In vivo recycling of nitrate (NO 3 − ) and nitrite (NO 2 − ) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate–nitrite–NO balance. More than 25% of the circulating NO 3 − is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO 3 − to NO 2 − , which enters circulation and leads to NO generation. The transporters for NO 3 − in salivary glands have not yet been identified. Here we report that sialin ( SLC17A 5 ), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO 3 − /H + cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO 3 − or sialic acid (SA), but not by Br − , and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO 3 − -induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H + -dependent NO 3 − conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO 3 − secretion in saliva after intake of a NO 3 − -rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO 3 − /H + transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis.

With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Malpighian tubule. Using assays and transgenic lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux., autophosphorylation of purified WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, WNK activity acutely increased. WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In kinase assays, Mo25 enhanced the activity of the WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type , with or without, did not affect transepithelial ion transport, overexpressed with chloride-insensitive increased ion flux. Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.

The electroneutral cation-chloride cotransporter gene family, SLC12, contains nine members in vertebrates. These include seven sodium and/or potassium-coupled chloride transporters and two membrane proteins of unknown function. Although SLC12 family members have been identified in a number of lower species, the functional properties of these proteins are unknown. There are five SLC12 homologues in Drosophila melanogaster, including at least one member on each of the four main branches of the vertebrate phylogenetic tree. We have employed in situ hybridization to study the expression patterns of the Drosophila SLC12 proteins during embryonic development. Our studies indicate that all five members of this family are expressed during early embryogenesis (stages 1-6), but that spatial and temporal expression patterns become more refined as development proceeds. Expression during late embryogenesis was seen predominantly in the ventral nerve cord, salivary gland, gut, and anal pad. In parallel studies, we have carried out transport assays on each of the five Drosophila homologues, expressed as recombinant proteins in the cultured insect cell line High Five. Under our experimental conditions, we found that only one of these proteins, CG4357, transported the potassium congener (86)Rb. Additional experiments established that rubidium transport via CG4357 was saturable (K(m) = 0.29 +/- 0.05 mM), sodium-dependent (half-saturation constant = 53 +/- 11 mM), chloride-dependent (half-saturation constant = 48 +/- 5 mM), and potently inhibited by bumetanide (inhibitor constant = 1.17 +/- 0.08 muM), a specific inhibitor of Na(+)-K(+)-2Cl(-) cotransporters. Taken together, our results provide strong evidence that CG4357 is an insect ortholog of the vertebrate Na(+)-K(+)-2Cl(-) cotransporters.

WNK (With No Lysine (K)) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low or high potassium diet. We therefore investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of Drosophila WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either ~13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na+/K+-ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule Drosophila WNK activity. In vitro, potassium increased the melting temperature of Drosophila WNK, WNK1 and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of Drosophila WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, SPAK (Ste20-related proline/alanine-rich kinase). The greatest sensitivity of WNK4 to potassium occurred in the range of 80 to 180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of Drosophila and mammalian WNK kinases through direct effects of potassium ion on the kinase.

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