Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti

Piermarini, Peter M.; Rouhier, Matthew F.; Schepel, Matthew; Kosse, Christin; Beyenbach, Klaus W.

doi:10.1016/j.ibmb.2012.09.009

Cited by 50 publications

(97 citation statements)

References 76 publications

(129 reference statements)

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“…As shown in Figure 3, blood-feeding elicits a sustained down-regulation of two transcripts encoding Kir channel subunits (AAEL008931-RA, AAEL013373-RA) and one transcript encoding a putative NKCC (AAEL006180-RA). We recently cloned and characterized AAEL008931-RA (Kir2B) from the Malpighian tubules of A. aegypti and have confirmed that it encodes a barium-sensitive Kir channel [41]. The NKCC is an ortholog of Drosophila melanogaster Ncc69, which has been shown to a play a key role in fluid and K + secretion in fruit fly Malpighian tubules [42].…”

Section: Resultsmentioning

confidence: 93%

Transcriptomic Evidence for a Dramatic Functional Transition of the Malpighian Tubules after a Blood Meal in the Asian Tiger Mosquito Aedes albopictus

2014

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BackgroundThe consumption of a vertebrate blood meal by adult female mosquitoes is necessary for their reproduction, but it also presents significant physiological challenges to mosquito osmoregulation and metabolism. The renal (Malpighian) tubules of mosquitoes play critical roles in the initial processing of the blood meal by excreting excess water and salts that are ingested. However, it is unclear how the tubules contribute to the metabolism and excretion of wastes (e.g., heme, ammonia) produced during the digestion of blood.Methodology/Principal FindingsHere we used RNA-Seq to examine global changes in transcript expression in the Malpighian tubules of the highly-invasive Asian tiger mosquito Aedes albopictus during the first 24 h after consuming a blood meal. We found progressive, global changes in the transcriptome of the Malpighian tubules isolated from mosquitoes at 3 h, 12 h, and 24 h after a blood meal. Notably, a DAVID functional cluster analysis of the differentially-expressed transcripts revealed 1) a down-regulation of transcripts associated with oxidative metabolism, active transport, and mRNA translation, and 2) an up-regulation of transcripts associated with antioxidants and detoxification, proteolytic activity, amino-acid metabolism, and cytoskeletal dynamics.Conclusions/SignificanceThe results suggest that blood feeding elicits a functional transition of the epithelium from one specializing in active transepithelial fluid secretion (e.g., diuresis) to one specializing in detoxification and metabolic waste excretion. Our findings provide the first insights into the putative roles of mosquito Malpighian tubules in the chronic processing of blood meals.

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Section: Resultsmentioning

confidence: 93%

Transcriptomic Evidence for a Dramatic Functional Transition of the Malpighian Tubules after a Blood Meal in the Asian Tiger Mosquito Aedes albopictus

2014

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“…Interestingly, no functional channel activity was observed with attempts at expression of Irk3 in Xenopus oocytes or Drosophila S2 cultured cells, whereas both Irk1 and Irk2 possessed inwardly rectifying K ϩ channel activity (7). Similarly, no channel activity was observed in Xenopus oocytes expressing the Aedes aegypti Irk3 homolog, AeKir3, although it is highly expressed in the mosquito tubule (29). In addition, recent immunolocalization data indicates that AeKir3 is expressed in intracellular punctae in the mosquito tubule (28).…”

Section: Inwardly Rectifying Kmentioning

confidence: 99%

“…Examples include the yellow fever mosquito Aedes aegypti (22,41), the forest ant Formica polyctena (17), the Chagas vector Rhodnius prolixus (12), the agricultural pest Locusta migratoria (14), and the mealworm Tenebrio molitor (45). In addition, transcripts for inwardly rectifying K ϩ channels are expressed in the Malpighian tubules of Aedes aegypti, the vector for yellow fever, dengue, and chikungunya (29); Anopheles gambiae, the malaria vector (30); and the bed bug Cimex lectularius (21). Drugs targeting renal tubule inwardly rectifying K ϩ channels are currently being developed as mosquitocidal insecticides in Aedes aegypti (31,32,38).…”

Section: Inwardly Rectifying Kmentioning

confidence: 99%

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Two inwardly rectifying potassium channels,Irk1andIrk2, play redundant roles inDrosophilarenal tubule function

Baum

Huang

et al. 2015

American Journal of Physiology-Regulatory, Integrative and Comp

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Inwardly rectifying potassium channels play essential roles in renal physiology across phyla. Bariumsensitive K ϩ conductances are found on the basolateral membrane of a variety of insect Malpighian (renal) tubules, including Drosophila melanogaster. We found that barium decreases the lumen-positive transepithelial potential difference in isolated perfused Drosophila tubules and decreases fluid secretion and transepithelial K ϩ flux. In those insect species in which it has been studied, transcripts from multiple genes encoding inwardly rectifying K ϩ channels are expressed in the renal (Malpighian) tubule. In Drosophila melanogaster, this includes transcripts of the Irk1, Irk2, and Irk3 genes. The role of each of these gene products in renal tubule function is unknown. We found that simultaneous knockdown of Irk1 and Irk2 in the principal cell of the fly tubule decreases transepithelial K ϩ flux, with no additive effect of Irk3 knockdown, and decreases barium sensitivity of transepithelial K ϩ flux by ϳ50%. Knockdown of any of the three inwardly rectifying K ϩ channels individually has no effect, nor does knocking down Irk3 simultaneously with Irk1 or Irk2. Irk1/Irk2 principal cell double-knockdown tubules remain sensitive to the kaliuretic effect of cAMP. Inhibition of the Na ϩ /K ϩ -ATPase with ouabain and Irk1/Irk2 double knockdown have additive effects on K ϩ flux, and 75% of transepithelial K ϩ transport is due to Irk1/Irk2 or ouabain-sensitive pathways. In conclusion, Irk1 and Irk2 play redundant roles in transepithelial ion transport in the Drosophila melanogaster renal tubule and are additive to Na ϩ /K ϩ -ATPase-dependent pathways.Malpighian tubule; epithelial ion transport; barium; Kir; Ir; Dir; dKirIII; ion-specific electrode; Ramsay assay INWARDLY RECTIFYING POTASSIUM CHANNELS play key roles in vertebrate and invertebrate renal physiology and epithelial ion transport (13, 27, 28, 31, 32, 36 -38, 43, 44, 47). Mutations in KCNJ1/Kir1.1 (also known as renal outer medullary K ϩ channel, or ROMK) and KCNJ10/Kir4.1 result in human diseases characterized by salt-losing tubulopathies and electrolyte disturbances (1,40,42). Inwardly rectifying K ϩ channels also play important roles in insect iono-and osmoregulation. For example, in mosquitoes, pharmacological inhibition of inwardly rectifying K ϩ channels decreases renal tubule fluid secretion and alters both transepithelial ion fluxes in the tubule as well as whole-body ion clearances and urine excretion (28,31,32,36,38,41). This results in decreased viability and flight capacity (31,32,38). As such, inwardly rectifying K ϩ channels are potential targets for insecticidal agents.

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“…We hypothesized that the Kir subunit immunoreactivity would be expressed primarily on the basolateral membrane of principal cells given the results of our previous physiological studies on mosquito Malpighian tubules (Beyenbach and Masia, 2002;Masia et al, 2000;Piermarini et al, 2013;Scott et al, 2004). Moreover, a previous in situ hybridization study in Malpighian tubules of Drosophila melanogaster by the Dow laboratory found the expression of Kir subunit mRNAs to occur only in principal cells (Evans et al, 2005).…”

Section: Introductionmentioning

confidence: 94%

Localization and role of inward rectifier K+ channels in Malpighian tubules of the yellow fever mosquito Aedes aegypti

Piermarini

Dunemann

Rouhier

et al. 2015

Insect Biochemistry and Molecular Biology

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Malpighian tubules of adult female yellow fever mosquitoes Aedes aegypti express three inward rectifier K(+) (Kir) channel subunits: AeKir1, AeKir2B and AeKir3. Here we 1) elucidate the cellular and membrane localization of these three channels in the Malpighian tubules, and 2) characterize the effects of small molecule inhibitors of AeKir1 and AeKir2B channels (VU compounds) on the transepithelial secretion of fluid and electrolytes and the electrophysiology of isolated Malpighian tubules. Using subunit-specific antibodies, we found that AeKir1 and AeKir2B localize exclusively to the basolateral membranes of stellate cells and principal cells, respectively; AeKir3 localizes within intracellular compartments of both principal and stellate cells. In isolated tubules bathed in a Ringer solution containing 34 mM K(+), the peritubular application of VU590 (10 μM), a selective inhibitor of AeKir1, inhibited transepithelial fluid secretion 120 min later. The inhibition brings rates of transepithelial KCl and fluid secretion to 54% of the control without a change in transepithelial NaCl secretion. VU590 had no effect on the basolateral membrane voltage (Vbl) of principal cells, but it significantly reduced the cell input conductance (gin) to values 63% of the control within ∼90 min. In contrast, the peritubular application of VU625 (10 μM), an inhibitor of both AeKir1 and AeKir2B, started to inhibit transepithelial fluid secretion as early as 60 min later. At 120 min after treatment, VU625 was more efficacious than VU590, inhibiting transepithelial KCl and fluid secretion to ∼35% of the control without a change in transepithelial NaCl secretion. Moreover, VU625 caused the Vbl and gin of principal cells to respectively drop to values 62% and 56% of the control values within only ∼30 min. Comparing the effects of VU590 with those of VU625 allowed us to estimate that AeKir1 and AeKir2B respectively contribute to 46% and 20% of the transepithelial K(+) secretion when the tubules are bathed in a Ringer solution containing 34 mM K(+). Thus, we uncover an important role of AeKir1 and stellate cells in transepithelial K(+) transport under conditions of peritubular K(+) challenge. The physiological role of AeKir3 in intracellular membranes of both stellate and principal cells remains to be determined.

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Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti

Cited by 50 publications

References 76 publications

Transcriptomic Evidence for a Dramatic Functional Transition of the Malpighian Tubules after a Blood Meal in the Asian Tiger Mosquito Aedes albopictus

Transcriptomic Evidence for a Dramatic Functional Transition of the Malpighian Tubules after a Blood Meal in the Asian Tiger Mosquito Aedes albopictus

Two inwardly rectifying potassium channels,Irk1andIrk2, play redundant roles inDrosophilarenal tubule function

Localization and role of inward rectifier K+ channels in Malpighian tubules of the yellow fever mosquito Aedes aegypti

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