Search

Abstract: Inward-rectifier potassium (Kir) channels play key roles in nerve, muscle, and epithelial cells in mammals, but their physiological roles in insects remain to be determined. The yellow fever mosquito (Aedes aegypti) possesses five different genes encoding Kir channel subunits: Kir1, Kir2A, Kir2B, Kir2B′, and Kir3. We have recently cloned and characterized the Kir1, Kir2B, and Kir3 cDNAs in the renal (Malpighian) tubules of adult female Ae. aegypti. Here we characterize the expression of the Kir2A gene in Ae. aegypti, which was not abundantly expressed in Malpighian tubules. We find that the 1) Kir2A gene is expressed primarily in the midgut and hindgut of adult female mosquitoes, and 2) Kir2A mRNAs are alternatively spliced into three distinct variants (Kir2A-a, -b, and -c). The deduced Kir2A proteins from these splice forms share a completely conserved transmembrane domain (a pore-forming domain flanked by two transmembrane-spanning segments), but possess novel NH2-terminal and/or COOH-terminal domains. Semi-quantitative RT-PCR analyses indicate that the splice variants exhibit both developmental- and tissue-specific expression. Lastly, we provide insights into the conservation of alternative splicing among the Kir2A genes of dipterans, which may add molecular diversity that compensates for the relatively limited number of Kir channel genes in insects compared to mammals. [Copyright &y& Elsevier]

Abstract: Inward-rectifying K+ (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K+ currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na+. Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl+ > K+ > Rb+ > NH4 +) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K+. [Copyright &y& Elsevier]

Malpighian tubules of adult female yellow fever mosquitoes Aedes aegypti express three inward rectifier K + (Kir) channel subunits: Ae Kir1, Ae Kir2B and Ae Kir3. 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 Ae Kir1 and Ae Kir2B 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 Ae Kir1 and Ae Kir2B localize exclusively to the basolateral membranes of stellate cells and principal cells, respectively; Ae Kir3 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 Ae Kir1, 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 (V bl ) of principal cells, but it significantly reduced the cell input conductance (g in ) to values 63% of the control within ∼90 min. In contrast, the peritubular application of VU625 (10 μM), an inhibitor of both Ae Kir1 and Ae Kir2B, 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 V bl and g in 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 Ae Kir1 and Ae Kir2B 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 Ae Kir1 and stellate cells in transepithelial K + transport under conditions of peritubular K + challenge. The physiological role of Ae Kir3 in intracellular membranes of both stellate and principal cells remains to be determined. [ABSTRACT FROM AUTHOR]