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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).
Beyenbach,
Membrane conductances of principal cells in Malpighian tubules of Aedes aegypti.
2002, Pubmed
Beyenbach,
Membrane conductances of principal cells in Malpighian tubules of Aedes aegypti.
2002,
Pubmed
Beyenbach,
Transcellular and paracellular pathways of transepithelial fluid secretion in Malpighian (renal) tubules of the yellow fever mosquito Aedes aegypti.
2011,
Pubmed
Blaesse,
Cation-chloride cotransporters and neuronal function.
2009,
Pubmed
Bowles,
Characterization of Rb uptake into Sf9 cells using cation chromatography: evidence for a K-Cl cotransporter.
2001,
Pubmed
Breitwieser,
Osmotic stimulation of Na(+)-K(+)-Cl- cotransport in squid giant axon is [Cl-]i dependent.
1990,
Pubmed
Cater,
The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter.
2016,
Pubmed
Choi,
An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel.
2000,
Pubmed
,
Xenbase
Coast,
Mosquito natriuretic peptide identified as a calcitonin-like diuretic hormone in Anopheles gambiae (Giles).
2005,
Pubmed
Coast,
Neuroendocrine control of ionic homeostasis in blood-sucking insects.
2009,
Pubmed
Darman,
A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter, NKCC1.
2002,
Pubmed
Delpire,
Human and murine phenotypes associated with defects in cation-chloride cotransport.
2002,
Pubmed
Dürr,
Measuring cation transport by Na,K- and H,K-ATPase in Xenopus oocytes by atomic absorption spectrophotometry: an alternative to radioisotope assays.
2013,
Pubmed
,
Xenbase
Fiumelli,
An ion transport-independent role for the cation-chloride cotransporter KCC2 in dendritic spinogenesis in vivo.
2013,
Pubmed
Gadsby,
Ion channels versus ion pumps: the principal difference, in principle.
2009,
Pubmed
Hekmat-Scafe,
Mutations in the K+/Cl- cotransporter gene kazachoc (kcc) increase seizure susceptibility in Drosophila.
2006,
Pubmed
Horn,
Premature expression of KCC2 in embryonic mice perturbs neural development by an ion transport-independent mechanism.
2010,
Pubmed
Ianowski,
Basolateral ion transport mechanisms during fluid secretion by Drosophila Malpighian tubules: Na+ recycling, Na+:K+:2Cl- cotransport and Cl- conductance.
2004,
Pubmed
Kaila,
Cation-chloride cotransporters in neuronal development, plasticity and disease.
2014,
Pubmed
Kinne,
Ammonium transport in medullary thick ascending limb of rabbit kidney: involvement of the Na+,K+,Cl(-)-cotransporter.
1986,
Pubmed
Kwon,
Role in diuresis of a calcitonin receptor (GPRCAL1) expressed in a distal-proximal gradient in renal organs of the mosquito Aedes aegypti (L.).
2012,
Pubmed
Laemmli,
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
1970,
Pubmed
Leiserson,
Drosophila glia use a conserved cotransporter mechanism to regulate extracellular volume.
2011,
Pubmed
Linsdell,
Cystic fibrosis transmembrane conductance regulator (CFTR): Making an ion channel out of an active transporter structure.
2018,
Pubmed
Lytle,
Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl.
1996,
Pubmed
Nessler,
Evidence for activation of endogenous transporters in Xenopus laevis oocytes expressing the Plasmodium falciparum chloroquine resistance transporter, PfCRT.
2004,
Pubmed
,
Xenbase
Orlov,
Cation-chloride cotransporters: regulation, physiological significance, and role in pathogenesis of arterial hypertension.
2014,
Pubmed
Payne,
Molecular operation of the cation chloride cotransporters: ion binding and inhibitor interaction.
2012,
Pubmed
Payne,
Molecular characterization of a putative K-Cl cotransporter in rat brain. A neuronal-specific isoform.
1996,
Pubmed
Pedersen,
Intramolecular and intermolecular fluorescence resonance energy transfer in fluorescent protein-tagged Na-K-Cl cotransporter (NKCC1): sensitivity to regulatory conformational change and cell volume.
2008,
Pubmed
Petzel,
Preliminary isolation of mosquito natriuretic factor.
1985,
Pubmed
Petzel,
Hormone-controlled cAMP-mediated fluid secretion in yellow-fever mosquito.
1987,
Pubmed
Piermarini,
NHE8 is an intracellular cation/H+ exchanger in renal tubules of the yellow fever mosquito Aedes aegypti.
2009,
Pubmed
,
Xenbase
Piermarini,
Role of an apical K,Cl cotransporter in urine formation by renal tubules of the yellow fever mosquito (Aedes aegypti).
2011,
Pubmed
,
Xenbase
Piermarini,
Differential expression of putative sodium-dependent cation-chloride cotransporters in Aedes aegypti.
2017,
Pubmed
Ponce-Coria,
Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases.
2008,
Pubmed
,
Xenbase
Pond,
The chloride transporter Na(+)-K(+)-Cl- cotransporter isoform-1 contributes to intracellular chloride increases after in vitro ischemia.
2006,
Pubmed
Prael,
Use of chemical probes to explore the toxicological potential of the K+/Cl- cotransporter (KCC) as a novel insecticide target to control the primary vector of dengue and Zika virus, Aedes aegypti.
2018,
Pubmed
Rodan,
The Drosophila NKCC Ncc69 is required for normal renal tubule function.
2012,
Pubmed
Sawyer,
Dibutyryl-cAMP increases basolateral sodium conductance of mosquito Malpighian tubules.
1985,
Pubmed
Sun,
Developmental and functional studies of the SLC12 gene family members from Drosophila melanogaster.
2010,
Pubmed
Walters,
NKCC1 (SLC12a2) induces a secondary axis in Xenopus laevis embryos independently of its co-transporter function.
2009,
Pubmed
,
Xenbase
Worrell,
Ammonium effects on colonic Cl- secretion: anomalous mole fraction behavior.
2004,
Pubmed