The bestrophin-1 chloride channel is required in the Malpighian tubules and hindgut for osmoregulation in response to high salt diet
Authors: John Pleinis; Sima Jonusaite; Katherine Beebe; Daryl Morrison; Forest Streeter; Jacob Hudac; Austin Goodwin; Ashlee Roberts; Jeffrey Schellinger; Adrian Rothenfluh; Aylin Rodan
Affiliation: University of Utah
Keywords: j. physiology of adult organs; j. physiology of adult organs
The mechanisms allowing Drosophila melanogaster to tolerate a high-salt diet are poorly understood. Here, we study flies carrying a mutation in the chloride channel, Bestrophin-1 (Best1). Flies with a loss-of-function mutation in Best1, Best1c04106, have increased lethality when fed a high-salt (0.3 M NaCl) diet. High salt lethality is rescued in mutant flies carrying a genomic rescue construct, or with expression of wild-type Best1 in the Malpighian tubules and the rectal pads of the hindgut, the iono- and osmoregulatory epithelia of insects. Malpighian tubules from Best1 mutants have impaired fluid secretion and ion flux. This is likely due to impaired ability to recycle chloride entering through the sodium-potassium-2-chloride cotransporter (NKCC), as there is no effect of Best1 knockdown in flies carrying a loss-of-function mutation in the NKCC-encoding Ncc69. Best1 mutants have lower hemolymph osmolality when fed a normal diet, and fail to increase osmolality as much as controls when fed a high-salt diet. This phenotype is also rescued by the genomic rescue construct, as well as by expression of Best1 in the Malpighian tubule and rectal pads. High salt diet leads to dehydration in control flies, and this is further exacerbated in the Best1 mutants, likely due to greater osmotic loss of water from the hemolymph into the high osmolar gut lumen, as suggested by increased excretion and excreta osmolality in Best1 mutants. Analysis using specific GAL4 lines indicates that coordinated action of the Malpighian tubules and the hindgut is required to modulate excretion and dehydration. Metabolomic analysis indicates that urea is the most upregulated solute in control flies, whereas Best1 mutants fail to upregulate urea. Accordingly, feeding Best1 mutants urea partially rescues high salt lethality, while having no effect on survival in control flies. Best1 mutants have seizures, as occurs in patients with tonicity disorders. Best1 expression in the Malpighian tubules and rectal pads rescues the seizure phenotype, indicating that it is due to the osmoregulatory defects, whereas Best1 knockdown in neurons or glia does not result in seizures. Finally, Best1 mutants sleep less, likely due to searching for water, as water supplementation restores sleep. Thus, Best1 plays an essential role in the Malpighian tubules and hindgut to allow Drosophila to cope with salt stress.