Cryoelectron microscopy of Na+,K+-ATPase in the two E2P states with and without cardiotonic steroids.

Cryoelectron microscopy (cryo-EM) was applied to Na+,K+-ATPase (NKA) to determine the structures of two E2P states, one (E2PATP) formed by ATP and Mg2+ in the forward reaction, and the other (E2PPi) formed by inorganic phosphate (Pi) and Mg2+ in the backward reaction, with and without ouabain or istaroxime, representatives of classical and new-generation cardiotonic steroids (CTSs). These two E2P states exhibit different biochemical properties. In particular, K+-sensitive acceleration of the dephosphorylation reaction is not observed with E2PPi, attributed to the presence of a Mg2+ ion in the transmembrane cation binding sites. The cryo-EM structures of NKA demonstrate that the two E2P structures are nearly identical but Mg2+ in the transmembrane binding cavity is identified only in E2PPi, corroborating the idea that it should be denoted as E2PPi·Mg2+. We can now explain why the absence of transmembrane Mg2+ in E2PATP confers the K+ sensitivity in dephosphorylation. In addition, we show that ATP bridges the actuator (A) and nucleotide binding (N) domains, stabilizing the E2PATP state; CTS binding causes hardly any changes in the structure of NKA, both in E2PATP and E2PPi·Mg2+, indicating that the binding mechanism is conformational selection; and istaroxime binds to NKA, extending its aminoalkyloxime group deep into the cation binding site. This orientation is upside down compared to that of classical CTSs with respect to the steroid ring. Notably, mobile parts of NKA are resolved substantially better in the electron microscopy (EM) maps than in previous X-ray structures, including sugars sticking out from the ß-subunit and many phospholipid molecules.

Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin.

Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin, which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of Na+ pump rhodopsin from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.

ATP1A1 is a promising new target for melanoma treatment and can be inhibited by its physiological ligand bufalin to restore targeted therapy efficacy.

Despite advancements in treating metastatic melanoma, many patients exhibit resistance to targeted therapies. Our study focuses on ATP1A1, a sodium pump subunit associated with cancer development. We aimed to assess ATP1A1 prognostic value in melanoma patients and examine the impact of its ligand, bufalin, on melanoma cell lines in vitro and in vivo. High ATP1A1 expression (IHC) correlated with reduced overall survival in melanoma patients. Resistance to BRAF inhibitor was linked to elevated ATP1A1 levels in patient biopsies (IHC, qPCR) and cell lines (Western blot, qPCR). Additionally, high ATP1A1 mRNA expression positively correlated with differentiation/pigmentation markers based on data from The Cancer Genome Atlas (TCGA) databases and Verfaillie proliferative gene signature analysis. Bufalin specifically targeted ATP1A1 in caveolae, (proximity ligation assay) and influenced Src phosphorylation (Western blot), thereby disrupting multiple signaling pathways (phosphokinase array). In vitro, bufalin induced apoptosis in melanoma cell lines by acting on ATP1A1 (siRNA experiments) and, in vivo, significantly impeded melanoma growth using a nude mouse xenograft model with continuous bufalin delivery via an osmotic pump. In conclusion, our study demonstrates that ATP1A1 could serve as a prognostic marker for patient survival and a predictive marker for response to BRAF inhibitor therapy. By targeting ATP1A1, bufalin inhibited cell proliferation, induced apoptosis in vitro, and effectively suppressed tumor development in mice. Thus, our findings strongly support ATP1A1 as a promising therapeutic target, with bufalin as a potential agent to disrupt its tumor-promoting activity.

Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state.

The sodium pump (Na+, K+-ATPase, NKA) is vital for animal cells, as it actively maintains Na+ and K+ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF3- complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K+-antagonism and suggest a route to isoform specific CTSs.

Na,K-ATPase Expression Can Be Limited Post-Transcriptionally: A Test of the Role of the Beta Subunit, and a Review of Evidence.

The Na,K-ATPase is an α-ß heterodimer. It is well known that the Na,K-ATPase ß subunit is required for the biosynthesis and trafficking of the α subunit to the plasma membrane. During investigation of properties of human ATP1A3 mutations in 293 cells, we observed a reciprocal loss of endogenous ATP1A1 when expressing ATP1A3. Scattered reports going back as far as 1991 have shown that experimental expression of one subunit can result in reduction in another, suggesting that the total amount is strictly limited. It seems logical that either α or ß subunit should be rate-limiting for assembly and functional expression. Here, we present evidence that neither α nor ß may be limiting and that there is another level of control that limits the amount of Na,K-ATPase to physiological levels. We propose that α subunits compete for something specific, like a private chaperone, required to finalize their biosynthesis or to prevent their degradation in the endoplasmic reticulum.

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

Real-time identification of two substrate-binding intermediates for the light-driven sodium pump rhodopsin.

Membrane transport proteins undergo critical conformational changes during substrate uptake and release, as the substrate-binding site is believed to switch its accessibility from one side of the membrane to the other. Thus, at least two substrate-binding intermediates should appear during the process, that is, after uptake and before the release of the substrate. However, this view has not been verified for most transporters because of the difficulty in detecting short-lived intermediates. Here, we report real-time identification of these intermediates for the light-driven outward current-generating Na+-pump rhodopsin. We triggered the transport cycle of Na+-pump rhodopsin using a short laser pulse, and subsequent formation and decay of various intermediates was detected by time-resolved measurements of absorption changes. We used this method to analyze transport reactions and elucidated the sequential formation of the Na+-binding intermediates O1 and O2. Both intermediates exhibited red-shifted absorption spectra and generated transient equilibria with short-wavelength intermediates. The equilibria commonly shifted toward O1 and O2 with increasing Na+ concentration, indicating that Na+ is bound to these intermediates. However, these equilibria were formed independently; O1 reached equilibrium with preceding intermediates, indicating Na+ uptake on the cytoplasmic side. In contrast, O2 reached equilibrium with subsequent intermediates, indicating Na+ release on the extracellular side. Thus, there is an irreversible switch in "accessibility" during the O1 to O2 transition, which could represent one of the key processes governing unidirectional Na+ transport.

Whither digitalis? What we can still learn from cardiotonic steroids about heart failure and hypertension.

Cloning of the "Na+ pump" (Na+,K+-ATPase or NKA) and identification of a circulating ligand, endogenous ouabain (EO), a cardiotonic steroid (CTS), triggered seminal discoveries regarding EO and its NKA receptor in cardiovascular function and the pathophysiology of heart failure (HF) and hypertension. Cardiotonic digitalis preparations were a preferred treatment for HF for two centuries, but digoxin was only marginally effective in a large clinical trial (1997). This led to diminished digoxin use. Missing from the trial, however, was any consideration that endogenous CTS might influence digitalis' efficacy. Digoxin, at therapeutic concentrations, acutely inhibits NKA but, remarkably, antagonizes ouabain's action. Prolonged treatment with ouabain, but not digoxin, causes hypertension in rodents; in this model, digoxin lowers blood pressure (BP). Furthermore, NKA-bound ouabain and digoxin modulate different protein kinase signaling pathways and have disparate long-term cardiovascular effects. Reports of "brain ouabain" led to the elucidation of a new, slow neuromodulatory pathway in the brain; locally generated EO and the α2 NKA isoform help regulate sympathetic drive to the heart and vasculature. The roles of EO and α2 NKA have been studied by EO assay, ouabain-resistant mutation of α2 NKA, and immunoneutralization of EO with ouabain-binding Fab fragments. The NKA α2 CTS binding site and its endogenous ligand are required for BP elevation in many common hypertension models and full expression of cardiac remodeling and dysfunction following pressure overload or myocardial infarction. Understanding how endogenous CTS impact hypertension and HF pathophysiology and therapy should foster reconsideration of digoxin's therapeutic utility.

Plasticity in Na+/K+-ATPase thermal kinetics drives variation in the temperature of cold-induced neural shutdown of adult Drosophila melanogaster.

Most insects can acclimate to changes in their thermal environment and counteract temperature effects on neuromuscular function. At the critical thermal minimum, a spreading depolarization (SD) event silences central neurons, but the temperature at which this event occurs can be altered through acclimation. SD is triggered by an inability to maintain ion homeostasis in the extracellular space in the brain and is characterized by a rapid surge in extracellular K+ concentration, implicating ion pump and channel function. Here, we focused on the role of the Na+/K+-ATPase specifically in lowering the SD temperature in cold-acclimated Drosophila melanogaster. After first confirming cold acclimation altered SD onset, we investigated the dependency of the SD event on Na+/K+-ATPase activity by injecting the inhibitor ouabain into the head of the flies to induce SD over a range of temperatures. Latency to SD followed the pattern of a thermal performance curve, but cold acclimation resulted in a left-shift of the curve to an extent similar to its effect on the SD temperature. With Na+/K+-ATPase activity assays and immunoblots, we found that cold-acclimated flies have ion pumps that are less sensitive to temperature, but do not differ in their overall abundance in the brain. Combined, these findings suggest a key role for plasticity in Na+/K+-ATPase thermal sensitivity in maintaining central nervous system function in the cold, and more broadly highlight that a single ion pump can be an important determinant of whether insects can respond to their environment to remain active at low temperatures.

The 370-Da Inhibitor of the Sodium Pump in the Plasma of Haemodialysis Patients.

BACKGROUND: Previous studies have shown that a molecule of mass 370 Da that inhibits the sodium pump can be extracted from human placentas and from the concentrated plasma or ultrafiltrate of volume-expanded patients. AIM: This study aimed to study the abundance of the 370-Da molecule and its changes across dialysis in a population of patients with renal failure treated by haemodialysis. METHODS: Four millilitres of pre- and post-dialysis blood samples (2 mL plasma) were taken from patients receiving intermittent haemodialysis and analysed by high-performance liquid chromatography coupled to high sensitivity mass spectrometry. RESULTS: In over half of the study population, the 370-Da molecule was present in abundance that exceeded the limit of quantitation. Most patients experienced a marked fall in the abundance of the molecule over a haemodiafiltration session, though exceptions were seen in 2 individuals, both of whom showed clear evidence for the presence of 2 structural isomers of the 370-Da molecule. CONCLUSIONS: Advanced renal failure is frequently accompanied by an increased abundance of a 370-Da inhibitor of the sodium pump and that abundance is strongly impacted by haemodialysis. The technique described here could readily be applied to other clinical situations where sodium pump inhibition might be anticipated, such as hypertension, pregnancy, and foetal medicine, and thereby lead to a better understanding of the physiology and pathophysiology of these conditions.

With a grain of salt: Sodium elevation and metabolic remodelling in heart failure.

Elevated intracellular Na (Nai) and metabolic impairment are interrelated pathophysiological features of the failing heart (HF). There have been a number of studies showing that myocardial sodium elevation subtly affects mitochondrial function. During contraction, mitochondrial calcium (Camito) stimulates a variety of TCA cycle enzymes, thereby providing reducing equivalents to maintain ATP supply. Nai elevation has been shown to impact Camito; however, whether metabolic remodelling in HF is caused by increased Nai has only been recently demonstrated. This novel insight may help to elucidate the contribution of metabolic remodelling in the pathophysiology of HF, the lack of efficacy of current HF therapies and a rationale for the development of future metabolism-targeting treatments. Here we review the relationship between Na pump inhibition, elevated Nai, and altered metabolic profile in the context of HF and their link to metabolic (in)flexibility and mitochondrial reprogramming.

Muscarinic regulation of the neuronal Na+ /K+ -ATPase in rat hippocampus.

KEY POINTS: Stimulation of postsynaptic muscarinic receptors was shown to excite principal hippocampal neurons by modulating several membrane ion conductances. We show here that activation of postsynaptic muscarinic receptors also causes neuronal excitation by inhibiting Na+ /K+ -ATPase activity. Muscarinic Na+ /K+ -ATPase inhibition is mediated by two separate signalling pathways that lead downstream to enhanced Na+ /K+ -ATPase phosphorylation by activating protein kinase C and protein kinase G. Muscarinic excitation through Na+ /K+ -ATPase inhibition is probably involved in cholinergic modulation of hippocampal activity and may turn out to be a widespread mechanism of neuronal excitation in the brain. ABSTRACT: Stimulation of muscarinic cholinergic receptors on principal hippocampal neurons enhances intrinsic neuronal excitability by modulating several membrane ion conductances. The electrogenic Na+ /K+ -ATPase (NKA; the 'Na+ pump') is a ubiquitous regulator of intrinsic neuronal excitability, generating a hyperpolarizing current to thwart excessive neuronal firing. Using electrophysiological and pharmacological methodologies in rat hippocampal slices, we show that neuronal NKA pumping activity is also subjected to cholinergic regulation. Stimulation of postsynaptic muscarinic, but not nicotinic, cholinergic receptors activates membrane-bound phospholipase C and hydrolysis of membrane-integral phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3 ). Along one signalling pathway, DAG activates protein kinase C (PKC). Along a second signalling pathway, IP3 causes Ca2+ release from the endoplasmic reticulum, facilitating nitric oxide (NO) production. The rise in NO levels stimulates cGMP synthesis by guanylate-cyclase, activating protein kinase G (PKG). The two pathways converge to cause partial NKA inhibition through enzyme phosphorylation by PKC and PKG, leading to a marked increase in intrinsic neuronal excitability. This novel mechanism of neuronal NKA regulation probably contributes to the cholinergic modulation of hippocampal activity in spatial navigation, learning and memory.

Effect of handling on ATP utilization of cerebral Na,K-ATPase in rats with trimethyltin-induced neurodegeneration.

Previously it was shown that for reduction of anxiety and stress of experimental animals, preventive handling seems to be one of the most effective methods. The present study was oriented on Na,K-ATPase, a key enzyme for maintaining proper concentrations of intracellular sodium and potassium ions. Malfunction of this enzyme has an essential role in the development of neurodegenerative diseases. It is known that this enzyme requires approximately 50% of the energy available to the brain. Therefore in the present study utilization of the energy source ATP by Na,K-ATPase in the frontal cerebral cortex, using the method of enzyme kinetics was investigated. As a model of neurodegeneration treatment with trimethyltin (TMT) was applied. Daily handling (10 min/day) of healthy rats and rats suffering neurodegeneration induced by administration of TMT in a dose of (7.5 mg/kg), at postnatal days 60-102 altered the expression of catalytic subunits of Na,K-ATPase as well as kinetic properties of this enzyme in the frontal cerebral cortex of adult male Wistar rats. In addition to the previously published beneficial effect on spatial memory, daily treatment of rats was accompanied by improved maintenance of sodium homeostasis in the frontal cortex. The key system responsible for this process, Na,K-ATPase, was able to utilize better the energy substrate ATP. In rats, manipulation of TMT-induced neurodegeneration promoted the expression of the α2 isoform of the enzyme, which is typical for glial cells. In healthy rats, manipulation was followed by increased expression of the α3 subunit, which is typical of neurons.

Diversity, Mechanism, and Optogenetic Application of Light-Driven Ion Pump Rhodopsins.

Ion-transporting microbial rhodopsins are widely used as major molecular tools in optogenetics. They are categorized into light-gated ion channels and light-driven ion pumps. While the former passively transport various types of cations and anions in a light-dependent manner, light-driven ion pumps actively transport specific ions, such as H+, Na+, Cl-, against electrophysiological potential by using light energy. Since the ion transport by these pumps induces hyperpolarization of membrane potential and inhibit neural firing, light-driven ion-pumping rhodopsins are mostly applied as inhibitory optogenetics tools. Recent progress in genome and metagenome sequencing identified more than several thousands of ion-pumping rhodopsins from a wide variety of microbes, and functional characterization studies has been revealing many new types of light-driven ion pumps one after another. Since light-gated channels were reviewed in other chapters in this book, here the rapid progress in functional characterization, molecular mechanism study, and optogenetic application of ion-pumping rhodopsins were reviewed.

Thermal acclimation alters Na+/K+-ATPase activity in a tissue-specific manner in Drosophila melanogaster.

Insects, like the model species Drosophila melanogaster, lose neuromuscular function and enter a state of paralysis (chill coma) at a population- and species-specific low temperature threshold that is decreased by cold acclimation. Entry into this coma is related to a spreading depolarization in the central nervous system, while recovery involves restoration of electrochemical gradients across muscle cell membranes. The Na+/K+-ATPase helps maintain ion balance and membrane potential in both the brain and hemolymph (surrounding muscles), and changes in thermal tolerance traits have therefore been hypothesized to be closely linked to variation in the expression and/or activity of this pump in multiple tissues. Here, we tested this hypothesis by measuring activity and thermal sensitivity of the Na+/K+-ATPase at the tagma-specific level (head, thorax and abdomen) in warm- (25 °C) and cold-acclimated (15 °C) flies by measuring Na+/K+-ATPase activity at 15, 20, and 25 °C. We relate differences in pump activity to differences in chill coma temperature, spreading depolarization temperature, and thermal dependence of muscle cell polarization. Differences in pump activity and thermal sensitivity induced by cold acclimation varied in a tissue-specific manner: While thermal sensitivity remained unchanged, cold-acclimated flies had decreased Na+/K+-ATPase activity in the thorax (mainly muscle) and head (mainly composed of brain). We argue that these changes may assist in maintenance of K+ homeostasis and membrane potential across muscle membranes, and discuss how reduced Na+/K+-ATPase activity in the brain may counterintuitively help insects delay coma onset in the cold.

Regulation of Neuronal Na+/K+-ATPase by Specific Protein Kinases and Protein Phosphatases.

The Na+/K+-ATPase (NKA) is a ubiquitous membrane-bound enzyme responsible for generating and maintaining the Na+ and K+ electrochemical gradients across the plasmalemma of living cells. Numerous studies in non-neuronal tissues have shown that this transport mechanism is reversibly regulated by phosphorylation/dephosphorylation of the catalytic α subunit and/or associated proteins. In neurons, Na+/K+ transport by NKA is essential for almost all neuronal operations, consuming up to two-thirds of the neuron's energy expenditure. However, little is known about its cellular regulatory mechanisms. Here we have used an electrophysiological approach to monitor NKA transport activity in male rat hippocampal neurons in situ We report that this activity is regulated by a balance between serine/threonine phosphorylation and dephosphorylation. Phosphorylation by the protein kinases PKG and PKC inhibits NKA activity, whereas dephosphorylation by the protein phosphatases PP-1 and PP-2B (calcineurin) reverses this effect. Given that these kinases and phosphatases serve as downstream effectors in key neuronal signaling pathways, they may mediate the coupling of primary messengers, such as neurotransmitters, hormones, and growth factors, to the NKAs, through which multiple brain functions can be regulated or dysregulated.SIGNIFICANCE STATEMENT The Na+/K+-ATPase (NKA), known as the "Na+ pump," is a ubiquitous membrane-bound enzyme responsible for generating and maintaining the Na+ and K+ electrochemical gradients across the plasma membrane of living cells. In neurons, as in most types of cells, the NKA generates the negative resting membrane potential, which is the basis for almost all aspects of cellular function. Here we used an electrophysiological approach to monitor physiological NKA transport activity in single hippocampal pyramidal cells in situ We have found that neuronal NKA activity is oppositely regulated by phosphorylation and dephosphorylation, and we have identified the main protein kinases and phosphatases mediating this regulation. This fundamental form of NKA regulation likely plays a role in multiple brain functions.

Coordinate adaptations of skeletal muscle and kidney to maintain extracellular [K+] during K+-deficient diet.

Extracellular fluid (ECF) potassium concentration ([K+]) is maintained by adaptations of kidney and skeletal muscle, responses heretofore studied separately. We aimed to determine how these organ systems work in concert to preserve ECF [K+] in male C57BL/6J mice fed a K+-deficient diet (0K) versus 1% K+ diet (1K) for 10 days (n = 5-6/group). During 0K feeding, plasma [K+] fell from 4.5 to 2 mM; hindlimb muscle (gastrocnemius and soleus) lost 28 mM K+ (from 115 ± 2 to 87 ± 2 mM) and gained 27 mM Na+ (from 27 ± 0.4 to 54 ± 2 mM). Doubling of muscle tissue [Na+] was not associated with inflammation, cytokine production or hypertension as reported by others. Muscle transporter adaptations in 0K- versus 1K-fed mice, assessed by immunoblot, included decreased sodium pump α2-ß2 subunits, decreased K+-Cl- cotransporter isoform 3, and increased phosphorylated (p) Na+,K+,2Cl- cotransporter isoform 1 (NKCC1p), Ste20/SPS-1-related proline-alanine rich kinase (SPAKp), and oxidative stress-responsive kinase 1 (OSR1p) consistent with intracellular fluid (ICF) K+ loss and Na+ gain. Renal transporters' adaptations, effecting a 98% reduction in K+ excretion, included two- to threefold increased phosphorylated Na+-Cl- cotransporter (NCCp), SPAKp, and OSR1p abundance, limiting Na+ delivery to epithelial Na+ channels where Na+ reabsorption drives K+ secretion; and renal K sensor Kir 4.1 abundance fell 25%. Mass balance estimations indicate that over 10 days of 0K feeding, mice lose ~48 µmol K+ into the urine and muscle shifts ~47 µmol K+ from ICF to ECF, illustrating the importance of the concerted responses during K+ deficiency.

Partial Reactions of the Na,K-ATPase: Determination of Activation Energies and an Approach to Mechanism.

Kinetic experiments were performed with preparations of kidney Na,K-ATPase in isolated membrane fragments or reconstituted in vesicles to obtain information of the activation energies under turnover conditions and for selected partial reactions of the Post-Albers pump cycle. The ion transport activities were detected with potential or conformation sensitive fluorescent dyes in steady-state or time-resolved experiments. The activation energies were derived from Arrhenius plots of measurements in the temperature range between 5 °C and 37 °C. The results were used to elaborate indications of the respective underlying rate-limiting reaction steps and allowed conclusions to be drawn about possible molecular reaction mechanisms. The observed consequent alteration between ligand-induced reaction and conformational relaxation steps when the Na,K-ATPase performs the pump cycle, together with constraints set by thermodynamic principles, provided restrictions which have to be met when mechanistic models are proposed. A model meeting such requirements is presented for discussion.

Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant.

Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state. In addition, 50% of the KR2 13C and 15N resonances could be assigned by multidimensional high-field solid-state NMR experiments. Assigned residues include part of the NDQ motif as well as sodium binding sites. Based on these data, the structural effects of the H30A mutation, which seems to shift the ion selectivity of KR2 primarily to Na+, could be analysed. Our data show that it causes long-range effects within the retinal binding pocket and at the extracellular Na+ binding site, which can be explained by perturbations of interactions across the protomer interfaces within the KR2 complex. This study is complemented by data from time-resolved optical spectroscopy.