The V-type H+-ATPase is targeted in anti-diuretic hormone control of the Malpighian ‘renal’ tubules

Like other insects, secretion by mosquito Malpighian tubules (MTs) is driven by the V-type H+-ATPase (VA) localized in the apical membrane of principal cells. The anti-diuretic neurohormone CAPA inhibits secretion by MTs stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we demonstrate that the VA inhibitor bafilomycin selectively inhibits serotonin (5HT)- and calcitonin-related diuretic hormone (DH31)-stimulated secretion. VA activity increases in DH31-treated MTs, whereas CAPA abolishes this increase through a NOS/cGMP/PKG signaling pathway. A critical feature of VA activation involves the reversible association of the cytosolic (V1) and membrane (Vo) complexes. Indeed, higher V1 protein abundance was found in membrane fractions of DH31-treated MTs whereas CAPA significantly decreased V1 abundance in membrane fractions while increasing it in cytosolic fractions. Immunolocalization of V1 was observed strictly in the apical membrane of MTs treated with DH31 alone whereas immunoreactivity was dispersed following CAPA treatment. VA complexes colocalized apically in female MTs shortly after a blood-meal consistent with the peak and post-peak phases of diuresis. Comparatively, V1 immunoreactivity in MTs was more dispersed and did not colocalize with the Vo complex in the apical membrane at 3 hours post blood-meal, representing a timepoint after the late phase of diuresis has concluded. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target in hormone-mediated inhibition of MTs countering diuresis that provides a deeper understanding of this critical physiological process necessary for hydromineral balance. Significance Statement The V-type H+ ATPase (VA), also known as the proton pump, provides the driving force for transepithelial ion and fluid secretion in insect Malpighian tubules (MTs). While studies have shown that diuretic stimulation activates various signaling pathways promoting increased VA activity, our understanding of anti-diuretic signaling and its potential regulation of the VA remains rudimentary. Here we show that a CAPA neuropeptide acts through the NOS/cGMP/PKG pathway to inhibit VA activity, supporting the notion that the anti-diuretic regulation is achieved by promoting dissociation of the VA complexes. These results demonstrate a critical role of VA inhibition and trafficking necessary for anti-diuretic signaling and advances our understanding of the complex neuroendocrine control of the MTs in this important human disease-vector mosquito.