Two inwardly rectifying potassium channels,Irk1andIrk2, play redundant roles inDrosophilarenal tubule function

Abstract

Inwardly rectifying potassium channels play essential roles in renal physiology across phyla. Bariumsensitive K ϩ conductances are found on the basolateral membrane of a variety of insect Malpighian (renal) tubules, including Drosophila melanogaster. We found that barium decreases the lumen-positive transepithelial potential difference in isolated perfused Drosophila tubules and decreases fluid secretion and transepithelial K ϩ flux. In those insect species in which it has been studied, transcripts from multiple genes encoding inwardly rectifying K ϩ channels are expressed in the renal (Malpighian) tubule. In Drosophila melanogaster, this includes transcripts of the Irk1, Irk2, and Irk3 genes. The role of each of these gene products in renal tubule function is unknown. We found that simultaneous knockdown of Irk1 and Irk2 in the principal cell of the fly tubule decreases transepithelial K ϩ flux, with no additive effect of Irk3 knockdown, and decreases barium sensitivity of transepithelial K ϩ flux by ϳ50%. Knockdown of any of the three inwardly rectifying K ϩ channels individually has no effect, nor does knocking down Irk3 simultaneously with Irk1 or Irk2. Irk1/Irk2 principal cell double-knockdown tubules remain sensitive to the kaliuretic effect of cAMP. Inhibition of the Na ϩ /K ϩ -ATPase with ouabain and Irk1/Irk2 double knockdown have additive effects on K ϩ flux, and 75% of transepithelial K ϩ transport is due to Irk1/Irk2 or ouabain-sensitive pathways. In conclusion, Irk1 and Irk2 play redundant roles in transepithelial ion transport in the Drosophila melanogaster renal tubule and are additive to Na ϩ /K ϩ -ATPase-dependent pathways.Malpighian tubule; epithelial ion transport; barium; Kir; Ir; Dir; dKirIII; ion-specific electrode; Ramsay assay INWARDLY RECTIFYING POTASSIUM CHANNELS play key roles in vertebrate and invertebrate renal physiology and epithelial ion transport (13, 27, 28, 31, 32, 36 -38, 43, 44, 47). Mutations in KCNJ1/Kir1.1 (also known as renal outer medullary K ϩ channel, or ROMK) and KCNJ10/Kir4.1 result in human diseases characterized by salt-losing tubulopathies and electrolyte disturbances (1,40,42). Inwardly rectifying K ϩ channels also play important roles in insect iono-and osmoregulation. For example, in mosquitoes, pharmacological inhibition of inwardly rectifying K ϩ channels decreases renal tubule fluid secretion and alters both transepithelial ion fluxes in the tubule as well as whole-body ion clearances and urine excretion (28,31,32,36,38,41). This results in decreased viability and flight capacity (31,32,38). As such, inwardly rectifying K ϩ channels are potential targets for insecticidal agents.

Chemical inhibition of Kir channels reduces salivary secretions and phloem feeding of the cotton aphid, Aphis gossypii (Glover)

“…[27][28][29][30] Similarly, several recent lines of genetic and pharmacological evidence suggest Kir channels play important physiological roles in exocrine systems of dipteran insects as well. For instance, genetic depletion or pharmacological inhibition of Kir channels have been shown to reduce the fluid and ion secretion of dipteran Malpighian tubules 24,[31][32][33][34] and previous work has indicated Kir channels are critical for the salivary gland function and feeding of arthropods pests. 25,26,35 The development of insect Kir channel pharmacology 23,34,36,37 has enabled researchers to begin to characterize the physiological role of these channels in various arthropod tissue systems as well as determine the toxicological relevance to pest species.…”

An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria

“…In Drosophila melanogaster , embryonic depletion of Kir1, Kir2, or Kir3 mRNA levels leads to death or defects in wing development12. Knocking down Kir1 and Kir2 mRNA expression in the heart and Malpighian (renal) tubules of Drosophila , respectively, inhibits the immune response against cardiotropic viruses13 and transepithelial secretion of fluid and K + 14. In yellow fever mosquitoes (i.e.…”

When two cells are better than one: specialized stellate cells provide a privileged route for uniquely rapid water flux inDrosophilarenal tubule

“…The main, principal cell has long apical microvilli (40), each containing a mitochondrion (41), and loaded with proton-pumping V-ATPase and is thought to drive an exchanger from the NHA family to produce a net K + flux. Basolaterally, the infoldings contain high levels of Na + , K + -ATPase (46), inward rectifier K + channels (10, 11), and Na + /K + /Cl − cotransporters (14). This metabolically active cell is likely the route for excretion of a wide range of solutes via ABC transporters and other organic solute transporters, many of which hare abundantly expressed in the tubule (24).…”

“…Remarkably, the insect Malpighian (renal) tubule is capable of secreting fluid faster (on a per cell volume basis) than any other epithelium known (1, 2). In Drosophila , the renal tubule has two major cell types (3-5); the mitochondria-rich principal cell actively transports protons via an apical, plasma membrane V-ATPase (6), setting up a gradient which is exchanged primarily for potassium (7, 8) and enters the cell basolaterally through a combination of Na + , K + -ATPase (9), potassium channels (10, 11) and cotransports (12-14). The smaller stellate cell (15, 16) provides a route for hormone-stimulated (17-20) chloride conductance through a basolateral ClC-a chloride channel (21), and secCl, an apical cys-loop chloride channel (22), to balance the lumen-positive charge, and so effect a net movement of salt.…”