutation of a calmodulin binding site on Ca2+ regulation of inositol trisphosphate receptors.
Several studies have shown that calmodulin (CaM) modulates d-myo-inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) channel activity and ligand binding to IP(3)Rs. It has been proposed that CaM may act as the Ca(2+) sensor for mediating Ca(2+) inhibition of IP(3)R channel activity. However, the functional role of CaM binding sites and the mechanism by which CaM regulates IP(3)R activities remains unclear. Tryptophan at position 1577 of type I IP(3)R has been shown to be part of a motif that is responsible for CaM binding to IP(3)Rs and we have mutated this residue to alanine in the long (neuronal) and short (peripheral) SII splice variants of the type I IP(3)R. CaM-Sepharose binding assays using COS-7 cell lysates confirmed that the W1577A mutant in both splice variants completely eliminated CaM binding. Functional measurements of IP(3)-mediated (45)Ca(2+) fluxes indicated that there was no change in the IP(3) sensitivity of the channel induced by the W1577A mutation. Such measurements also indicated that the W1577A mutants of both splice variants have a dependence on external [Ca(2+)] that was indistinguishable from the corresponding wild-types. Although subtle differences in the Ca(2+) and CaM sensitivity of [(3)H]IP(3) binding were noted between wild-type and mutant receptors, our data suggest that the CaM binding motif involving the W1577A locus does not play a role in Ca(2+) regulation of IP(3)R channel activity.
Zhang
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Department of Pathology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Joseph
S K
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D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D015220:Calcium Channels / Q000235:genetics* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008861:Microsomes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014364:Tryptophan / 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; D015544:Inositol 1,4,5-Trisphosphate; D014364:Tryptophan; D000409:Alanine; D002118:Calcium