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

摘要

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 IP(3)R 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 IP3R1 and IP3R3 in Spodoptera frugiperda (Sf9) insect cells. Thimerosal (100 microM) 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 Kd for IP3 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 Bmax. Similarly, IP3 binding to microsomes of Sf9 insect cells overexpressing the full-length IP3R1 was 1.2-fold stimulated by thimerosal. Thimerosal, however, did not affect IP3 binding to Sf9-IP3R3 microsomes, suggesting that in situ thimerosal will only directly affect ligand binding to the type 1 isoform. cADPR (50 microM) stimulated IP3 binding to Sf9-IP3R1 microsomes (1.5-fold), but not to Sf9-IP3R3 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 IP3R1 and 2, but not of IP3R3.