Electrophysiological Analysis of the Mutated Na,K-ATPase Cation Binding Pocket

Abstract

؉ . Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the currentvoltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na ؉ /Na ؉ exchange currents are rather voltage-independent and at potentials above ؊60 mV faster than the wild type. Thus, the characteristic voltagedependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn 776 and Asp 804 , respectively, decreases the extracellular Na ؉ affinity.

Altered Na + transport after an intracellular α-subunit deletion reveals strict external sequential release of Na + from the Na/K pump

“…The change in selectivity of the inward current from H ϩ in normal pumps to Na ϩ in truncated pumps is consistent with such a perturbation. However, evaluation of this possibility will require future examination of ion-binding site mutants with reported reduced Na o ϩ affinity (29).…”

ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

“…Immunoprecipitated proteins were subsequently removed from the beads by incubation with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, fractionated by 8% SDS-PAGE, and transferred to Protran nitrocellulose transfer membranes (Schleicher & Schuell BioScience). The membranes were incubated in a block buffer (phosphate-buffered saline/5% milk powder/0.05% Tween 20) for 1 hour at room temperature and incubated with rabbit polyclonal anti-ATP8B1 (2K), mouse monoclonal anti-HA (clone 12CA5), rabbit polyclonal antibody to Atp1a1 (C356-M09), 26 or mouse monoclonal antibody to green fluorescent protein (GFP; JL-8, Clontech Laboratories) in a block buffer for 2 hours at room temperature. Immune complexes were visualized with peroxidaseconjugated goat-anti-rabbit immunoglobulin G (IgG) or goat-anti-mouse IgG2b (anti-HA and anti-GFP).…”

Conversion of the Low Affinity Ouabain-binding Site of Non-gastric H,K-ATPase into a High Affinity Binding Site by Substitution of Only Five Amino Acids

“…The rat Na,K-ATPase ␣ 1 -and ␤ 1 -subunits were cloned into the pTLN vector as described earlier (17). This vector is suitable for the Xenopus laevis oocyte expression system (18).…”