Janne Petersen

Crystal structure of the sodium-potassium pump.

The Na+,K+-ATPase generates electrochemical gradients for sodium and potassium that are vital to animal cells, exchanging three sodium ions for two potassium ions across the plasma membrane during each cycle of ATP hydrolysis. Here we present the X-ray crystal structure at 3.5 Å resolution of the pig renal Na+,K+-ATPase with two rubidium ions bound (as potassium congeners) in an occluded state in the transmembrane part of the α-subunit. Several of the residues forming the cavity for rubidium/potassium occlusion in the Na+,K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of sarco(endo)plasmic reticulum. The β- and γ-subunits specific to the Na+,K+-ATPase are associated with transmembrane helices αM7/αM10 and αM9, respectively. The γ-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the α-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.

Purification and characterization of (Na+, K+)-ATPase. V. Conformational changes in the enzyme. Transitions between the Na-form and the K-form studied with tryptic digestion as a tool

1. Purified (Na+, K+)-ATPase consisting of membrane fragments was digested with trypsin. The time course of enzyme inactivation was related to the electrophoretic pattern of native and cleaved proteins remaining in the membrane. 2. Differences in both the inactivation kinetics and the cleavage of the large chain (mol. wt 98 000) allow distinction of two patterns of tryptic digestion of (Na+, K+)-ATPase seen with Na+ or K+ in the medium. 3. With K+, the inactivation of (Na+, K+)-ATPase is linear with time in semilogarithmic plots and the activity is lost in parallel with cleavage of the large chain to fragments with molecular weights 58 000 and 48 000. 4. With Na+, the inactivation curves are biphasic. In the initial phase of rapid inactivation, 50% of the activity is lost with minor changes in the composition of the large chain. In the final phase, the large chain is cleaved at a low rate to a fragment with a molecular weight of 78 000. 5. It is concluded that the regions of the large chain exposed in the presence of K+ are distinct from the regions exposed in presence of Na+ and that two conformations of (Na+, K+)-ATPase can be sensed with trypsin, a (t)K-form and a (t)Na-form. 6. Reaction of the (t)K-form with ATP cause transition to the (t)Na-form. Relatively high concentrations of ATP are required and Mg2+ is not necessary. Phosphorylation of (Na+, K+)-ATPase is accompanied by transition from the (t)Na-form to the (t)K-form. Previous kinetic data suggest that these conformational changes are accompanied by shifts in the affinities of the enzyme for Na+ and K+.

Mutation I810N in the α3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS

In a mouse mutagenesis screen, we isolated a mutant, Myshkin (Myk), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk/+ mice carry a mutation (I810N) which renders the normally expressed Na+,K+-ATPase α3 isoform inactive. Total Na+,K+-ATPase activity was reduced by 42% in Myk/+ brain. The epilepsy in Myk/+ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na+,K+-ATPase α3 by transgenesis, which also rescued Na+,K+-ATPase activity. Our findings reveal the functional significance of the Na+,K+-ATPase α3 isoform in the control of epileptiform activity and seizure behavior.

Mania-like behavior induced by genetic dysfunction of the neuron-specific Na+,K+-ATPase α3 sodium pump.

Bipolar disorder is a debilitating psychopathology with unknown etiology. Accumulating evidence suggests the possible involvement of Na+,K+-ATPase dysfunction in the pathophysiology of bipolar disorder. Here we show that Myshkin mice carrying an inactivating mutation in the neuron-specific Na+,K+-ATPase α3 subunit display a behavioral profile remarkably similar to bipolar patients in the manic state. Myshkin mice show increased Ca2+ signaling in cultured cortical neurons and phospho-activation of extracellular signal regulated kinase (ERK) and Akt in the hippocampus. The mood-stabilizing drugs lithium and valproic acid, specific ERK inhibitor SL327, rostafuroxin, and transgenic expression of a functional Na+,K+-ATPase α3 protein rescue the mania-like phenotype of Myshkin mice. These findings establish Myshkin mice as a unique model of mania, reveal an important role for Na+,K+-ATPase α3 in the control of mania-like behavior, and identify Na+,K+-ATPase α3, its physiological regulators and downstream signal transduction pathways as putative targets for the design of new antimanic therapies.

Decreased neuronal Na + ,K + -ATPase activity in Atp1a3 heterozygous mice increases susceptibility to depression-like endophenotypes by chronic variable stress

Unipolar depression and bipolar depression are prevalent and debilitating diseases in need of effective novel treatments. It is becoming increasingly evident that depressive disorders manifest from a combination of inherited susceptibility genes and environmental stress. Genetic mutations resulting in decreased neuronal Na+,K+‐ATPase (sodium‐potassium adenosine triphosphatase) activity may put individuals at risk for depression given that decreased Na+,K+‐ATPase activity is observed in depressive disorders and animal models of depression. Here, we show that Na+,K+‐ATPase α3 heterozygous mice (Atp1a3+/−), with 15% reduced neuronal Na+,K+‐ATPase activity, are vulnerable to develop increased depression‐like endophenotypes in a chronic variable stress (CVS) paradigm compared to wild‐type littermates (Atp1a3+/+). In Atp1a3+/+ mice CVS did not decrease Na+,K+‐ATPase activity, however led to despair‐like behavior in the tail suspension test (TST), anhedonia in a sucrose preference test and a minimal decrease in sociability, whereas in Atp1a3+/− mice CVS decreased neuronal Na+,K+‐ATPase activity to 33% of wild‐type levels, induced despair‐like behavior in the TST, anhedonia in a sucrose preference test, anxiety in the elevated plus maze, a memory deficit in a novel object recognition task and sociability deficits in a social interaction test. We found that a mutation that decreases neuronal Na+,K+‐ATPase activity interacts with stress to exacerbate depression. Furthermore, we observed an interesting correlation between Na+,K+‐ATPase activity and mood that may relate to both unipolar depression and bipolar disorder. Pharmaceuticals that increase Na+,K+‐ATPase activity or block endogenous Na+, K+‐ATPase inhibition may provide effective treatment for depressive disorders and preclude depression in susceptible individuals.

Chymotryptic cleavage of α-subunit in E1-forms of renal (Na+ + K +)-ATPase: effects on enzymatic properties, ligand binding and cation exchange

Chymotrypsin in NaCl medium at low ionic strength rapidly cleaves a bond in the N-terminal half of the alpha-subunit of pure membrane-bound (Na+ + K+)-ATPase from outer renal medulla. Secondary cleavage is very slow and the alpha-subunit can be converted almost quantitatively to a 78 kDa fragment. The sensitive bond is exposed to cleavage when the protein is stabilized in the E1 form by binding of Na+ or nucleotides. The bond is protected in medium containing KCl (E2K form), but it is exposed when ADP or ATP are added (E1KATP form). Fluorescence analysis and examination of ligand binding and enzymatic properties of the cleaved protein demonstrate that cleavage of the bond stabilizes the protein in the E1 form with sites for tight binding of nucleotides and cations exposed to the medium. About two 86Rb ions are bound per cleaved alpha-subunit with normal affinity (Kd = 9 microM). The bound Rb+ is not displaced by ATP or ADP. The nucleotide-potassium antagonism is abolished and ATP is bound with high affinity both in NaCl and in KCl media. Na+-dependent phosphorylation is quantitatively recovered in the 78 kDa fragment, but the affinity for binding of [48V]vanadate is very low after cleavage. ADP-ATP exchange is stimulated 4-5-fold by cleavage; while nucleotide dependent Na+-Na+, K+-K+, or Na+-K+ exchange are abolished. Cleavage with chymotrypsin in NaCl at the N-terminal side of the phosphorylated residue thus stabilizes the E1 form of the protein and abolishes cation exchange and conformational transitions in the protein although binding of cations, nucleotides and phosphate is preserved. In contrast, cleavage with trypsin in KCl at the C-terminal side of the phosphorylated residue does not interfere with E1-E2 transitions and Na+-Na+ or K+-K+ exchange. This data support the notion that cation exchange and E1-E2 transitions are thightly coupled.

High-affinity 86Rb-binding and structural changes in the α-subunit of Na+,K+ -atpase as detected by tryptic digestion and fluorescence analysis

High-affinity 86Rb-binding has been related to tryptic cleavage and fluorescence from intrinsic and extrinsic probes in order to examine the relationship of cation binding to structural transitions in the alpha-subunit of pure membrane-bound Na+,K+-ATPase from the outer renal medulla. Native Na+,K+-Atpase binds two Rb+ ions per alpha-subunit (12.3 nmol/mg protein) with high affinity (Kd = 7.5 microM) in 25 mM Tris-HCl, pH 7.5. Enzyme with one molecule of covalently attached fluorescein per alpha-subunit has the same capacity (12.8 nmol/mg protein) but a much lower affinity for Rb+ (Kd = 29.2 microM). The changes in conformational state of the protein are correlated with occupancy of the high-affinity sites for Rb+, also at concentrations of Rb+ below the Kd. Titration at varying ionic strength suggests that the E2-form is the relaxed or native conformation of the alpha-subunit. Changes in tryptic digestion pattern and in fluorescence are parallel events both in the conditions of the binding assay and at physiological ionic strength. Reversible blocking of sulfhydryl groups with Thimerosal (ethylmercurythiosalicylate) abolishes the fluorescence responses to K+ or Rb+ without affecting the capacity or the affinity for binding of 86Rb. The demonstration of high-affinity binding of Rb+ without coupling to a conformational change suggests that the E1-form of the protein exposes sites for tight binding of K+ or Rb+ at the cytoplasmic membrane surface.

Identification of A Na+, K+, Cl−-cotransport protein of Mr 34000 from kidney by photolabeling with [3H]bumethanide. The protein is associated with cytoskeleton components

A polypeptide of Mr 34 000 is photolabeled with [3H]bumethanide after binding of this drug to membranes from the outer renal medulla and irradiation at 345 nm, a wavelength where bumethanide has an absorption maximum. Our data show that the polypeptide of Mr 34 000 is a component of the Na+/K+/Cl--cotransport system. The [3H]bumethanide binding protein is not extracted by concentrations of the nonionic detergent C12E8 that solubilizes 67% of the protein of the membranes including (Na+ + K+)-ATPase. This step increases the capacity for binding of [3H]bumethanide to 681 pmol/mg protein. Extraction of the binding protein requires high ionic strength suggesting that the Na+/K+/Cl--cotransport protein is associated with cytoskeleton components. This association may be important for control of the entry of NaCl into the cytoplasm and for cellular regulation of the rate of active transport of NaCl across the tubule cells in the thick ascending limb of Henles loop.

Purification of Ca2+‐activated K+ channel protein on calmodulin affinity columns after detergent solubilization of luminal membranes from outer renal medulla

A method is developed for purification of the protein of the Ca2+‐activated K+ channel from outer renal medulla of pig kidney. The response of this K+ channel to physiological concentrations of Ca2+ is important for regulation of transtubular NaCl transport. In reconstituted vesicles direct addition of calmodulin doubles Ca2+ activation with sufficient affinity (K 0.1 nM) for chromatographic purification of the protein. For purification luminal plasma membrane vesicles are isolated on metrizamide density gradients and solubilized in CHAPS. The fraction of soluble protein retained on calmodulin‐Sepharose 4B columns in the presence of Ca2+ and eluted by EGTA is 0.7%. The purified protein has high Ca2+‐activated K+ channel activity after reconstitution into phospholipid vesicles. It distributes on two bands of 51 and 36 kDa after gel electrophoresis in SDS. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation of the channel. Phosphorylation from cAMP‐dependent protein kinase strongly stimulates Ca2+‐activated K+ channel activity and labels the 51 kDa band suggesting that this protein is involved in regulation of K+ channel opening.

Purification and characterization of (Na+ + K+)-ATPase. VII. Tryptic degradation of the Na-form of the enzyme protein resulting in selective modification of dephosphorylation reactions of the (Na+ + K+)-ATPase

Abstract 1. 1. We have examined the effect of partial tryptic digestion of purified ( Na + + K + )-ATPase in the presence of NaCl on the formation and degradation of the phosphoenzyme formed from ATP. The digestion to a ( Na + + K + )-ATPase activity of 40% barely affects ATP binding and the steady-state level of phosphorylation, but increases the rate of the Na + -ADP-ATP exchange to 150% of control and reduces the K + -phosphatase activity to 15–20% of control (Jorgensen, P.L. (1977) Biochim. Biophys. Acta 466, 97–108). 2. 2. After this tryptic cleavage in the presence of NaCl, the fraction of ADP sensitive phosphoenzyme was 2–3-fold higher than control levels. The presence of KCl depressed the phosphoenzyme level 2–3-fold less than that of the control and the K + -dependent increment of the rate constant for dephosphorylation was less than half that of the control. 3. 3. The results show that partial tryptic digestion of the purified ( Na + + K + )-ATPase in the presence of NaCl selectively affects protein areas involved both in the transformation of the phosphoenzyme from an ADP-sensitive to an ADP-insensitive form and in the stimulation by K + of the dephosphorylation process and the K + -phosphatase activity. The protein areas that are involved in these reactions seem therefore to be close to one another, or identical, and they appear to be spatially separated from the ATP binding area.