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Insects
2019 Mar 14;103:. doi: 10.3390/insects10030071.
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Heterologous Expression of Aedes aegypti Cation Chloride Cotransporter 2 (aeCCC2) in Xenopus laevis Oocytes Induces an Enigmatic Na⁺/Li⁺ Conductance.
Kalsi M
,
Gillen C
,
Piermarini PM
.
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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.
Figure 1. Heterologous expression of aeCCC2 in Xenopus oocytes. Western blot of total membrane fractions prepared from oocytes 3 days after injection with aeCCC2 cRNA (80 ng) or nuclease-free H2O (80 nL). Molecular mass markers (kDa) are on the left. Note that an immunoreactive band of ~100 kDa appeared in membrane fractions of both H2O-injected and aeCCC2 oocytes, which presumably indicates reaction of the aeCCC2 antibody with an endogenous protein in Xenopus oocytes.
Figure 2. Rates of Li+ (A) and Rb+ (B) uptake measured in Xenopus oocytes three days after injection of aeCCC2 cRNA (80 ng) or nuclease-free H2O (80 nL). The * represents a significant difference using a Student’s t-test (p < 0.05). Effects of substituting extracellular Cl− with gluconate (C) or iodide (D) on Li+ uptake rates in aeCCC2 or H2O-injected oocytes. The lowercase letters indicate statistical categorization of the means as determined by an ANOVA with a Tukey post-test (p < 0.05). In all panels, values are means ± SEM based on the number of oocytes in parentheses.
Figure 3. Pharmacology of the Li+ uptake in Xenopus oocytes heterologously expressing aeCCC2. Rates of Li+ uptake was measured in Xenopus oocytes 2–3 days after injection with aeCCC2 cRNA (80 ng) or H2O (80 nL). Inhibitor concentrations are indicated on the x-axis. DMSO (0.1%) served as the control. Values are means ± SEM based on the number of oocytes in parentheses. A one-way ANOVA was performed, which was not significant (p > 0.05). EIPA: ethyl isopropyl amiloride; HTZ: hydrochlorothiazide.
Figure 4. The mean current–voltage (I–V) relationships of Xenopus oocytes in ND96 2–3 days after injection with aeCCC2 cRNA (80 ng) or H2O (80 nL) as determined by TEVC. Values are means ± SEM; N = 3 oocytes each for aeCCC2 and H2O.
Figure 5. (A) Representative tracings of membrane current (Im) in aeCCC2 or H2O-injected oocytes that were clamped at −85 mV and −100 mV, respectively. The bidirectional arrows signify the change in membrane current (ΔIm); (B) Summary of relative effects of Na+, K+, and Li+ on the ΔIm in aeCCC2 oocytes. For each oocyte and cation, the ΔIm values were normalized to the ΔIm elicited by Na+ and corrected for the average ΔIm in H2O-injected oocytes (N = 5) associated with that cation. Values are means ± SEM, based on four aeCCC2 oocytes. Lowercase letters indicate statistical categorization of the means based on a one-way repeated-measures ANOVA and Tukey’s post-test (p < 0.05).
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