title: A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.
abstract: In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.
authors.affiliation: Zoological Institute, University of Munich, Federal Republic of Germany.
authors.forename: H
authors.initials: H
authors.lastname: Wieczorek
authors.forename: M
authors.initials: M
authors.lastname: Putzenlechner
authors.forename: W
authors.initials: W
authors.lastname: Zeiske
authors.forename: U
authors.initials: U
authors.lastname: Klein
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006859:Hydrogen / Q000378:metabolism*; D018942:Macrolides; D008564:Membrane Potentials; D009036:Moths; D011188:Potassium / Q000378:metabolism*; D027961:Potassium-Hydrogen Antiporters; D006180:Proton-Translocating ATPases / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D018942:Macrolides; D027961:Potassium-Hydrogen Antiporters; C057620:bafilomycin A; D000584:Amiloride; D006859:Hydrogen; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D011188:Potassium