Function and regulation of the insect NaCCC2 sodium transport proteins

Abstract

NaCCC2 transport proteins, including those from Drosophila melanogaster (Ncc83) and Aedes aegypti (aeCCC2), are an insect-specific clade with sequence similarity to Na⁺-K⁺-2Cl⁻ cotransporters. Whereas the Na⁺-K⁺-2Cl⁻ cotransporters and other cation-chloride cotransporters are electroneutral, recent work indicates that Ncc83 and aeCCC2 carry charge across membranes. Here, we further characterize the regulation and transport properties of Ncc83 and aeCCC2 expressed in Xenopus oocytes. In cation uptake experiments, Li⁺ was used as a tracer for Na⁺ and Rb⁺ was used as a tracer for K⁺. Li⁺ uptake of oocytes expressing either aeCCC2 or Ncc83 was greater than uptake in water-injected controls, activated by hypotonic swelling, and not inhibited by ouabain or ethyl cinnamate. Rb⁺ uptake of oocytes expressing either aeCCC2 or Ncc83 was not different than water injected controls. In oocytes expressing either aeCCC2 or Ncc83, Li⁺ uptake plateaued with increasing Li⁺ concentrations, with apparent K_m values in the range of 10 to 20 mM. Following exposure to ouabain, intracellular [Na⁺] was greater in oocytes expressing aeCCC2 than in controls. Elevating intracellular cAMP (via 8-bromo-cAMP) in Ncc83 oocytes significantly stimulated both Li⁺ uptake and membrane conductances. Elevating intracellular cAMP in aeCCC2 oocytes did not affect Li⁺ uptake, but stimulated membrane conductances. Overall, these results confirm that the NaCCC2s resemble other cation-chloride cotransporters in their regulation and some transport properties. However, unlike other cation-chloride cotransporters, they carry charge across membranes.