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

Abstract

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...

The Regulation of Calcium/Calmodulin-dependent Protein Kinase II during Oocyte Activation in the Rat

“…However, the effect of KN93 on other aspects of the Ca 2+ pathways cannot be discounted as KN93 has also been suggested to inhibit Ca 2+ release directly through binding to the calmodulin-binding site of the inositol triphosphate receptor [36]. It is possible that the effects of KN93 on the phosphorylation of CaMKII observed in this study may be due to the actions of KN93 on several steps of the pathway that lead to the activation of the enzyme.…”

Ca2+-Binding Protein-1 Facilitates and Forms a Postsynaptic Complex with Cav1.2 (L-Type) Ca2+Channels

“…This later effect of KN-93 suggests that CaM kinase II may oppose the effects of CaBP1 on I Ca facilitation. Alternatively, enhanced Ca 2ϩ -dependent facilitation with KN-93 may be related to nonspecific actions independent of CaM kinase II, which have been reported previously (Smyth et al, 2002). Regardless of a potential modulatory role for CaM kinase II, these results clearly show that CaM kinase II activation is not required for CaBP1-dependent facilitation of Ca v 1.2, which may rely instead on direct Ca I Ca in cells cotransfected with CaBP1 similarly depended on the IQ, we analyzed channels in which the first two residues of this site (I1624 and Q1625) were replaced with either alanine (Ca v 1.2 IQ-AA ) or glutamate (Ca v 1.2 IQ-EE ).…”

Phosphorylation of IP3R1 and the regulation of[Ca2+]i responses at fertilization: a role for the MAP kinase pathway

“…Sperm entry triggers a series of increases in the intracellular free-Ca 2+ concentration ([Ca 2+ ] i ), termed [Ca 2+ ] i oscillations, which enable exit from the MII arrest and induce egg activation (Miyazaki et al, 1993). Egg activation entails the orderly initiation of several events, including cortical granule exocytosis, extrusion of the second polar body (2PB), pronuclear (PN) formation and progression into interphase, the completion of which is required for the normal initiation of development (Ducibella et al, 2002;Schultz and Kopf, 1995).…”