Diazoxide, a selective opener of the mitochondrial ATP-sensitive K+ channel (mitoK(ATP)), has been reported to enhance F(0)F(1)ATP-synthase inhibition during ischemia, but the underlying mechanisms are still unclear. Here, we demonstrate that diazoxide directly interacts with the F-1 sector of beef heart F(0)F(1)ATPsynthase markedly promoting the binding of the inhibitor protein (IF1) to beta subunit. More specifically, the treatment of soluble F-1 with one equivalent of diazoxide was sufficient to decrease the K-d of IF1-F-1 complex at low pH. Such effect was revealed only on the cycling enzyme, while no effect was observed in the absence of Mg-ATP. However, diazoxide binding occurred independently from the catalysis, as shown by the structural changes induced by the drug in not catalytically active F-1 and revealed by CD spectra. In addition, kinetic analysis of ATP hydrolysis demonstrated that diazoxide exerts a stabilising role on Mg-ADP bound in the catalytic site of the beta subunit adopting the tight conformation (beta(DP)). In accordance, a stabilising effect of Mg-ADP at the nucleotide binding domain (NBD) has been reported also for K-ATP channel. These results suggest that diazoxide binds to beta subunit at NBD, which is highly conserved in the ATP-binding cassette protein family, thus inducing nucleotide stabilisation and favouring F-1 conformation suitable for IF, binding. Finally, diazoxide also increased IF1 binding to membrane bound F1, while it did not influence the energisation-dependent IF1 release. As IF1 binding mediates the F(0)F(1)ATPsynthase inhibition, we suggest that such mechanism may contribute to cardioprotection during ischemia.

Diazoxide affects the IF1 inhibitor protein binding to F1 sector of beef heart F0F1ATPsynthase

CONTESSI, Stefania;METELLI, Giuliana;MAVELLI, Irene;LIPPE, Giovanna
2004

Abstract

Diazoxide, a selective opener of the mitochondrial ATP-sensitive K+ channel (mitoK(ATP)), has been reported to enhance F(0)F(1)ATP-synthase inhibition during ischemia, but the underlying mechanisms are still unclear. Here, we demonstrate that diazoxide directly interacts with the F-1 sector of beef heart F(0)F(1)ATPsynthase markedly promoting the binding of the inhibitor protein (IF1) to beta subunit. More specifically, the treatment of soluble F-1 with one equivalent of diazoxide was sufficient to decrease the K-d of IF1-F-1 complex at low pH. Such effect was revealed only on the cycling enzyme, while no effect was observed in the absence of Mg-ATP. However, diazoxide binding occurred independently from the catalysis, as shown by the structural changes induced by the drug in not catalytically active F-1 and revealed by CD spectra. In addition, kinetic analysis of ATP hydrolysis demonstrated that diazoxide exerts a stabilising role on Mg-ADP bound in the catalytic site of the beta subunit adopting the tight conformation (beta(DP)). In accordance, a stabilising effect of Mg-ADP at the nucleotide binding domain (NBD) has been reported also for K-ATP channel. These results suggest that diazoxide binds to beta subunit at NBD, which is highly conserved in the ATP-binding cassette protein family, thus inducing nucleotide stabilisation and favouring F-1 conformation suitable for IF, binding. Finally, diazoxide also increased IF1 binding to membrane bound F1, while it did not influence the energisation-dependent IF1 release. As IF1 binding mediates the F(0)F(1)ATPsynthase inhibition, we suggest that such mechanism may contribute to cardioprotection during ischemia.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11390/879984
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