Abstract
The yellow fever mosquito Aedes aegypti possesses three genes encoding putative Na+-coupled cation chloride cotransporters (CCCs): aeNKCC1, aeCCC2, and aeCCC3. To date, none of the aeCCCs have been functionally characterized. Here we expressed aeCCC2 heterologously in Xenopus oocytes and measured the uptake of Li+ (a tracer for Na+) and Rb+ (a tracer for K+). Compared to control (H2O-injected) oocytes, the aeCCC2-expressing oocytes exhibited significantly greater uptake of Li+, but not Rb+. However, the uptake of Li+ was neither Cl−-dependent nor inhibited by thiazide, loop diuretics, or amiloride, suggesting unconventional CCC activity. To determine if the Li+-uptake was mediated by a conductive pathway, we performed two-electrode voltage clamping (TEVC) on the oocytes. The aeCCC2 oocytes were characterized by an enhanced conductance for Li+ and Na+, but not K+, compared to control oocytes. It remains to be determined whether aeCCC2 directly mediates the Na+/Li+ conductance or whether heterologous expression of aeCCC2 stimulates an endogenous cation channel in the oocyte plasma membrane.
Highlights
The sodium potassium chloride cotransporters (NKCCs) belong to the solute carrier-12(SLC-12) superfamily of cation chloride cotransporters (CCCs), which includes sodium chloride cotransporters (NCCs) and potassium chloride cotransporters (KCCs) [1]
In larval and adult female Ae. aegypti, aeNKCC1 and aeCCC2 mRNAs were highly expressed in the Malpighian tubules and hindgut, whereas aeCCC3 mRNAs were primarily expressed in the anal papillae of larvae [16]
Our results suggest that heterologous expression of aeCCC2 stimulates an endogenous cation channel in the oocyte plasma membrane and/or that aeCCC2 possesses channel-like properties independent of conventional CCC activity
Summary
(SLC-12) superfamily of cation chloride cotransporters (CCCs), which includes sodium chloride cotransporters (NCCs) and potassium chloride cotransporters (KCCs) [1]. CCCs have not been as extensively studied, but pharmacological and genetic studies in fruit flies and mosquitoes suggest critical roles in neuronal function and transepithelial fluid secretion [9,10,11,12,13,14,15]. The genome of Ae. aegypti contains three putative Na-coupled CCCs, which we have designated as aeNKCC1 ( known as aeCCC1), aeCCC2, and aeCCC3. The aeNKCC1 gene contains substantial similarity to vertebrate bumetanide-sensitive NKCCs and Drosophila melanogaster NCC69 (CG4357), a bonafide bumetanide-sensitive NKCC that contributes to transepithelial fluid secretion in Malpighian
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