Inhibition of chloroplast ATPase by the K⁺ channel blocker α‐dendrotoxin

Abstract

Possible structural and functional similarities between the channel part, CFo, of chloroplast ATPase (CFoCFl) and ion channels permeable to monovalent cations were investigated using highaffinity toxins mainly targeted against voltage-sensitive K + channels. In particular, the effect of the K+-channel blocker a-dendrotoxin and the crude scorpion venom of Leiurus quinquestriutus hebraeus (LQ venom) on ATP synthesis in thylakoid membranes and in CFoCFl-containing liposomes was characterised.a-dendrotoxin (Ki z 5.05 pM) and the LQ venom (Ki z 1.55 pg/ml) specifically inhibited ATP synthesis in thylakoid membranes and in CFoCF1-containing liposomes. Our results show that adendrotoxin and peptides of the LQ venom with an apparent molecular mass of about 4.0 kDa, probably isoforms of charybdotoxin, specifically bind to CFoCFl. This binding was reversible and induced a high leak conductance for H + through CFo. The Ca2+-dependent ATPase activity of the isolated soluble part of CFoCF1 (CF,) was completely inhibited by 1 pM a-dendrotoxin, while the crude LQ venom, at concentrations up to 10 Fg/ml, had no affect on ATPase activity. The concentration dependence of the inhibition by a-dendrotoxin indicates that approximately 2 mol adendrotoxin bind/mol CFoCFl and 1 mol a-dendrotoxin/mol CF1. Known inhibitors of H+-flowthrough CFo acted in the presence of a-dendrotoxin synergistically. Dicyclohexylcarbodiimide and venturicidin, in contrast to their known effect of blocking H+-flow-through CFo, increased the leak conductance through CFo in the presence of a-dendrotoxin drastically. This uncoupling effect indicates that their normal mode of blocking is a secondary effect. Binding of the inhibitors to their respective sites apparently does not affect the proton pathway in CFo, but induces a conformation which closes the channel part for H + . channel. According to secondary-structure predictions, the homologous sequences in subunit 111 and the shaker K + channel represent putative hydrophilic loops connecting two transmembrane a-helices. Apparently the shaker K + channel and subunit I11 share significant topological features in these hydrophilic loops which may be part of the respective channel entrance.The key enzymes of energy conversion in the membrane of bacteria, mitochondria and chloroplasts, the H+-translocating F-type ATPases, are composed of two moieties: the water-soluble part F,, containing the catalytic sites, and the intrinsic membrane part, Fo, involved in H + translocation

Study of the Effect of Ion Channel Modulators on Photosynthetic Oxygen Evolution

“…However, the use of specific channel inhibitors for the study of the ion-fluxphotosynthesis relationship has been undertaken only in a few cases. [12][13][14][15][16] Several chloride-, potassium-, and divalent cation-selective ion channels [17][18][19][20][21][22][23][24][25][26][27][28][29] and light-induced currents [30][31][32][33] have been recorded from all three chloroplast membranes. Patch-clamp experiments were performed on intact chloroplasts 18,19 and isolated thylakoid membranes.…”

“…A role for cationic channels in counterbalancing and, in general, the existence of ion channels in the thylakoid membrane were hypothesized in the 1970s, thanks to the pioneer works of several groups. − It is, indeed, well-established by now that mainly K + , Mg 2+ , and small anions take part in counterbalancing and that changes in the concentrations of all these ions in the stroma are accompanied by passive H + , K + , Cl - , and probably Na + transport across the envelope of intact chloroplasts. However, the use of specific channel inhibitors for the study of the ion-flux−photosynthesis relationship has been undertaken only in a few cases. − …”