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The Journal of Experimental Biology 205, 463-474 (2002)
© 2002 The Company of Biologists Limited

Insect midgut K⁺ secretion: concerted run-down of apical/basolateral transporters with extra-/intracellular acidity

Wolfgang Zeiske^*, Heiko Meyer and Helmut Wieczorek

Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, 49069 Osnabrück, Germany

*Author for correspondence (e-mail: zeiske{at}biologie.uni-osnabrueck.de)

Accepted 27 November 2001

In lepidopteran larvae, three transport mechanisms are involved in the active and electrogenic K⁺ secretion that occurs in the epithelial goblet cells of the midgut. These consist of (i) basolateral K⁺ channels, allowing K⁺ entry from the haemolymph into the cytosol, (ii) apical electrogenic K⁺/2H⁺ antiporters, which are responsible for secondary active extrusion of K⁺ from the cell into the gut lumen via the goblet cavity and (iii) apical V-ATPase-type proton pumps. The latter energize apical K⁺ exit by building up a large, cavity-positive electrical potential that drives the antiporters. Net K⁺ secretion (I_K) can be measured as short-circuit current (I_sc) across the in vitro midgut mounted in an Ussing chamber. We investigated the influence of protons on the transepithelial I_K and the partial reactions of the basolateral K⁺ permeability (P_K) and the apical, lumped ‘K⁺ pump’ current (I_P) at various extra- and intracellular pH values. In particular, we wanted to know whether increased cellular acidity could counteract the reversible dissociation of the V-ATPase into its V₁ and V_o parts, as occurs in yeast after glucose deprivation and in the midgut of Manduca sexta during starvation or moulting, thus possibly enhancing K⁺ transport.When intact epithelia were perfused with high-[K⁺] (32 mmol l^–1) salines with different pH values, I_K was reversibly reduced when pH values fell below 6 on either side of the epithelium. Attempts to modify the intracellular pH by pulsing with NH₄⁺ or propionate showed that intracellular acidification caused a reduction in I_K similar to that obtained in response to application of external protons. Treatment with azide, a well-known inhibitor of the mitochondrial ATP synthase, had the same effect as pulsing with ammonium or propionate with, however, much faster kinetics and higher reversibility. Breakdown of the basolateral or apical barrier using the antibiotic nystatin allowed the intracellular pH to be clamped to that of the saline facing the nystatin-treated epithelial border. Cell acidification achieved by this manipulation led to a reduction in both apical I_P and basolateral P_K. The transepithelial I_K showed an approximately half-maximal reduction at external pH values close to 5 in intact tissues, and a similar reduction in I_P and P_K values was seen at an intracellular pH of 5 in nystatin-permeabilised epithelia. Thus, the hypothesized V₁V_o stabilization by cell acidity is not reflected in the pH-sensitivity of I_P. Moreover, all components that transport K⁺ are synchronously inhibited below pH 6. The significance of our findings for the midgut in vivo is discussed.

Key words: Manduca sexta, tobacco hornworm, K⁺ secretion, V-ATPase, vacuolar, pump current, K⁺ channel, intracellular pH.

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