The chloride channel Bestrophin‐1 acts in the Malpighian tubule and hindgut of Drosophila melanogaster to maintain salt and water balance

Abstract

ObjectiveThe Malpighian tubule and hindgut are iono‐ and osmoregulatory epithelia in insects. The tubule secretes KCl‐rich fluid, and ions and water are subsequently reabsorbed in the hindgut. We characterized the role of the chloride channel, Bestrophin‐1 (Best1), in the tubule and hindgut of Drosophila melanogaster.MethodsWe studied flies carrying a germline mutation in Best1, Best1c04106, as well as Best1c04106 flies with genomic rescue, and flies with tissue‐specific knockdown (quantified by qRT‐PCR) or rescue of Best1. Malpighian tubule ion flux was measured ex vivo, and immunostaining was performed using antibodies to HA‐tagged Bestrophin‐1. Flies were assayed for lethality on normal vs. 0.3 M‐supplemented NaCl (high‐salt) diet and after water restriction. Excretion was quantified by counting excretory drops. Hemolymph and excretory drop osmolality were measured using a vapor pressure osmometer. Water weight was determined by measuring flies’ weight before and after baking at 60 °C.ResultsBest1c04106 mutant Malpighian tubules had decreased fluid secretion and transepithelial ion flux that was rescued by expression of wild‐type Best1 in the K+‐secreting principal cell. Principal cell Best1 knockdown decreased transcript levels by 80% and decreased fluid secretion and ion flux. We previously showed that NKCC is required for principal cell K+ secretion. Here, we immunolocalized both Best1 and NKCC to the principal cell basolateral membrane. There was no further decrease in ion transport in Best1 knockdown tubules when NKCC was mutated, suggesting that Best1 allows Cl− entering through the NKCC to recycle across the basolateral membrane. Best1c04106 mutant flies also had increased lethality on high‐salt diet. Hemolymph osmolality increased in control flies fed high‐salt diet, but Best1c04106 mutants had decreased osmolality on both normal and high salt diet, and increased number and osmolality of excretory drops. As a consequence, Best1c04106 mutant flies were dehydrated and died when water restricted. The Best1c04106 mutant phenotypes were rescued by genomic rescue (which recapitulates normal Best1 expression throughout the body), or targeted rescue in the Malpighian tubule and hindgut.ConclusionBest1 is required for normal ion transport in the Drosophila Malpighian tubule, and in the tubule and hindgut for iono‐ and osmoregulation, where ion reabsorption through Best1 in high‐salt conditions allows an increase in hemolymph osmolality to prevent osmotic water loss and dehydration.Support or Funding InformationNIH DK110358