Basolateral ion transport mechanisms during fluid secretion byDrosophilaMalpighian tubules: Na+ recycling,Na+:K+:2Cl– cotransport and Cl– conductance

Abstract

SUMMARY Mechanisms of ion transport during primary urine formation by the Malpighian tubule of Drosophila melanogaster were analyzed through measurements of fluid secretion rate, transepithelial ion flux, basolateral membrane potential (Vbl) and intracellular activities of K+ (aKi) and Cl–(aCli). Calculation of the electrochemical potentials for both ions permitted assessment of the possible contributions of K+ channels, Na+:K+:2Cl–cotransport, and K+:Cl– cotransport, to net transepithelial ion secretion across the basolateral membrane. The data show that passive movement of both K+ and Cl– from cell to bath is favoured across the basolateral membrane, indicating that both ions are actively transported into the cell. Contributions of basolateral K+ channels or K+:Cl– cotransporters to net transepithelial ion secretion can be ruled out. After prior exposure of tubules to ouabain, subsequent addition of bumetanide reduced fluid secretion rate, K+ flux and Na+ flux, indicating a role for a Na+:K+:2Cl– cotransporter in fluid secretion. Addition of the K+ channel blocker Ba2+ had no effect on aKi or aCli. Addition of Ba2+ unmasked a basolateral Cl– conductance and the hyperpolarization of Vbl in response to Ba2+ was Cl–-dependent. A new model for fluid secretion proposes that K+ and Cl– cross the basolateral membrane through a Na+-driven Na+:K+:2Cl–cotransporter and that most of the Na+ that enters the cells is returned to the bath through the Na+/K+-ATPase.

Random Stimulation of Spider Mechanosensory Neurons Reveals Long-Lasting Excitation by GABA and Muscimol

“…Therefore the simulations suggest that the depolarizing role of GABA dominates its effects on VS-3 neurons stimulated by randomly varying membrane current. This finding is consistent with a previous report where a simulated model of spinal cord neurons revealed that shunting was a local effect while the depolarization was longer lasting, spreading electrotonically in the somatodendritic tree leading to excitation (Jean-Xavier et al 2007). …”

“…To learn whether the excitatory GABA response in VS-3 neurons could also be regulated by Cl Ϫ transporters we used the "loop" diuretics furosemide and bumetanide. In addition to NKCC1 these diuretics also inhibit KCC1, a K ϩ -Cl Ϫ cotransporter that actively extrudes Cl Ϫ from cells in vertebrate and invertebrate preparations (Cheung et al 2006;Deisz and Lux 1982;Gillen et al 2006;Hille and Walz 2006;Ianowski and O'Donnell 2004;Theander et al 1999). Furosemide has about equal potency for NKCC1 and KCC1, but bumetanide has a significantly higher affinity for NKCC1 (Payne et al 2003) and would therefore be more likely to affect the response of VS-3 neurons.…”

“…However, this is unlikely since both antagonists have previously been extensively used in many other invertebrate species, including Drosophila (Ianowski and O'Donnell 2004), moth (Gillen et al 2006), cockroach (Hille and Waltz 2006), crayfish (Deisz and Lux 1982), and lobster (Theander et al 1999), suggesting that similar Cl Ϫ transporters in a number of different species are sensitive to these drugs. However, it is also important to remember that in addition to Cl Ϫ , ionotropic GABA receptor channels are permeable to other anions, such A: an original recording shows that although the membrane potential was clamped to Ϫ84 mV, the spike amplitude decreased from 44 to 8 mV in response to application of 100 M muscimol.…”

Hypotonicity Stimulates Potassium Flux through the WNK-SPAK/OSR1 Kinase Cascade and the Ncc69 Sodium-Potassium-2-Chloride Cotransporter in the Drosophila Renal Tubule

“…Thus, a decrease in intracellular chloride occurring during cell swelling and regulatory volume decrease could stimulate WNK through relief of chloride inhibition, followed by Fray and Ncc69 activation, stimulating an increase in transepithelial ion and water flux. Indeed, intracellular Cl Ϫ concentration in the fly tubule is 30 mM in isotonic conditions (72), slightly higher than the apparent median inhibitory concentration of 20 mM for Cl Ϫ inhibition of mammalian WNK1 autophosphorylation (71). This suggests that tubule WNK-Fray signaling is poised to respond to changes in intracellular Cl Ϫ .…”

Transport of H+, Na+ and K+ across the posterior midgut of blood-fed mosquitoes (Aedes aegypti)

“…Bumetanide is well-known as an inhibitor of basolateral Na + :K + :2Cl -cotransport in the Malpighian tubules of dipterans (Scott et al, 2004;Ianowski & O'Donnell, 2004) and other species (e.g. crickets; Coast, 2012).…”