Ca2+ differentially regulates the ligand-affinity states of type 1 and type 3 inositol 1,4,5-trisphosphate receptors.
To elucidate the functional difference between type 1 and type 3 Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 respectively] we studied the effect of Ca2+ on the ligand-binding properties of both Ins(1,4,5)P3R types. We expressed full-length human Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 from cDNA species in insect ovary Sf9 cells, and the membrane fractions were used for Ins(1,4,5)P3-binding assays. The binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 was differentially regulated by Ca2+. With increasing concentrations of free Ca2+ ([Ca2+]), Ins(1,4,5)P3 binding to Ins(1,4,5)P2R1 decreased, whereas that to Ins(1,4,5)P3R3 increased. Alteration of Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 was observed at [Ca2+] ranging from less than 1 nM to more than 10 microM. The EC50 of Ins(1,4,5)P3 binding was 100 nM Ca2+ for Ins(1,4,5)P3R1. In contrast, Ins(1,4,5)P3 binding to Ins(1,4,5)P3R3 was changed at high [Ca2+] with an EC50 value of 872 nM, and steeply between 100 nM and 10 microM. These Ca2+-dependent alterations of Ins(1,4,5)P3 binding to both Ins(1,4,5)P3R types were reversible. Scatchard analyses revealed that Ca2+ changed the affinity of both Ins(1,4,5)P3R types but not the total number of Ins(1,4,5)P3-binding sites. The Kd values of Ins(1,4,5)P3R1 for Ins(1,4,5)P3 were 78.5 nM with 3 nM free Ca2+, and 312 nM with 1.4 microM free Ca2+. In contrast, Ins(1,4,5)P3R3 exhibited an affinity for Ins(1,4,5)P3 with Kd values of 116 nM with 3 nM free Ca2+, and 62.2 nM with 1.4 microM free Ca2+. These results indicate that (1) both Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 have at least two affinity states, (2) Ca2+ regulates interconversions between these states, and (3) Ca2+ regulates the binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 in opposite manners.
Yoneshima
H
H
Department of Molecular Neurobiology, University of Tokyo, Japan.
Miyawaki
A
A
Michikawa
T
T
Furuichi
T
T
Mikoshiba
K
K
D002118:Calcium / Q000494:pharmacology*; D015220:Calcium Channels / Q000096:biosynthesis / Q000145:classification / Q000187:drug effects*; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000145:classification / Q000187:drug effects*; D011994:Recombinant Proteins / Q000187:drug effects
D015220:Calcium Channels; D002614:Chelating Agents; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium