The Inner Workings of Proton Slippage through the Sodium Pump

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In Na⁺/K⁺-ATPase research, important findings sometimes need a long breathing space to be properly acknowledged. While the latter is true for the 1997 Nobel prize in Chemistry awarded to J. C. Skou for the discovery of the Na⁺/K⁺-ATPase ∼40 years earlier, the former can equally be said regarding the peculiar ouabain-sensitive leak currents passing through the Na⁺/K⁺-ATPase. First described in 1991 by Rakowski et al. (1), this somewhat irritating finding suffered a shadowy existence for many years. Only a few researchers gave it due attention, and despite the fact that Wang and Horisberger prophetically titled their 1995 article “A Conformation of Na⁺-K⁺ Pump is Permeable to Proton” (2), it took another 20 years until threads from human disease, crystal structures, and electrophysiology converged into a fundamental understanding of the interactions of protons with the Na⁺/K⁺-ATPase. The work of Stanley et al. (3) in this issue of the Biophysical Journal finally establishes the intracellular requirements of proton currents through the Na⁺/K⁺-ATPase and adds an essential cornerstone to the Na⁺/K⁺-ATPase literature.

Having established the extracellular conditions needed for proton currents through the Na⁺/K⁺-ATPase in previous work (4), the Artigas lab needed to take a deep breath before tackling the intracellular requirements, because the only technique that gathers about a billion Na⁺ pumps in a single patch of membrane (required to record picoampere currents) involves drawing giant excised inside-out patches from Xenopus laevis oocytes (5). To this end, the results from an exhaustive series of rigorous experiments presented by the team (3) eliminate another blank area on the map of ion transporter research with cross-disciplinary implications into other fields of biomedical research. Stanley et al. (3) set out to study the effects of intracellularly applied ligands on the proton current (I_H) and compared these to the properties of the so-called transient currents of the Na⁺/K⁺-ATPase. The latter, which occur in the phosphorylated enzyme upon voltage steps at high extracellular [Na⁺] in the absence of extracellular K⁺, are integrated to give the moved charge (Q_Na) as a measure of the voltage dependence of Na⁺ transport. Although the intracellular [Na⁺] dependence of I_H and Q_Na were quite similar, I_H needed 100-fold higher MgATP concentrations than required for the high (submicromolar) affinity phosphorylation that facilitates Na⁺ shuttling, suggesting that low-affinity ATP binding without hydrolysis is required for I_H on top of high-affinity phosphorylation. In line with this, addition of the nonhydrolysable ATP analog AMPPNP to low-micromolar [ATP] required for phosphorylation also stimulated I_H. These results conform well with previous extracellular ligand studies that had already delineated the importance of Na⁺-dependent pump phosphorylation on I_H (4, 6).

However, the finding that ATP and ADP activated I_H even in the presence of intracellular K⁺ certainly must have given the authors a headache. But, from long training in the field, the team knew about the process of back-door phosphorylation of the Na⁺/K⁺-ATPase, which occurs in reverse direction of the pumping cycle (see Fig. 1) if enough inorganic phosphate (P_i), Mg²⁺ and K⁺ is present on the intracellular side (7). Scrutinizing their ATP-containing buffer solutions for P_i revealed unavoidable impurities of some tenths of a percent of free P_i formed by spontaneous hydrolysis. Nevertheless, these micromolar P_i concentrations in ATP-containing buffers were sufficient to activate I_H, and further augmentation of I_H was seen upon deliberate addition of P_i to the solution. Because the process also required Mg²⁺ as a general prerequisite for Na⁺/K⁺-ATPase phosphorylation, it is now established that back-door phosphorylation is also sufficient to bring about I_H through the enzyme.

Figure 1.

Na⁺/K⁺-ATPase states permitting proton leak currents. The enzyme in (A) assumes a phosphorylated E₂ state, with extracellular-open cation binding sites, ATP bound with low affinity (E₂P/ATP) and cation binding sites not fully occupied by Na⁺ (●) or K⁺ (▪) ions, therefore, the extracellular cation occlusion gate (shaded bar) cannot fully close. Protons (⊗) enter and leave the third cation binding site exclusive for Na⁺ from the extracellular side by a different pathway than the normal transport substrates (thick dashed line). The E₂P/ATP state can be reached through Na⁺-dependent, high-affinity phosphorylation via E₁P (forward direction of pump cycle) or via back-door phosphorylation by inorganic phosphate in the presence of intracellular K⁺ and Mg²⁺ (reverse direction of pump cycle). The remaining reaction cycle intermediates are only schematically indicated at the bottom. Alternatively (B), an E₂P state permitting proton leak currents is formed upon binding of BeF₃^–, which stabilizes an E₂P-like intermediate with extracellular-open cation binding sites.

A beryllium fluoride experiment provided the final piece complementing the puzzle. Like fluoride complexes of Mg²⁺ and Al³⁺, beryllium fluoride (BeF₃^–) is known to act as phosphate analog that mimics phosphorylation by binding to the conserved aspartate residue in the Na⁺/K⁺-ATPase’s phosphorylation domain. It enforces an externally open E₂P-like conformation, which is sufficiently rigid to even enable crystallization. Whereas BeF₃^– abolishes Na⁺/K⁺ pump function (6), I_H in the presence of BeF₃^– is even larger than when it is induced by MgATP. Moreover, the effect is irreversible and does not require further ATP binding. Taken together, Stanley et al. (3) convincingly demonstrate that I_H occurs whenever the Na⁺ pump assumes an extracellularly open, necessarily phosphorylated conformation at high ATP concentrations in the cell, and when extracellular Na⁺ and K⁺ concentrations are nonsaturating (Fig. 1). This state may be reached via two pathways: in forward pumping direction upon Na⁺-dependent phosphorylation and low-affinity ATP binding; and by back-door phosphorylation with sufficient intracellular K⁺, Mg²⁺, and P_i present. Knowledge of these prerequisites is pivotal for defining the parameters, under which proton leak currents may play a physiological role in cells, tissues, and organs.

Ouabain-sensitive leak currents were first observed at negative voltages in Na⁺/K⁺-ATPase deprived of extracellular Na⁺ and K⁺ ions (1). These currents were strongly inwardly rectifying, blocked by extracellular [Na⁺] and [K⁺], and the augmentation by extracellular acidification (2) suggested that protons carried the current. However, due to the strongly inwardly rectifying nature of I_H, the ion selectivity and the passive conduction mechanism (usually inferred from reversal potential measurements) were difficult to assess, which caused some confusion about whether the currents are purely passive and H⁺-selective, whether there is a contribution of active transport, or if importing of Na⁺ ions is also involved. To this end, a comprehensive mechanistic explanation has been established only recently, which also integrates the peculiar dependence of I_H on low and high Na⁺ concentrations based on reaction cycle intermediates of the Na⁺,K⁺-ATPase (4, 6).

The concept of a critical role for protons in the transport cycle has been revived by the elucidation of the Na⁺/K⁺ pump’s crystal structure, which highlighted an as yet unrecognized intracellular C-terminal pathway, through which protons gain access to the cation binding pocket and bring about stoichiometric 3Na⁺/2K⁺ transport (8). Consequently, mutations interfering with this C-terminal pathway strongly augment I_H (9), and, because several mutations in Na⁺ pump isoforms implicated in human disease produced such phenotypes, I_H also found its way into human pathophysiology.

Recently, it was demonstrated that I_H is an inherent property of Na⁺,K⁺-ATPase that does not require the absence of external Na⁺ and K⁺, but also flows at physiological K⁺ and Na⁺ concentrations and membrane potentials (4, 6). I_H utilizes the reversibility of a series of partial reactions associated with extracellular Na⁺ release from the phosphorylated enzyme (Fig. 1). Although such a reverse step of phosphorylated Na⁺,K⁺-ATPase bringing about proton import is not needed to complete the transport cycle, it readily occurs during Na⁺/K⁺ transport when external Na⁺ and K⁺ ion binding and occlusion are impeded (6). It was shown that protons presumably pass through the third, uniquely Na⁺-selective binding site (10, 11); however, the proton pathway is different from the route taken by Na⁺ and K⁺ ions (6). Importantly, Mitchell et al. (4) and Vedovato and Gadsby (6) showed that Na⁺/K⁺ transport and H⁺ import occur simultaneously during the same conformational cycle of a single pump molecule, which eventually classifies the Na⁺,K⁺-ATPase as a hybrid cation transporter (6). Mitchell et al. (4) also delineated a previously unrecognized inhibitory action of low extracellular pH (pH_o) on I_H, which occurs in addition to the acid-induced I_H stimulation. Based on the strong voltage dependence of the pH_o-induced leak stimulation, these authors concluded that protons leak through the third Na⁺ binding site, which is responsible for the strong voltage dependence of cation transport. Because inhibition of I_H by external protons was only weakly dependent upon voltage, similar to the inhibition by low concentrations of Na⁺ and K⁺, it is likely that the inhibitory action of external cations (H⁺, Na⁺, or K⁺) on I_H requires binding to the two common sites that are shared during Na⁺ and K⁺ transport (9).

It remains to be determined whether Na⁺/K⁺ pump-mediated proton uptake plays a physiological role, or if it is simply “the price paid by nature” (6) to bring about efficient 3Na⁺/2K⁺ transport across cell membranes. Proton leak must significantly accompany Na⁺/K⁺ pumping at the normal negative resting potentials of excitable cells, if pH_o is sufficiently low, e.g., during ischemia, heavy neuronal activity, or vigorous heart or muscle activity (11). Sorting out the role of all facets of the Na⁺/K⁺-ATPase hybrid transporter in human health and disease will be the task for the next generation of physiologists and biophysicists, and may eventually pave the way for novel means of clinical intervention.

Editor: Ian Forster.