The Midgut as a Model System for Insect K⁺-Transporting Epithelia: Immunocytochemical Localization of a Vacuolar-Type H⁺ Pump

In Manduca sexta midgut, a vacuolar-type H⁺-ATPase and a K⁺/nH⁺ antiport represent the functional elements of the electrogenic K⁺ pump. This vacuolar-type ATPase was localized by immunofluorescence and immunogold staining using monoclonal antibodies to defined subunits of the midgut ATPase. The antibodies labelled the membrane projections of the goblet cell apical plasma membrane, the site of active K⁺ transport. Furthermore, an immunologically related epitope was localized in the apical brush border of Manduca Malpighian tubules. In addition, cross-reactivity of protein bands corresponding to the major subunits of the midgut vacuolar-type ATPase was found in crude homogenates of Malpighian tubules and antennal sensory epithelium of Manduca by immunostaining with a polyclonal anti-holoenzyme serum to the midgut ATPase. This comparative immunological approach suggests that a vacuolar-type ATPase may be a common constituent of insect plasma membranes bearing the insect K⁺ pump. These results support the hypothesis that a vacuolar-type H⁺-ATPase provides an alternative to the Na⁺/K⁺-ATPase in energizing animal plasma membranes.