Micromolar calcium decreases affinity of inositol trisphosphate receptor in vascular smooth muscle

Abstract

The mechanism by which Ca2+ inhibits InsP3-induced Ca2+ release from sarcoplasmic reticulum of vascular smooth muscle was investigated. InsP3 binding to sarcoplasmic-reticulum vesicles from dog aortic smooth muscle was inhibited by 51 +/- 6% by 2 microM Ca2+ in the presence of 10 nM [3H]InsP3. Scatchard analysis indicated the presence of two InsP3-binding sites in the absence of Ca2+ (Kd = 2.5 +/- 0.9 and 49 +/- 8 nM InsP3), though the low-affinity site was more prevalent (representing 92 +/- 3% of the total number of binding sites). Ca2+ (2 microM) did not alter InsP3 binding to the high-affinity site (P > 0.05), but increased the Kd of the low-affinity site 3-fold (Kd = 155 +/- 4 nM InsP3; P < 0.001). The possibility that the apparent decrease in InsP3 affinity was caused by Ca(2+)-dependent activation of an endogenous phospholipase C could be excluded, because the Ca(2+)-dependent inhibition of InsP3 binding was completely reversible and insensitive to an inhibitor of phospholipase C. Moreover, Ca2+ did not inhibit InsP3 binding to InsP3 receptor partially purified by heparin-Sepharose chromatography, though another fraction (devoid of InsP3 receptor) restored Ca(2+)-sensitivity of the partially purified InsP3 receptor. Thus Ca2+ binding to a Ca(2+)-sensitizing factor associated with the InsP3 receptor decreases the affinity of the receptor complex for InsP3. This Ca(2+)-sensitizing factor may provide a negative-feedback mechanism for regulating the rise in cytosolic Ca2+ concentration in vascular smooth muscle after hormone activation of the phosphoinositide cascade.

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

“…This agrees with most previous studies in the cerebellum ( [17,21,29,33], but see [28]) and in other cells or tissues containing a high proportion of the InsP $ R1 isoform [27,38,39]. However, a high-affinity site was also detected in the presence of Ca# + , which accounted for a small fraction (4 %) of the total number of binding sites.…”

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

“…Up to now, complex effects of Ca 2ϩ on the binding of InsP 3 to its receptor have been reported. An inhibition of the binding was observed in several (14,45,46) but not in all (47) studies. In hepatocytes Ca 2ϩ converts the InsP 3 R from a low affinity to a high affinity conformational state, thereby increasing the binding (10,48).…”

“…Sr 2ϩ could only mimic the inhibitory effect on the cooperativity but not on the EC 50 itory sites were involved. The increase in EC 50 may be related to the Ca 2ϩ -induced inhibition of InsP 3 binding (14,45,46), especially since the inhibition by Ca 2ϩ in A7r5 cells (11,23) and in cerebellum (58,59) (49,50).…”

“…Although these kinetically distinct effects are believed to be crucial for the generation of Ca 2ϩ oscillations and waves (12), little is known about how Ca 2ϩ interacts with the InsP 3 R. Regulation might be exerted by direct binding of Ca 2ϩ to the InsP 3 R and/or via Ca 2ϩ -sensitive protein(s), e.g. 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).…”

IP3 Receptors: Toward Understanding Their Activation

“…Ca 2þ -mediated inhibition of IP 3 R is widely assumed to contribute to termination of local cytosolic Ca 2þ signals, but it remains far from clear whether such inhibition is mediated by Ca 2þ binding directly to IP 3 R or to an associated protein (Taylor and Laude 2002 (Worley et al 1987;Supattapone et al 1988;Joseph et al 1989;Varney et al 1990;Richardson and Taylor 1993;Benevolensky et al 1994;Cardy et al 1997;Yoneshima et al 1997), but the effects of Ca 2þ on IP 3 binding to IP 3 R from cells expressing predominantly IP 3 R2 or IP 3 R3 are confused (Pietri et al 1990;Mohr et al 1993;Marshall and Taylor 1994;Cardy et al 1997;Yoneshima et al 1997;Lin et al 2000;Swatton and Taylor 2002). These conflicting results, and evidence that purified IP 3 R1 is not inhibited by Ca 2þ (Danoff et al 1988;Richardson and Taylor 1993;Benevolensky et al 1994;Lin et al 2000), lend some support to the idea that Ca 2þ inhibition may be mediated by an accessory protein.…”