Isolation and cloning of the K+-independent, ouabain-insensitive Na+-ATPase

Abstract

Primary Na+ transport has been essentially attributed to Na+/K+ pump. However, there are functional and biochemical evidences that suggest the existence of a K+-independent, ouabain-insensitive Na+ pump, associated to a Na+-ATPase with similar characteristics, located at basolateral plasma membrane of epithelial cells. Herein, membrane protein complex associated with this Na+-ATPase was identified. Basolateral membranes from guinea-pig enterocytes were solubilized with polyoxyethylene-9-lauryl ether and Na+-ATPase was purified by concanavalin A affinity and ion exchange chromatographies. Purified enzyme preserves its native biochemical characteristics: Mg2+ dependence, specific Na+ stimulation, K+ independence, ouabain insensitivity and inhibition by furosemide (IC50: 0.5 mM) and vanadate (IC50: 9.1 μM). IgY antibodies against purified Na+-ATPase did not recognize Na+/K+-ATPase and vice versa. Analysis of purified Na+-ATPase by SDS-PAGE and 2D-electrophoresis showed that is constituted by two subunits: 90 (α) and 50 (β) kDa. Tandem mass spectrometry of α-subunit identified three peptides, also present in most Na+/K+-ATPase isoforms, which were used to design primers for cloning both ATPases by PCR from guinea-pig intestinal epithelial cells. A cDNA fragment of 1148 bp (atna) was cloned, in addition to Na+/K+-ATPase α1-isoform cDNA (1283 bp). In MDCK cells, which constitutively express Na+-ATPase, silencing of atna mRNA specifically suppressed Na+-ATPase α-subunit and ouabain-insensitive Na+-ATPase activity, demonstrating that atna transcript is linked to this enzyme. Guinea-pig atna mRNA sequence (2787 bp) was completed using RLM-RACE. It encodes a protein of 811 amino acids (88.9 kDa) with the nine structural motifs of P-type ATPases. It has 64% identity and 72% homology with guinea-pig Na+/K+-ATPase α1-isoform. These structural and biochemical evidences identify the K+-independent, ouabain-insensitive Na+-ATPase as a unique P-type ATPase.

Altered signaling pathways linked to angiotensin II underpin the upregulation of renal Na+-ATPase in chronically undernourished rats

“…Ang II is one of the most prominent activators of renal Na + -ATPase [11][12][13], and the intense increase in the number of Ang II-positive cells in the interstitium neighboring proximal tubule cells (Fig. 4) reveals hyperactivity of the system in the vicinity of the basolateral membranes where the pump is located [6,7]. As the result of the increased Na + efflux across the basolateral membrane mediated by the Na + -ATPase, it is 5.…”

“…2), which culminates in an overall augmented pumping turnover. The remarkably increased affinity of the α-subunit of the pump for Na + would favor its binding in the E 1 conformation at sites of coordination formed by the transmembrane segments, M1, M2, M4 and M6 [6], followed by the Na + -dependent binding of ATP and phosphoryl transfer at the highly conserved D 364 . This proposed sequence of events would result in increased P i release from Na + -ATPase of BRD rats (Fig.…”

“…A further issue was to see whether phosphorylatable domains exist in the recently identified sequence of Na + -ATPase [6,7], which could be targeted by PKC or PKA. With the NetPhos 2 tool [35], we first found 15 Ser and 5 Thr cytosolic potentially phosphorylatable residues, which were located in the center of sequences containing 9 amino acids with a phosphorylation score ≥0.70.…”

“…Primary compromise of kidney functioning by chronic malnutrition impacts the cardiovascular system, increasing the risk of developing hypertension and atherosclerosis [reviewed in 4]. We recently demonstrated that chronically undernourished rats present simultaneous losartan-sensitive alterations in renal and cardiac Na + handling [5], in which upregulation of the recently cloned and purified ouabain-resistant, furosemide-inhibitable Na + -ATPase is pivotal [6,7]. This pump is considered to be the fine-tune mechanism of Na + reabsorption by proximal tubule cells [8], and its altered kinetic and regulatory properties constitute a hallmark of kidney alterations that involve deregulated local angiotensin II (Ang II) levels, inflammation and reactive oxygen species (ROS) signaling [9,10].…”

The Drosophila NKCC Ncc69 is required for normal renal tubule function

“…The Ncc83 gene sequence is homologous to the NCC/NKCC branch of SLC12 transporters (42,75), but the putative cotransporter encoded by this gene has not been studied so far. Additional targets of high-dose bumetanide that have been demonstrated in other systems include potassium-chloride cotransporters (28,29,48), parallel Na ϩ /H ϩ and Cl Ϫ /HCO 3 Ϫ exchange in cortical thick ascending limb (22)(23)(24), a K ϩ -independent NaCl uptake pathway in rabbit medullary thick ascending limb (2,21), and the ouabain-insensitive Na ϩ -ATPase (69). Whether any of these mechanisms are operative in the fly renal tubule remains to be determined.…”