title: Inhibition of chloroplast ATPase by the K+ channel blocker alpha-dendrotoxin.
abstract: Possible structural and functional similarities between the channel part, CF0, of chloroplast ATPase (CF0CF1) and ion channels permeable to monovalent cations were investigated using high-affinity toxins mainly targeted against voltage-sensitive K+ channels. In particular, the effect of the K(+)-channel blocker alpha-dendrotoxin and the crude scorpion venom of Leiurus quinquestriatus hebraeus (LQ venom) on ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes was characterised. Alpha-dendrotoxin (K(i) approximately 5.05 microM) and the LQ venom (K(i) approximately 1.55 micrograms/ml) specifically inhibited ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes. Our results show that alpha-dendrotoxin and peptides of the LQ venom with an apparent molecular mass of about 4.0 kDa, probably isoforms of charybdotoxin, specifically bind to CF0CF1. This binding was reversible and induced a high leak conductance for H+ through CF0. The Ca(2+)-dependent ATPase activity of the isolated soluble part of CF0CF1 (CF1) was completely inhibited by 1 microM alpha-dendrotoxin, while the crude LQ venom, at concentrations up to 10 micrograms/ml, had no affect on ATPase activity. The concentration dependence of the inhibition by alpha-dendrotoxin indicates that approximately 2 mol alpha-dendrotoxin bind/mol CF0CF1 and 1 mol alpha-dendrotoxin/mol CF1. Known inhibitors of H(+)-flow-through CF0 acted in the presence of alpha-dendrotoxin synergistically. Dicyclohexylcarbodiimide and venturicidin, in contrast to their known effect of blocking H(+)-flow-through CF0, increased the leak conductance through CF0 in the presence of alpha-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 CF0, but induces a conformation which closes the channel part for H+. Protein sequence comparison between the known binding site of charybdotoxin in the shaker K+ channel from Drosophila [MacKinnon, R. & Heginbotham, L. (1990) Neuron 5, 767-771] and the choroplast ATPase showed that subunit III reveals a significant similarity (64%) in parts of its sequence (Gln28-Leu53) to the helix 5 and helix 6 (S5-S6) linker region (Ala413-Cys462; the charybdotoxin-binding site) of the shaker K+ channel. According to secondary-structure predictions, the homologous sequences in subunit III and the shaker K+ channel represent putative hydrophilic loops connecting two transmembrane alpha-helices. Apparently the shaker K+ channel and subunit III share significant topological features in these hydrophilic loops which may be part of the respective channel entrance.
authors.affiliation: Biophysik, Universität Osnabrück, FB Biologie/Chemie, Federal Republic of Germany.
authors.forename: P
authors.initials: P
authors.lastname: Schulenburg
authors.forename: M
authors.initials: M
authors.lastname: Schwarz
authors.forename: R
authors.initials: R
authors.lastname: Wagner
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D002463:Cell Membrane Permeability / Q000187:drug effects; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D004546:Elapid Venoms / Q000494:pharmacology*; D004579:Electron Transport; D007425:Intracellular Membranes / Q000378:metabolism; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000494:pharmacology*; D015221:Potassium Channels / Q000187:drug effects*; D011510:Proteolipids; D012604:Scorpion Venoms / Q000633:toxicity; D017386:Sequence Homology, Amino Acid
chemical_list: D004546:Elapid Venoms; D008051:Lipid Bilayers; D009498:Neurotoxins; D015221:Potassium Channels; D011510:Proteolipids; D012604:Scorpion Venoms; C020485:proteoliposomes; C020273:dendrotoxin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases