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

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 IP₃ binding. 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.