Two calcium-binding sites mediate the interconversion of liver inositol 1,4,5-trisphosphate receptors between three conformational states

Abstract

Cytosolic Ca2+ biphasically regulates Ins(1,4,5)P3-stimulated Ca2+ mobilization in liver [Marshall and Taylor (1993) J. Biol. Chem. 268, 13214-13220]. We have investigated the mechanisms underlying this biphasic control of Ca2+ mobilization in permeabilized hepatocytes by comparing the effects of Sr2+, Ba2+ and Ca2+ on the liver Ins(1,4,5)P3 receptor. Both Ca2+ and Sr2+ increased the binding of [3H]Ins(1,4,5)P3 to liver membranes by converting receptors from a low-affinity (KD approximately 35 nM) to a high-affinity (KD approximately 5 nM) state. Ba2+ (< or = 20 microM) did not affect [3H]Ins(1,4,5)P3 binding. At concentrations similar to those that caused an enhancement of [3H]Ins(1,4,5)P3 binding, Sr2+ (EC50 = 570 nM) and Ca2+ (EC50 = 200 nM) increased the sensitivity of the intracellular Ca2+ stores to Ins(1,4,5)P3. Further modest elevations in [Ca2+] (EC50 = 1.5 microM) inhibited Ins(1,4,5)P3-stimulated Ca2+ mobilization, whereas Sr2+ caused inhibition only when its concentration was very substantially increased (EC50 approximately 900 microM). Sr2+ is therefore only 3-fold less potent than Ca2+ in causing sensitization of Ins(1,4,5)P3-stimulated Ca2+ release, but 600-fold less potent in causing inhibition. Ba2+ neither sensitized ([Ba2+] < or = 20 microM) nor inhibited ([Ba2+] < or = 1 mM) Ins(1,4,5)P3-stimulated Ca2+ release, and did not inhibit either the sensitization of Ca2+ release evoked by Sr2+ or the inhibition of Ca2+ release evoked by Ca2+. Our results suggest that two distinct Ca(2+)-binding sites, which differ in their selectivities for bivalent cations, mediate the interconversion of Ins(1,4,5)P3 receptors between at least three different conformational states. These two Ca(2+)-binding sites, which may reside either on the Ins(1,4,5)P3 receptor itself or on distinct regulatory proteins, can be distinguished by their different selectivities for bivalent cations.

Regulation of cerebellar Ins(1,4,5)P3 receptor by interaction between Ins(1,4,5)P3 and Ca2+

“…The order of potency for this site (Ca# + Mn# + Sr# + Ba# + ) was identical and the relative affinities very similar to those determined for the site inhibiting InsP $ R activity. These characteristics were different from those of the activatory site [20][21][22]. The dissociation constants for Ca# + and Mn# + determined here are very close to those determined for InsP $ R channel inhibition in electrophysiological experiments [22].…”

“…Other experimental conditions were as described in the legend to Figure 1 for Ca# + . However, these sites exhibit different affinities for Mn# + and Sr# + [20][21][22]. In the present study we determined the selectivity of the Ca# + -binding site responsible for the inhibition of [$H]InsP $ binding to cerebellar membranes by comparing the effects of these cations on this binding.…”

“…The selectivities of stimulation and inhibition of IICR for bivalent cations differ [20][21][22], suggesting that different sites are involved. Ca# + -binding regions have been identified in type 1 [23][24][25] and type 2 InsP $ Rs [23] but their function is unknown.…”

IP3 Receptors: Toward Understanding Their Activation

“…The balance of opinion, supported by numerous studies of all three IP 3 R subtypes and using both single-channel and Ca 2þ -efflux studies, is that all three IP 3 R subtypes are biphasically regulated by cytosolic Ca 2þ (Marshall and Taylor 1993;Oancea and Meyer 1996;Dufour et al 1997;Missiaen et al 1998;Miyakawa et al 1999;Swatton et al 1999;Boehning and Joseph 2000b;Mak et al 2000;Mak et al 2001;Tu et al 2005a). Two independent Ca 2þ -binding sites, which differ in their interactions with different bivalent cations and in their affinities for Ca 2þ , mediate the stimulatory and inhibitory effects of cytosolic Ca 2þ (Marshall and Taylor 1994;Striggow and Ehrlich 1996;Hajnó czky and Thomas 1997). Both sites are essential elements of many models proposed to explain regenerative Ca 2þ signals (Lechleiter et al 1991;Berridge 1997).…”

Molecular and Functional Evidence for Multiple Ca2+-binding Domains in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

“…via Ca 2ϩ /CaMdependent protein kinase II and protein phosphatase 2B (13) or via a Ca 2ϩ -sensitizing factor (14). Studies in which effects of Sr 2ϩ were compared with those of Ca 2ϩ revealed that at least two interaction sites must exist: a stimulatory site that is modestly sensitive to Sr 2ϩ and an inhibitory site that is nearly insensitive to Sr 2ϩ (15). One Ca 2ϩ -binding domain has been localized on the cytosolic side of the InsP 3 R-1 (16,17), but whether this domain represents a stimulatory or an inhibitory interaction site is still unclear.…”