Stabilization of the α2 Isoform of Na,K-ATPase by Mutations in a Phospholipid Binding Pocket

Abstract

Background:The ␣2 isoform of Na,K-ATPase is unstable compared with ␣1 and ␣3. Results: Mutations in TM8 -10 strongly stabilize ␣2. A novel phospholipid antagonist selectively inactivates ␣2, and mutations in TM8 -10 protect against inactivation. Conclusion: A phosphatidylserine binding pocket within TM8 -10 has been identified. Significance: Mechanistic insights into ␣2 instability and a possible physiological role have been obtained.

Digoxin derivatives with selectivity for the α2β3 isoform of Na,K-ATPase potently reduce intraocular pressure

“…Purified isoform complexes (0.3-0.5 mg/mL) were eluted from the BD-Talon beads in a solution containing 200 mM Imidazole, 100 mM NaCl, 20 mM Tricine•HCl pH 7.4, 0.1 mg/mL C 12 E 8 , 0.07 mg/mL 1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-LSerine], 0.01 mg/mL cholesterol, and 25% (vol/vol) glycerol, by gravity column. The isoforms were reconstituted together with purified FXYD1 on the BD-Talon beads before elution, as described (13,14). Proteins were stored at -80°C.…”

“…To screen for isoform selectivity of the digoxin derivatives, we compared inhibition of Na,K-ATPase activity of purified detergentsoluble human isoform complexes α1β1FXYD1, α2β1FXYD1 described previously (11,13,14), and α2β3FXYD1, which is described briefly in Methods. Although all of the preparations were reconstituted with FXYD1 to stabilize them (14), for simplicity the FXYD1 is omitted in naming of isoform complexes.…”

Stimulation, Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

“…pastoris transformation, yeast growth, membrane preparation, and His tag purification of recombinant human ␣1␤1FXYD1 Na,K-ATPase were done essentially as described previously (27)(28)(29).…”

“…Recombinant Human FXYD1 Expression, Purification, and Reconstitution-Human FXYD1 (phospholemman (PLM)) was expressed in Escherichia coli CD41 cells from pET28-TevH vector (29). FXYD1 purification was described in detail previously (27).…”

“…In the presence of SOPS, cholesterol causes strong additional stabilization of the Na,K-ATPase (28). Using a mutant of the thermo-labile ␣ 2 isoform (␣ 2 VFP), we have recently shown that SOPS is bound between ␣TM8 and -10 and interacts with the FXYD protein (29). Using the same mutant, we provide here evidence for a direct interaction of cholesterol and SOPS, making the C-terminal helices of the ␣-subunit, the FXYD protein, and the lipids bound between ␣TM8 and -10 a stability "hotspot."…”