Modulation of inositol 1,4,5-trisphosphate binding to the various inositol 1,4,5-trisphosphate receptor isoforms by thimerosal and cyclic ADP-ribose

Three different genes encode the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), an intracellular Ca2+ channel involved in cellular Ca2+ signaling. The IP3-binding characteristics of the various IP3R isoforms differ, but until now no specific activators or inhibitors of IP3 binding have been described. We compared the effects of oxidizing reagents, in particular thimerosal, and of cyclic ADP-ribose (cADPR) on IP3 binding to the various IP3R isoforms. We therefore expressed the N-terminal 581 amino acids of the three IP3R isoforms as recombinant proteins in the soluble fraction of Escherichia coli (ligand-binding sites [Ibs] 1, 2, and 3) as well as the full-length IP(3)R1 and IP(3)R3 in Spodoptera frugiperda (Sf9) insect cells. Thimerosal (100 muM) stimulated IP3 binding to lbs-1 (1.4-fold) and lbs-3 (2.5-fold), but had no effect on lbs-2. Thimerosal acted on lbs-1 and lbs-3 by decreasing the K-d for IP3 binding (from 46 +/- 4 nM to 20 +/- 2 nM and from 54 +/- 21 nM to 19 +/- 7 nhl for lbs-1 and -3, respectively) without modifying the B-max. Similarly, IP3 binding to microsomes of Sf9 insect cells overexpressing the full-length IP(3)R1 was 1.2-fold stimulated by thimerosal. Thimerosal, however, did not affect IP3 binding to Sf9-IP(3)R3 microsomes, suggesting that in situ thimerosal will only directly affect ligand binding to the type 1 isoform. cADPR (50 muM) stimulated IP3 binding to Sf9-IP(3)R1 microsomes (1.5-fold), but not to Sf9-IP(3)R3 microsomes. In addition, cADPR inhibited IP3 binding to lbs-1 and lbs-2 by decreasing the affinity for IP3 1.8- and 2.8-fold, respectively, while IP3 binding to lbs-3 was not affected. These results suggest that a regulatory site for cADPR is present in the ligand-binding domain of IP(3)R1 and 2, but not of IP(3)R3. (C) 2001 Elsevier Science Inc. All rights reserved.