Modulation of Inositol 1,4,5-Trisphosphate Binding to the Recombinant Ligand-binding Site of the Type-1 Inositol 1,4,5-Trisphosphate Receptor by Ca2+ and Calmodulin

A 592-amino acid segment of the regulatory domain of the neuronal type-I inositol 1,4,5-trisphosphate receptor (IP 3 R) isoform (type-I long, amino acids1314 -1905) and the corresponding 552-amino acid alternatively spliced form present in peripheral tissues (type-I short, amino acids 1693-1733 deleted) were expressed as glutathione S-transferase fusion proteins. These domains encompass a putative calmodulin (CaM) binding domain and two protein kinase A phosphorylation sites. Both long and short fusion proteins retained the ability to bind CaM in a Ca 2؉ -dependent manner as measured by CaMSepharose chromatography or a dansyl-CaM fluorescence assay. Both assays indicated that the short fusion protein bound twice the amount of CaM than the long form at saturating concentrations of CaM. In addition, the binding of the short form to CaM-Sepharose was inhibited by phosphorylation with protein kinase A, whereas the binding of the long form was unaffected. [3][4][5]. The functional role of isoform diversity has not been established. The gene encoding the type-I isoform is subject to alternative splicing and gives rise to three splice variants that have been denoted as S1, S2, and S3 (6 -8). The S1 splice site is located in the ligand binding domain, whereas the S2 and S3 splice sites are in the regulatory domain. Functional studies comparing the S1(Ϫ) and S1(ϩ) forms of the type-I IP 3 R have not revealed any marked differences in ligand binding or channel function (9, 10), and the functional consequences of the S3 insertion/deletion have not been investigated. Both short and long forms of S1 and S3 appear to co-exist in the same tissues (7,8,11). In contrast, the expression of the S2 insert is more stringent with the S2 insert being present in neurons and absent from the type-I IP 3 Rs of peripheral tissues (7,12).The S2 splice site encodes a region of 40 amino acids and is located between serine 1589 and serine 1756. Both serine residues can be phosphorylated by protein kinase A (13), and serine 1756 in cerebellum IP 3 R is phosphorylated by G-kinase (14). In addition, binding sites for Ca 2ϩ (15), CaM (16), ATP (17), and FKBP-12 (18) are all found in the vicinity of the S2 splice site (see Fig. 1A). Hence, the presence or absence of the S2 splice site may modify regulation of type-I IP 3 Rs. For example, there is evidence that the deletion of the S2 region can alter the serine that is preferentially phosphorylated by protein kinase A (12). There is also experimental evidence to suggest that the effects of protein kinase A phosphorylation on IP 3 -gated channels in neuronal and peripheral tissues are different (reviewed in Joseph et al. (19) (S2(ϩ)) is also different from that observed in a number of tissues that express high amounts of the S2(Ϫ) form of the type-I IP 3 R, e.g. vas deferens (20). However, it is difficult to definitively attribute such differences solely to the presence of the alternatively spliced forms of the type-I IP 3 R, and the basis for these differences is not well understood. In the presen...

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

The Interaction of Calmodulin with Alternatively Spliced Isoforms of the Type-I Inositol Trisphosphate Receptor

Lin

Widjaja

Joseph

2000

“…3 H]IP 3 binding disappeared at alkaline pH, probably due to a conformational change of calmodulin (9,11). The apparent discrepancy between the functional data reported in the present study and the [ 3 H]IP 3 binding data (9 -11) may be due to the existence of multiple calmodulin-binding or Ca 2ϩ -binding sites that play a role and/or to the fact that IP 3 binding, which is only one step, albeit a crucial step, for channel opening, is not equivalent to the channel activity itself.…”

Section: Resultsmentioning

confidence: 99%

“…Calmodulin, a Ca 2ϩ -binding protein abundantly present in many cell types (6), binds to the modulatory region of the IP 3 R in a Ca 2ϩ -dependent way (7,8). Calmodulin also interacts with the IP 3 R in a Ca 2ϩ -independent way (9 -11), with one of the interaction sites located within the IP 3 -binding domain (11 (14). The association constants for BAPTA were: H-BAPTA, 6.36; H-HBAPTA, 5.47; Ca-BAPTA, 6.97; and Sr-BAPTA, 5.13 (15).…”

Section: From the Laboratorium Voor Fysiologie Katholieke Universitementioning

confidence: 99%

The Bell-shaped Ca2+ Dependence of the Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Is Modulated by Ca2+/Calmodulin

Missiaen

Parys

Weidema

et al. 1999

Self Cite

1 is a second messenger used by many cell types to release Ca 2ϩ from internal stores (1). Cytosolic Ca 2ϩ has a bell-shaped effect on the IP 3 receptor (IP 3 R), with low concentrations stimulating the release and high concentrations inhibiting it (2-5). Calmodulin, a Ca 2ϩ -binding protein abundantly present in many cell types (6), binds to the modulatory region of the IP 3 R in a Ca 2ϩ -dependent way (7,8). Calmodulin also interacts with the IP 3 R in a Ca 2ϩ -independent way (9 -11), with one of the interaction sites located within the IP 3 -binding domain (11

“…in peripheral tissues) and is antagonized by cAMP-dependent phosphorylation of IP 3 R-1 (11). The consequences of CaM binding to IP 3 R-1 are inhibitory; apo-CaM inhibits IP 3 binding to its receptor (8,12), whereas the binding of Ca 2ϩ /CaM inhibits Ca 2ϩ release through the channel (13,14). The relation, however, between the nature of the CaMbinding sites and the functional effects of CaM remains to be established.…”

Section: Camentioning

confidence: 99%

Inhibition of the Inositol Trisphosphate Receptor of Mouse Eggs and A7r5 Cells by KN-93 via a Mechanism Unrelated to Ca2+/Calmodulin-dependent Protein Kinase II Antagonism

Smyth

Abbott²,

Lee

et al. 2002

Self Cite

3 binding by KN-93 and calmodulin (CaM), either separately or combined, was compatible with a specific interaction of KN-93 with a CaM-binding site on IP 3 R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o ؊ cells that predominantly express type 3 IP 3 R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP 3 R-1 directly and may therefore be a useful tool in the study of IP 3 R functional regulation. Ca2ϩ acts as a ubiquitous second messenger that mediates a wide array of cellular functions, including muscle contraction, neurotransmitter release, and egg activation (reviewed in Ref. 1). The spatial and temporal dynamics of Ca 2ϩ release are complex and may range from localized, brief "puffs" of Ca 2ϩ to regenerative oscillations of the global cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) that may last from several minutes to several hours. The nature of the Ca 2ϩ response elicited by a specific cell stimulus is highly regulated; thus, the specificity of the cellular response to a stimulus may be dictated by the precise dynamics of the Ca 2ϩ signal (2, 3). The complexity of Ca 2ϩ signaling mandates that the regulation of Ca 2ϩ release dynamics must be very intricate and precise. The phosphoinositide pathway is a major intracellular signaling pathway that is involved in the regulation of Ca 2ϩ homeostasis (1). The hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phospholipase C (PLC) 1 leads to the formation of diacylglycerol and inositol 1,4,5-trisphosphate (IP 3 ). IP 3 then induces Ca 2ϩ release from the endoplasmic reticulum by binding to and activating the IP 3 receptor (IP 3 R), which acts as a ligand-gated Ca 2ϩ channel. Three isoforms of the IP 3 R, IP 3 R-1, IP 3 R-2, and IP 3 R-3, have been characterized thus far (4). The convergence of multiple regulatory pathways at the IP 3 R is likely to directly influence the characteristic properties of various Ca 2ϩ signals (5). For example, Ca 2ϩ itself is known to regulate its own release through the IP 3 R, giving rise to a bell-shaped relationship between [Ca 2ϩ ] i and IP 3 -induced Ca 2ϩ release, whereby low [Ca 2ϩ ] i potentiates Ca 2ϩ release and high [Ca 2ϩ ] i inhibits it. Ca 2ϩ may influence IP 3 R function by directly binding to the receptor, resulting in conformational changes to the receptor, or it may act through Ca 2ϩ -binding proteins such as calmodulin (CaM) (reviewed in Refs. 6 and 7). Three CaM-binding sites have been described on the IP 3 R-1: a low affinity site near the N terminus of the IP 3 R-1 that essentially binds Ca 2ϩ -free CaM (apo-CaM) (8, 9), a high affinity site in the central portion of the regulatory domain that mainly binds Ca 2ϩ /CaM (9, 10), and a third site that only appears after splicing out of S2 (i.e. in peripheral tissues) and is antagonized by cAMP-dependent phosphorylation of IP 3 R-1 (11). The consequences of CaM binding to IP 3 R-1 are inhibitory; apo-CaM * This work was supported in part by a Lotta M. Crabtree Fello...