Pablo Artigas

Na+/K+-pump ligands modulate gating of palytoxin-induced ion channels

The Na+/K+ pump is a ubiquitous P-type ATPase that binds three cytoplasmic Na+ ions deep within its core where they are temporarily occluded before being released to the extracellular surface. The 3Na+/2K+-exchange transport cycle is completed when two extracellular K+ ions bind and become temporarily occluded within the protein and subsequently released to the cytoplasm. Coupling of Na+-ion occlusion to phosphorylation of the pump by ATP and of K+-ion occlusion to its dephosphorylation ensure the vectorial nature of net transport. The occluded-ion conformations, with binding sites inaccessible from either side, represent intermediate states in these alternating-access descriptions of transport. They afford protection against potentially catastrophic effects of inadvertently allowing simultaneous access from both membrane sides. The marine toxin, palytoxin, converts Na+/K+ pumps into nonselective cation channels, possibly by disrupting the normal strict coupling between opening of one access pathway in the Na+/K+ ATPase and closing of the other. We show here that gating of the channels in palytoxin-bound Na+/K+ pumps in excised membrane patches is modulated by the pumps physiological ligands: cytoplasmic application of ATP promotes opening of the channels, and extracellular replacement of Na+ ions by K+ ions promotes closing of the channels. This suggests that, despite the presence of bound palytoxin, certain partial reactions of the normal Na+/K+-transport cycle persist and remain capable of effecting the conformational changes that control access to the pumps cation-binding sites. These findings affirm the alternating-access model of ion pumps and offer the possibility of examining ion occlusion/deocclusion reactions in single pump molecules.

Large Diameter of Palytoxin-induced Na/K Pump Channels and Modulation of Palytoxin Interaction by Na/K Pump Ligands

Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pumps ion translocation pathway. We systematically analyzed palytoxins interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na+-containing (K+-free) external and internal solutions, lowest for K+-containing (Na+-free) external and internal solutions, and intermediate for the mixed external Na+/internal K+, and external K+/internal Na+ conditions; with Na+ solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na+ solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained ∼30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the same Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine+, suggesting that the narrowest section of the pore must be ∼7.5 Å wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle.

The ion pathway through the opened Na+,K+-ATPase pump

P-type ATPases pump ions across membranes, generating steep electrochemical gradients that are essential for the function of all cells. Access to the ion-binding sites within the pumps alternates between the two sides of the membrane to avoid the dissipation of the gradients that would occur during simultaneous access. In Na+,K+-ATPase pumps treated with the marine agent palytoxin, this strict alternation is disrupted and binding sites are sometimes simultaneously accessible from both sides of the membrane, transforming the pumps into ion channels (see, for example, refs 2, 3). Current recordings in these channels can monitor accessibility of introduced cysteine residues to water-soluble sulphydryl-specific reagents. We found previously that Na+,K+ pump-channels open to the extracellular surface through a deep and wide vestibule that emanates from a narrower pathway between transmembrane helices 4 and 6 (TM4 and TM6). Here we report that cysteine scans from TM1 to TM6 reveal a single unbroken cation pathway that traverses palytoxin-bound Na+,K+ pump-channels from one side of the membrane to the other. This pathway comprises residues from TM1, TM2, TM4 and TM6, passes through ion-binding site II, and is probably conserved in structurally and evolutionarily related P-type pumps, such as sarcoplasmic- and endoplasmic-reticulum Ca2+-ATPases and H+,K+-ATPases.

Protonation of key acidic residues is critical for the K+-selectivity of the Na/K pump

The sodium-potassium (Na/K) pump is a P-type ATPase that generates Na+ and K+ concentration gradients across the cell membrane. For each hydrolyzed ATP molecule, the pump extrudes three Na+ and imports two K+ by alternating between outward- and inward-facing conformations that preferentially bind K+ or Na+, respectively. Remarkably, the selective K+ and Na+ binding sites share several residues, and how the pump is able to achieve the selectivity required for the functional cycle is unclear. Here, free energy–perturbation molecular dynamics (FEP/MD) simulations based on the crystal structures of the Na/K pump in a K+-loaded state (E2·Pi) reveal that protonation of the high-field acidic side chains involved in the binding sites is crucial to achieving the proper K+ selectivity. This prediction is tested with electrophysiological experiments showing that the selectivity of the E2P state for K+ over Na+ is affected by extracellular pH.

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

The Na/K pump actively exports 3 Na+ in exchange for 2 K+ across the plasmalemma of animal cells. As in other P-type ATPases, pump function is more effective when the relative affinity for transported ions is altered as the ion binding sites alternate between opposite sides of the membrane. Deletion of the five C-terminal residues from the α-subunit diminishes internal Na+ (Nai+) affinity ≈25-fold [Morth et al. (2007) Nature 450:1043–1049]. Because external Na+ (Nao+) binding is voltage-dependent, we studied the reactions involving this process by using two-electrode and inside-out patch voltage clamp in normal and truncated (ΔKESYY) Xenopus-α1 pumps expressed in oocytes. We observed that ΔKESYY (i) decreased both Nao+ and Nai+ apparent affinities in the absence of Ko+, and (ii) did not affect apparent Nao+ affinity at high Ko+. These results support a model of strict sequential external release of Na+ ions, where the Na+-exclusive site releases Na+ before the sites shared with K+ and the ΔKESYY deletion only reduces Nao+ affinity at the shared sites. Moreover, at nonsaturating Ko+, ΔKESYY induced an inward flow of Na+ through Na/K pumps at negative potentials. Guanidinium+ can also permeate truncated pumps, whereas N-methyl-D-glucamine cannot. Because guanidiniumo+ can also traverse normal Na/K pumps in the absence of both Nao+ and Ko+ and can also inhibit Na/K pump currents in a Na+-like voltage-dependent manner, we conclude that the normal pathway transited by the first externally released Na+ is large enough to accommodate guanidinium+.

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

The Na/K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na/K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na/K pump and the site of its interaction with ouabain. Palytoxin uncouples the pump’s gates, which normally open strictly alternately, thus allowing both gates to sometimes be open, so transforming the pump into an ion channel. Palytoxin therefore permits electrophysiological analysis of even a single Na/K pump. We used outside-out patch recording of Xenopus α1β3 Na/K pumps, which were made ouabain-resistant by point mutation, after expressing them in Xenopus oocytes. Endogenous, ouabain-sensitive, Xenopus α1β3 Na/K pumps were silenced by continuous exposure to ouabain. We found that side-chain charge of two residues at either end of the α subunit’s first extracellular loop, known to make a major contribution to ouabain affinity, strongly influenced conductance of single palytoxin-bound pump-channels by an electrostatic mechanism. The effects were mimicked by modification of cysteines introduced at those two positions with variously charged methanethiosulfonate reagents. The consequences of these modifications demonstrate that both residues lie in a wide vestibule near the mouth of the pump’s ion pathway. Bound ouabain protects the site with the strongest influence on conductance from methanethiosulfonate modification, while leaving the site with the weaker influence unprotected. The results suggest a method for mapping the footprint of bound cardiotonic steroid on the extracellular surface of the Na/K pump.

Ion Occlusion/Deocclusion Partial Reactions in Individual Palytoxin‐Modified Na/K Pumps

Abstract: In P‐type ion‐motive ATPases, transported ions approach their binding sites from one membrane surface, become buried deep within ‘occluded’ conformations in which the sites are inaccessible from either membrane side, and are then deoccluded and released to the opposite membrane surface. This describes an alternating‐gate transport mechanism, in which the pump acts like an ion channel with two gates that open and close alternately. The occluded states ensure that one gate closes before the other can open, thus preventing the large electrodiffusive ion fluxes that would otherwise quickly undo the pumps electrochemical work. High‐resolution crystal structures of two conformations of the SERCA (sarcoplasmic and endoplasmic reticulum Ca2+) P‐type ATPase, together with mutagenesis results and analyses of structural models based on homology, have begun to provide a picture of the ion coordination sites in related P‐type ATPases, including the Na/K pump. However, in no P‐type ATPase are the structures and mechanisms of the gates known. The marine toxin, palytoxin (PTX), is known to bind to the Na/K pump and elicit a nonselective cation leak pathway, possibly by disrupting the strict coupling between the pumps inner and outer gates, allowing them to both be open. We recently found that ion flow through PTX‐modified Na/K pump‐channels appears to be modulated by two gates that can be regulated by the pumps physiological ligands in a manner suggesting that gating reflects underlying ion occlusion/deocclusion partial reactions. We review that work here and provide evidence that the pore of the PTX‐induced pump‐channel has a diameter > 6 Å.

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

The Na/K pump is a P-type ATPase that exchanges three intracellular Na+ ions for two extracellular K+ ions through the plasmalemma of nearly all animal cells. The mechanisms involved in cation selection by the pumps ion-binding sites (site I and site II bind either Na+ or K+; site III binds only Na+) are poorly understood. We studied cation selectivity by outward-facing sites (high K+ affinity) of Na/K pumps expressed in Xenopus oocytes, under voltage clamp. Guanidinium+, methylguanidinium+, and aminoguanidinium+ produced two phenomena possibly reflecting actions at site III: (i) voltage-dependent inhibition (VDI) of outwardly directed pump current at saturating K+, and (ii) induction of pump-mediated, guanidinium-derivative–carried inward current at negative potentials without Na+ and K+. In contrast, formamidinium+ and acetamidinium+ induced K+-like outward currents. Measurement of ouabain-sensitive ATPase activity and radiolabeled cation uptake confirmed that these cations are external K+ congeners. Molecular dynamics simulations indicate that bound organic cations induce minor distortion of the binding sites. Among tested metals, only Li+ induced Na+-like VDI, whereas all metals tested except Na+ induced K+-like outward currents. Pump-mediated K+-like organic cation transport challenges the concept of rigid structural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Furthermore, actions by guanidinium+ derivatives suggest that Na+ binds to site III in a hydrated form and that the inward current observed without external Na+ and K+ represents cation transport when normal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites.

Peering into an ATPase ion pump with single-channel recordings

In principle, an ion channel needs no more than a single gate, but a pump requires at least two gates that open and close alternately to allow ion access from only one side of the membrane at a time. In the Na+,K+-ATPase pump, this alternating gating effects outward transport of three Na+ ions and inward transport of two K+ ions, for each ATP hydrolysed, up to a hundred times per second, generating a measurable current if assayed in millions of pumps. Under these assay conditions, voltage jumps elicit brief charge movements, consistent with displacement of ions along the ion pathway while one gate is open but the other closed. Binding of the marine toxin, palytoxin, to the Na+,K+-ATPase uncouples the two gates, so that although each gate still responds to its physiological ligand they are no longer constrained to open and close alternately, and the Na+,K+-ATPase is transformed into a gated cation channel. Millions of Na+ or K+ ions per second flow through such an open pump–channel, permitting assay of single molecules and allowing unprecedented access to the ion transport pathway through the Na+,K+-ATPase. Use of variously charged small hydrophilic thiol-specific reagents to probe cysteine targets introduced throughout the pumps transmembrane segments allows mapping and characterization of the route traversed by transported ions.

2,3-Butanedione Monoxime Affects Cystic Fibrosis Transmembrane Conductance Regulator Channel Function through Phosphorylation-Dependent and Phosphorylation-Independent Mechanisms: The Role of Bilayer Material Properties

2,3-Butanedione monoxime (BDM) is widely believed to act as a chemical phosphatase. We therefore examined the effects of BDM on the cystic fibrosis transmembrane regulator (CFTR) Cl- channel, which is regulated by phosphorylation in a complex manner. In guinea pig ventricular myocytes, forskolin-activated whole-cell CFTR currents responded biphasically to external 20 mM BDM: a rapid ∼2-fold current activation was followed by a slower (τ ∼20 s) inhibition (to ∼20% of control). The inhibitory response was abolished by intracellular dialysis with the phosphatase inhibitor microcystin, suggesting involvement of endogenous phosphatases. The BDM-induced activation was studied further in Xenopus laevis oocytes expressing human epithelial CFTR. The concentration for half-maximal BDM activation (K0.5) was state-dependent, ∼2 mM for highly and ∼20 mM for partially phosphorylated channels, suggesting a modulated receptor mechanism. Because BDM modulates many different membrane proteins with similar K0.5 values, we tested whether BDM could alter protein function by altering lipid bilayer properties rather than by direct BDM-protein interactions. Using gramicidin channels of different lengths (different channel-bilayer hydrophobic mismatch) as reporters of bilayer stiffness, we found that BDM increases channel appearance rates and lifetimes (reduces bilayer stiffness). At 20 mM BDM, the appearance rates increase ∼4-fold (for the longer, 15 residues/monomer, channels) to ∼10-fold (for the shorter, 13 residues/monomer channels); the lifetimes increase ∼50% independently of channel length. BDM thus reduces the energetic cost of bilayer deformation, an effect that may underlie the effects of BDM on CFTR and other membrane proteins; the state-dependent changes in K0.5 are consistent with such a bilayer-mediated mechanism.