Flemming Cornelius

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

The sodium-potassium pump (Na+,K+-ATPase) is responsible for establishing Na+ and K+ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K+·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K+, in marked contrast to previous models. Due to antagonism between ouabain and K+, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.

Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state

Na+,K+-ATPase pumps three Na+ ions out of cells in exchange for two K+ taken up from the extracellular medium per ATP molecule hydrolysed, thereby establishing Na+ and K+ gradients across the membrane in all animal cells. These ion gradients are used in many fundamental processes, notably excitation of nerve cells. Here we describe 2.8 Å-resolution crystal structures of this ATPase from pig kidney with bound Na+, ADP and aluminium fluoride, a stable phosphate analogue, with and without oligomycin that promotes Na+ occlusion. These crystal structures represent a transition state preceding the phosphorylated intermediate (E1P) in which three Na+ ions are occluded. Details of the Na+-binding sites show how this ATPase functions as a Na+-specific pump, rejecting K+ and Ca2+, even though its affinity for Na+ is low (millimolar dissociation constant). A mechanism for sequential, cooperative Na+ binding can now be formulated in atomic detail.

Reversible Oxidative Modification. A Key Mechanism of Na+-K+ Pump Regulation

Angiotensin II (Ang II) inhibits the cardiac sarcolemmal Na+-K+ pump via protein kinase (PK)C-dependent activation of NADPH oxidase. We examined whether this is mediated by oxidative modification of the pump subunits. We detected glutathionylation of β1, but not α1, subunits in rabbit ventricular myocytes at baseline. β1 Subunit glutathionylation was increased by peroxynitrite (ONOO−), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased α1/β1 subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with β1 subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na+-K+ pump current (Ip). Cysteine (Cys46) of the β1 subunit was the likely candidate for glutathionylation. We expressed Na+-K+ pump α1 subunits with wild-type or Cys46-mutated β1 subunits in Xenopus oocytes. ONOO− induced glutathionylation of β1 subunit and a decrease in Na+-K+ pump turnover number. This was eliminated by mutation of Cys46. ONOO− also induced glutathionylation of the Na+-K+ ATPase β1 subunit from pig kidney. This was associated with a ≈2-fold decrease in the rate-limiting E2→E1 conformational change of the pump, as determined by RH421 fluorescence. We propose that kinase-dependent regulation of the Na+-K+ pump occurs via glutathionylation of its β1 subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na+ levels.

Identification of a Phospholemman-like Protein from Shark Rectal Glands EVIDENCE FOR INDIRECT REGULATION OF Na,K-ATPase BY PROTEIN KINASE C VIA A NOVEL MEMBER OF THE FXYDY FAMILY

The Na,K-ATPase provides the driving force for many ion transport processes through control of Na+and K+ concentration gradients across the plasma membranes of animal cells. It is composed of two subunits, α and β. In many tissues, predominantly in kidney, it is associated with a small ancillary component, the γ-subunit that plays a modulatory role. A novel 15-kDa protein, sharing considerable homology to the γ-subunit and to phospholemman (PLM) was identified in purified Na,K-ATPase preparations from rectal glands of the shark Squalus acanthias, but was absent in pig kidney preparations. This PLM-like protein from shark (PLMS) was found to be a substrate for both PKA and PKC. Antibodies to the Na,K-ATPase α-subunit coimmunoprecipitated PLMS. Purified PLMS also coimmunoprecipitated with the α-subunit of pig kidney Na,K-ATPase, indicating specific association with different α-isoforms. Finally, PLMS and the α-subunit were expressed in stoichiometric amounts in rectal gland membrane preparations. Incubation of membrane bound Na,K-ATPase with non-solubilizing concentrations of C12E8resulted in functional dissociation of PLMS from Na,K-ATPase and increased the hydrolytic activity. The same effects were observed after PKC phosphorylation of Na,K-ATPase membrane preparations. Thus, PLMS may function as a modulator of shark Na,K-ATPase in a way resembling the phospholamban regulation of the Ca-ATPase.

Regulation of Na,K-ATPase by PLMS, the Phospholemman-like Protein from Shark MOLECULAR CLONING, SEQUENCE, EXPRESSION, CELLULAR DISTRIBUTION, AND FUNCTIONAL EFFECTS OF PLMS

In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase α-subunit and PLMS showed the presence of α and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E1∼P → E2-P reaction, which is partially rate-limiting in shark, and the K+ deocclusion reaction, E2(K) → E1, are accelerated. The latter may explain the finding that the apparent Na+ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase α-subunit is important for the modulation of shark Na,K-ATPase activity.

First crystal structures of Na+,K+-ATPase: new light on the oldest ion pump.

Na(+),K(+)-adenosine triphosphatase (NKA) is the first P-type ion translocating adenosine triphosphatase (ATPase) ever identified, and the significance of this class of proteins was highlighted by the 1997 Nobel Prize in Chemistry awarded to Jens C. Skou for the discovery in 1957. More than half a century passed between the initial identification and the publication of a high-resolution crystal structure of NKA. Although the new crystal structures provided many surprises and insights, structural biology on this system remains challenging, as NKA is a very difficult protein to crystallize. Here we explain the reasons behind the challenges, introduce a mechanism that governs the function, and summarize current knowledge of NKA structure in comparison with another member of the P-type ATPase family, Ca(2+)-ATPase.

Na+Na+ exchange mediated by (Na+ + K+)-ATPase reconstituted into liposomes. Evaluation of pump stoichiometry and response to ATP and ADP

(Na+ + K+)-ATPase from shark rectal glands reconstituted into lipid vesicles and oriented inside out catalyses an ouabain-sensitive Na+-Na+ exchange in the absence of intravesicular K+ when ATP is added extravesicularly. Intravesicular ouabain inhibited the exchange completely. This was also the case with digitoxigenin added to the vesicles. Intravesicular oligomycin inhibited the Na+-Na+ exchange partly in a fashion which was ATP dependent. The exchange is accompanied by a net hydrolysis of ATP with an apparent Km of 2.5 microM. ADP was found to give no stimulation of the Na+-Na+ exchange, contrarily, ADP inhibited the ATP-dependent exchange of Na+ both at optimal and supraoptimal ATP concentrations. When initial influx and efflux of 22Na was measured and the hydrolysis of ATP concomitantly determined a coupling ratio of 2.8:1.3:1 was found, i.e. 2.8 moles of Na+ were taken up (cellular efflux) and 1.3 moles of Na+ extruded (cellular influx) for each mole of ATP hydrolyzed. The electrogenic Na+-Na+ exchange generated a transmembrane potential which was measured with the fluorescent probe ANS (8-anilino-1-naphthalenesulfonic acid) to be 60 mV positive inside the liposomes (extracellular).

FXYD proteins reverse inhibition of the Na-K pump mediated by glutathionylation of its β1 subunit

The seven members of the FXYD protein family associate with the Na+-K+ pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na+-K+ pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na+-K+ pump β1 subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β1 subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β1 subunit was increased in myocardium from FXYD1−/− mice. In conclusion, there is a dependence of Na+-K+ pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na+-K+ pump complex. By facilitating deglutathionylation of the β1 subunit, FXYD proteins reverse oxidative inhibition of the Na+-K+ pump and play a dynamic role in its regulation.

Cholesterol modulation of molecular activity of reconstituted shark Na+,K(+)-ATPase.

The cholesterol content of liposome bilayers has been varied between 0-40 mol% to study the effects on reconstituted Na+,K(+)-ATPase. The maximum hydrolytic activity of reconstituted Na+,K(+)-ATPase was increased by cholesterol at concentrations above 10 mol% for both the physiological Na+/K(+)-exchange reactions, as well as for the partial reactions Na+/Na(+)-exchange and uncoupled Na+ efflux. Omission of cholesterol from the liposome bilayer modified the activation by cytoplasmic Na+, indicating effects on both Vmax and on the Na(+)-affinity. Several other kinetic parameters were found to be strongly influenced as well, most notable the steady-state phosphorylation level, and the characteristics of the phosphorylation/dephosphorylation reactions. These results indicate that cholesterol interacts directly with the Na+,K(+)-ATPase as an essential effector perhaps by affecting its conformational mobility or monomer interaction.

Rate limitation of the Na+,K+-ATPase pump cycle

The kinetics of Na(+)-dependent phosphorylation of the Na(+),K(+)-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na(+)], and [Mg(2+)], pH 7.4, and 24 degrees C). The well-established effect of buffer composition on the E(2)-E(1) equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E(2)) yielded a 1/tau value of 59 s(-1). Addition of MgCl(2) (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/tau to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E(2) state to an E(1) or E(1)-like state. The results thus demonstrate conclusively that the E(2) --> E(1) transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E(2) --> E(1) transition under physiological conditions could be estimated to be approximately 65 s(-1) for pig kidney enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (approximately 48 s(-1) and approximately 43 s(-1) for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the alpha(1) isoform the E(2) --> E(1) conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.