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

Abstract

Three different genes encode the inositol 1,4,5-trisphosphate (IP 3) receptor (IP 3R), an intracellular Ca 2+ channel involved in cellular Ca 2+ signaling. The IP 3-binding characteristics of the various IP 3R isoforms differ, but until now no specific activators or inhibitors of IP 3 binding have been described. We compared the effects of oxidizing reagents, in particular thimerosal, and of cyclic ADP-ribose (cADPR) on IP 3 binding to the various IP 3R isoforms. We therefore expressed the N-terminal 581 amino acids of the three IP 3R isoforms as recombinant proteins in the soluble fraction of Escherichia coli (ligand-binding sites [lbs] 1, 2, and 3) as well as the full-length IP 3R1 and IP 3R3 in Spodoptera frugiperda (Sf9) insect cells. Thimerosal (100 μM) stimulated IP 3 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 IP 3 binding (from 46 ± 4 nM to 20 ± 2 nM and from 54 ± 21 nM to 19 ± 7 nM for lbs-1 and -3, respectively) without modifying the B max. Similarly, IP 3 binding to microsomes of Sf9 insect cells overexpressing the full-length IP 3R1 was 1.2-fold stimulated by thimerosal. Thimerosal, however, did not affect IP 3 binding to Sf9–IP 3R3 microsomes, suggesting that in situ thimerosal will only directly affect ligand binding to the type 1 isoform. cADPR (50 μM) stimulated IP 3 binding to Sf9–IP 3R1 microsomes (1.5-fold), but not to Sf9–IP 3R3 microsomes. In addition, cADPR inhibited IP 3 binding to lbs-1 and lbs-2 by decreasing the affinity for IP 3 1.8- and 2.8-fold, respectively, while IP 3 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 3R1 and 2, but not of IP 3R3.