The Third Sodium Binding Site of Na,K-ATPase Is Functionally Linked to Acidic pH-Activated Inward Current

Abstract

Sodium- and potassium-activated adenosine triphosphatases (Na,K-ATPase) is the ubiquitous active transport system that maintains the Na(+) and K(+) gradients across the plasma membrane by exchanging three intracellular Na(+) ions against two extracellular K(+) ions. In addition to the two cation binding sites homologous to the calcium site of sarcoplasmic and endoplasmic reticulum calcium ATPase and which are alternatively occupied by Na(+) and K(+) ions, a third Na(+)-specific site is located close to transmembrane domains 5, 6 and 9, and mutations close to this site induce marked alterations of the voltage-dependent release of Na(+) to the extracellular side. In the absence of extracellular Na(+) and K(+), Na,K-ATPase carries an acidic pH-activated, ouabain-sensitive "leak" current. We investigated the relationship between the third Na(+) binding site and the pH-activated current. The decrease (in E961A, T814A and Y778F mutants) or the increase (in G813A mutant) of the voltage-dependent extracellular Na(+) affinity was paralleled by a decrease or an increase in the pH-activated current, respectively. Moreover, replacing E961 with oxygen-containing side chain residues such as glutamine or aspartate had little effect on the voltage-dependent affinity for extracellular Na(+) and produced only small effects on the pH-activated current. Our results suggest that extracellular protons and Na(+) ions share a high field access channel between the extracellular solution and the third Na(+) binding site.

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

“…S2). It has been proposed that an increase in [H o ϩ ] makes the internal gate permissive to H ϩ and Na ϩ (at low Na o ϩ ) flow through the exclusive Na ϩ site (21). Thus, one interpretation of our results may be that the C-terminal deletion alters the intracellular gate and thus destabilizes ion occlusion.…”

“…S2). This H ϩ current has been described in endogenous (12), wild-type (19), and mutant Na/K pumps (20,21) expressed in Xenopus oocytes and has been proposed to represent downhill transport of H ϩ through the exclusive Na ϩ binding site when the pump is in E2P (21,22). As the two currents present similar voltage dependencies, TEVC was used to study the [Na o ϩ ] dependence of ouabain-sensitive steady-state current in the absence of K o ϩ in RD⌬KESYY pumps.…”

Selectivity of externally facing ion-binding sites in the Na/K pump to alkali metals and organic cations

“…S5), probably resulting from electrogenic uncoupled Na + efflux (a conclusive determination of its nature exceeds the scope of this report), and an uncoupled inward current observed during negative voltage pulses, which has been associated with an ion pathway through site III. The (19,20) is thought to represent electrochemically dissipative H + flow through site III of the Na,K-ATPase (9,12,13). This transport mode appears too slow and rate limited by protein conformational changes to represent simple ion flow through an ion channel (12,21).…”

“…S2). In addition, several studies have proposed a noncanonical mode of transport in which protons move in the inward direction down their electrochemical gradient at very negative voltages (11) through a pathway that passes through site III (9,10,12,13).…”

Molecular simulations and free-energy calculations suggest conformation-dependent anion binding to a cytoplasmic site as a mechanism for Na+/K+-ATPase ion selectivity

“…Poulsen et al (5) have previously suggested that a cavity between TM5, TM7 and TM8 provides cytoplasmic access in the E2P conformation for protonation of D926 and proton leak currents (30,31). Cytoplasmic access to this cavity was also postulated to be blocked in the E1P conformation by the C-terminal hydrogenbond network (5).…”