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 Ca2+-flux properties of IP3 (inositol 1,4,5-trisphosphate) receptor (IP3R) via an as yet unidentified mechanism [Parys, Missiaen, De Smedt, Droogmans and Casteels (1993) Pflügers 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 (IP3-induced Ca2+ release) and IP3-binding activity of IP3R1, expressed in triple IP3R-knockout R23-11 cells derived from DT40 chicken B lymphoma cells, but not of IP3R3 or [D1-225]-IP3R1, which lacks the N-terminal suppressor domain. Using a 45Ca2+-flux technique in permeabilized A7r5 smooth-muscle cells, we have shown that Ca2+ shifted the stimulatory effect of thimerosal on IICR to lower concentrations of thimerosal and thereby increased the extent of Ca2+ release. This suggests that Ca2+ and thimerosal synergetically regulate IP3R1. Glutathione S-transferase pull-down experiments elucidated an interaction between amino acids 1-225 (suppressor domain) and amino acids 226-604 (IP3-binding core) of IP3R1, and this interaction was strengthened by both Ca2+ and thimerosal. In contrast, calmodulin and sCaBP-1 (short Ca2+-binding protein-1), both having binding sites in the 1-225 region, weakened the interaction. This interaction was not found for IP3R3, in agreement with the lack of functional stimulation of this isoform by thimerosal. The interaction between the IP3-binding and transmembrane domains (amino acids 1-604 and 2170-2749 respectively) was not affected by thimerosal and Ca2+, but it was significantly inhibited by IP3 and adenophostin A. Our results demonstrate that thimerosal and Ca2+ induce isoform-specific conformational changes in the N-terminal part of IP3R1, leading to the formation of a highly IP3-sensitive Ca2+-release channel.
Bultynck
Geert
G
Laboratorium voor Fysiologie, K.U.Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium.
Szlufcik
Karolina
K
Kasri
Nael Nadif
NN
Assefa
Zerihun
Z
Callewaert
Geert
G
Missiaen
Ludwig
L
Parys
Jan B
JB
De Smedt
Humbert
H
D000818:Animals; D001011:Aorta / Q000737:chemistry / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects; D002645:Chickens; D002522:Chlorocebus aethiops; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017384:Sequence Deletion / Q000235:genetics / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013849:Thimerosal / Q000378:metabolism / Q000494:pharmacology*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010455:Peptides; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D013849:Thimerosal; D005982:Glutathione Transferase; D002118:Calcium