An Investigation of the Midgut K+ Pump of the Tobacco Hornworm (manduca Sexta) Using Specific Inhibitors and Amphotericin B

Active K+ secretion in isolated posterior midguts of Manduca sexta was studied by measuring the short-circuit current. One aim of this study was to verify the postulate from biochemical reports that the cooperative apical arrangement of a vacuolar-type H+-ATPase (V-ATPase) and a K+/H+ antiporter drive the short-circuit current. Hence, we tested several specific inhibitors of the V-ATPase on the in vitro midgut preparation. Nitrate, bafilomycin A1, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) and amiloride all reduced the short-circuit current. This suggests that the H+-ATPase is involved in transepithelial K+ secretion. However, even at relatively high doses of these inhibitors, the block of the short-circuit current was not complete. Two other agents, thallium ions (Tl+, at millimolar concentrations) and trimethyltin chloride (TMT, 50 µmol l-1), did abolish the short-circuit current. Apical, but not basal, use of the ionophore amphotericin B largely eliminated the short-circuit current. This supports the view that the current-generating source resides in the apical membranes. An apical (and probably intracellular) site of action for NO3-, Tl+ and TMT is suggested by the observation that basal amphotericin B is needed for blockage by NO3- but does not, however, influence the effect of Tl+ and TMT. Likely sites of action are the V-ATPase (for nitrate and TMT) and the K+/H+ antiporter (for Tl+).