Functional analysis of mosquito and Drosophila Na ⁺ ‐dependent cation‐chloride cotransporters

Dipteran Na + ‐dependent cation‐chloride cotransporters (CCCs) group into two clades. Drosophila melanogaster Ncc69 is a bona fide Na + ‐K + ‐Cl − cotransporter and has a single ortholog in Aedes aegypti (aeNKCC1). Drosophila Ncc83 has not been physiologically characterized and has two orthologs in Ae. aegypti (aeCCC2 and aeCCC3). Previous work showed that oocytes expressing aeCCC2 have higher Na + conductances than controls. Moreover, in flux assays using Li + as a Na + tracer, Li + transport by aeCCC2 was not dependent upon extracellular Cl − and not inhibited by the loop diuretics furosemide or bumetanide. These are surprising results, since previously characterized CCCs are electroneutral, Cl − ‐dependent, and sensitive to the loop diuretics. We have further characterized aeCCC2 and performed an initial analysis of Ncc83, using two‐electrode voltage clamp. Following a hypotonic pre‐incubation, changes in membrane potential upon Na + replacement were 20 ± 3 mV in Ncc83 and 26 ± 4 mV in aeCCC2, approximately two‐fold greater than before swelling. Na + ‐dependent currents in oocytes expressing aeCCC2 depended on extracellular Na + with saturable Michaelis‐Menton kinetics. The apparent affinity for Na + was 4.6 ± 0.5 mM (mean, SEM, n=5). To characterize the function of Ncc83 in Drosophila Malpighian tubules (MT), we developed procedures to assess the cation concentrations of secreted fluid in Ramsay secretion assays using cation‐exchange chromatography. The physiological cations were readily detectable in 10,000–50,000 fold dilutions of secreted fluid, and Li + and Rb + were also detectable when added to the bathing saline as tracers for Na + and K + . In w118‐control flies, fluid secreted by MT measured using these procedures had a [K + ] of 104.1 ± 8.8 mM (mean, SEM, n=7) and [Na + ] of 61.2 ± 7.8 mM. In preliminary experiments, [K + ] was 112.7 ± 1.3 mM (n=4) and [Na + ] was 48.2 ± 4.1 mM in fluid secreted by MTs of flies with MT‐specific knockdown of Ncc83 using the GAL4‐UAS RNAi system. Further work is needed to determine the physiological roles of Ncc83 and aeCCC2. Funding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College. Support or Funding Information Funding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.