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 Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP(3)R (IP(3)R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca(2+) signaling depended neither on effects on IP(3) metabolism nor on the filling grade of Ca(2+) stores, suggesting a direct action on the IP(3)R. 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 Ca(2+). Analysis of Ca(2+) release in A7r5 cells at varying [IP(3)], of IP(3)R-1 degradation in eggs, and of [(3)H]IP(3) binding in Sf9 microsomes all indicated that KN-93 did not affect IP(3) binding. Comparison of the inhibition of Ca(2+) release and of [(3)H]IP(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.
Molecular and Cellular Biology Program and Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Jeremy T
JT
Smyth
Allison L
AL
Abbott
Bora
B
Lee
Ilse
I
Sienaert
Nael Nadif
NN
Kasri
Humbert
H
De Smedt
Tom
T
Ducibella
Ludwig
L
Missiaen
Jan B
JB
Parys
Rafael A
RA
Fissore
D000818:Animals; D001596:Benzylamines / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism / Q000502:physiology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D005260:Female; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D010063:Ovum / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology*; D000096926:Benzenesulfonamides
D001596:Benzylamines; D002118:Calcium; D015220:Calcium Channels; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015544:Inositol 1,4,5-Trisphosphate; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D013449:Sulfonamides; C072105:KN 93; D000096926:Benzenesulfonamides