CA2-AFFINITY STATES OF TYPE-1 AND TYPE-3 INOSITOL 1,4,5-TRISPHOSPHATERECEPTORS( DIFFERENTIALLY REGULATES THE LIGAND)

To elucidate the functional difference between type 1 and type 3 Ins(1 ,4,5)P-3 receptors [Ins(1,4,5)P(3)R1 and Ins(1,4,5)P(3)R3 respectively ] we studied the effect of Ca2+ on the ligand-binding properties of bo th Ins(1,4,5)P(3)R types. We expressed full-length human Ins(1,4,5)P(3 )R1 and Ins(1,4,5)P(3)R3 from cDNA species in insect ovary Sf9 cells, and the membrane fractions were used for Ins(1,4,5)P-3-binding assays. The binding of Ins(1,4,5)P-3 to Ins(1,4,5)P(3)R1 and Ins(1,4,5)P(3)R3 was differentially regulated by Ca2+. With increasing concentrations of free Ca2+ ([Ca2+]), Ins(1,4,5)P-3 binding to Ins(1,4,5)P(3)R1 decre ased, whereas that to Ins(1,4,5)P(3)R3 increased. Alteration of Ins(1, 4,5)P-3 binding to Ins(1,4,5)P(3)R1 was observed at [Ca2+] ranging fro m less than 1 nM to more than 10 mu M. The EC(50) of Ins(1,4,5)P-3 bin ding was 100 nM Ca2+ for Ins(1,4,5)P(3)R1. In contrast, Ins(1,4,5)P-3 binding to Ins(1,4,5)P(3)R3 was changed at high [Ca2+] with an EC(50) value of 872 nM, and steeply between 100 nM and 10 rho M. These Ca2+ d ependent alterations of Ins(1,4,5)P-3 binding to both Ins(1,4,5)P(3)R types were reversible. Scatchard analyses revealed that Ca2+ changed t he affinity of both Ins(1,4,5)P(3)R types but not the total number of Ins(1,4,5)P-3-binding sites. The K-d values of Ins(1,4,5)P(3)R1 for In s(1,4,5)P-3 were 78.5 nM with 3 nM free Ca2+, and 312 nM with 1.4 mu M free Ca2+. In contrast, Ins(1,4,5)P(3)R3 exhibited an affinity for In s(1,4,5)P-3 with K-d values of 116 nM with 3 nM free Ca2+, and 62.2 nM with 1.4 mu M free Ca2+. These results indicate that (1) both Ins(1,4 ,5)P(3)R1 and Ins(1,4,5)P(3)R3 have at least two affinity states, (2) Ca2+ regulates interconversions between these states, and (3) Ca2+ reg ulates the binding of Ins(1,4,5)P-3 to Ins(1,4,5)P(3)R1 and Ins(1,4,5) P(3)R3 in opposite manners.