Diazoxide affects the IF1 inhibitor protein binding to F1 sector of beef heart F0F1ATPsynthase.
Diazoxide, a selective opener of the mitochondrial ATP-sensitive K+ channel (mitoK(ATP)), has been reported to enhance F(0)F(1)ATPsynthase 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 (IF(1)) to beta subunit. More specifically, the treatment of soluble F(1) with one equivalent of diazoxide was sufficient to decrease the K(d) of IF(1)-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(1) binding. Finally, diazoxide also increased IF(1) binding to membrane bound F(1), while it did not influence the energisation-dependent IF(1) release. As IF(1) binding mediates the F(0)F(1)ATPsynthase inhibition, we suggest that such mechanism may contribute to cardioprotection during ischemia.
Contessi
Stefania
S
Departmrnt of Biomedical Sciences and Technologies, University of Udine, Piazzale Kolbe 4, 33100 Udine, Italy.
Metelli
Giuliana
G
Mavelli
Irene
I
Lippe
Giovanna
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003981:Diazoxide / Q000494:pharmacology*; D006321:Heart / Q000187:drug effects*; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D000097795:ATPase Inhibitory Protein
D000255:Adenosine Triphosphate; D003981:Diazoxide; D011506:Proteins; D006180:Proton-Translocating ATPases; D000097795:ATPase Inhibitory Protein