Thimerosal stimulates Ca2+ flux through inositol 1,4,5-trisphosphate receptor type 1, but not type 3, via modulation of an isoform-specific Ca2+-dependent intramolecular interaction

Thiol-reactive agents such as thimerosal have been shown to modulate the Ca 2+ -flux properties of IP 3 (inositol 1,4,5-trisphosphate) receptor (IP 3 R) via an as yet unidentified mechanism [Parys, Missiaen, De Smedt, Droogmans and Casteels (1993) Pflugers Arch. 424 , 516–522; Kaplin, Ferris, Voglmaier and Snyder (1994) J. Biol. Chem. 269 , 28972–28978; Missiaen, Taylor and Berridge (1992) J. Physiol. (Cambridge, U.K.) 455 , 623–640; Missiaen, Parys, Sienaert, Maes, Kunzelmann, Takahashi, Tanzawa and De Smedt (1998) J. Biol. Chem. 273 , 8983–8986]. In the present study, we show that thimerosal potentiated IICR (IP 3 -induced Ca 2+ release) and IP 3 -binding activity of IP 3 R1, expressed in triple IP 3 R-knockout R23-11 cells derived from DT40 chicken B lymphoma cells, but not of IP 3 R3 or [Δ1–225]-IP 3 R1, which lacks the N-terminal suppressor domain. Using a 45 Ca 2+ -flux technique in permeabilized A7r5 smooth-muscle cells, we have shown that Ca 2+ shifted the stimulatory effect of thimerosal on IICR to lower concentrations of thimerosal and thereby increased the extent of Ca 2+ release. This suggests that Ca 2+ and thimerosal synergetically regulate IP 3 R1. Glutathione S -transferase pull-down experiments elucidated an interaction between amino acids 1–225 (suppressor domain) and amino acids 226–604 (IP 3 -binding core) of IP 3 R1, and this interaction was strengthened by both Ca 2+ and thimerosal. In contrast, calmodulin and sCaBP-1 (short Ca 2+ -binding protein-1), both having binding sites in the 1–225 region, weakened the interaction. This interaction was not found for IP 3 R3, in agreement with the lack of functional stimulation of this isoform by thimerosal. The interaction between the IP 3 -binding and transmembrane domains (amino acids 1–604 and 2170–2749 respectively) was not affected by thimerosal and Ca 2+ , but it was significantly inhibited by IP 3 and adenophostin A. Our results demonstrate that thimerosal and Ca 2+ induce isoform-specific conformational changes in the N-terminal part of IP 3 R1, leading to the formation of a highly IP 3 -sensitive Ca 2+ -release channel.