Jeremy T. Smyth, A. Abbott, Bora Lee
Sep 20, 2002
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Journal
The Journal of Biological Chemistry
Abstract
KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP3R)-mediated [Ca2+] i signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP3R (IP3R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca2+ signaling depended neither on effects on IP3 metabolism nor on the filling grade of Ca2+stores, suggesting a direct action on the IP3R. Inhibition was independent of CaMKII, since in identical conditions other CaMKII inhibitors (KN-62, peptide 281–309, and autocamtide-related inhibitory peptide) were ineffective and since CaMKII activation was precluded in permeabilized cells. Moreover, KN-93 was most effective in the absence of Ca2+. Analysis of Ca2+ release in A7r5 cells at varying [IP3], of IP3R-1 degradation in eggs, and of [3H]IP3 binding in Sf9 microsomes all indicated that KN-93 did not affect IP3binding. Comparison of the inhibition of Ca2+ release and of [3H]IP3 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 IP3R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o− cells that predominantly express type 3 IP3R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP3R-1 directly and may therefore be a useful tool in the study of IP3R functional regulation.