Anja Pernille Einholm

The C-terminus of Na+,K+-ATPase controls Na+ affinity on both sides of the membrane through Arg935

The Na+,K+-ATPase C terminus has a unique location between transmembrane segments, appearing to participate in a network of interactions. We have examined the functional consequences of amino acid substitutions in this region and deletions of the C terminus of varying lengths. Assays revealing separately the mutational effects on internally and externally facing Na+ sites, as well as E1-E2 conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr1017 and Tyr1018, as well as putative interaction partners, Arg935 in the loop between transmembrane segments M8 and M9 and Lys768 in transmembrane segment M5, as crucial to Na+ activation of phosphorylation of E1, a partial reaction reflecting Na+ interaction on the cytoplasmic side of the membrane. Tyr1017, Tyr1018, and Arg935 are furthermore indispensable to Na+ interaction on the extracellular side of the membrane, as revealed by inability of high Na+ concentrations to drive the transition from E1P to E2P backwards toward E1P and inhibit Na+-ATPase activity in mutants. Lys768 is not important for Na+ binding from the external side of the membrane but is involved in stabilization of the E2 form. These data demonstrate that the C terminus controls Na+ affinity on both sides of the membrane and suggest that Arg935 constitutes an important link between the C terminus and the third Na+ site, involving an arginine-π stacking interaction between Arg935 and the C-terminal tyrosines. Lys768 may interact preferentially with the C terminus in E1 and E1P forms and with the loop between transmembrane segments M6 and M7 in E2 and E2P forms.

A C-terminal mutation of ATP1A3 underscores the crucial role of sodium affinity in the pathophysiology of rapid-onset dystonia-parkinsonism

The Na(+)/K(+)-ATPases are ion pumps of fundamental importance in maintaining the electrochemical gradient essential for neuronal survival and function. Mutations in ATP1A3 encoding the alpha3 isoform cause rapid-onset dystonia-parkinsonism (RDP). We report a de novo ATP1A3 mutation in a patient with typical RDP, consisting of an in-frame insertion of a tyrosine residue at the very C terminus of the Na(+)/K(+)-ATPase alpha3-subunit-the first reported RDP mutation in the C terminus of the protein. Expression studies revealed that there is no defect in the biogenesis or plasma membrane targeting, although cells expressing the mutant protein showed decreased survival in response to ouabain challenge. Functional analysis demonstrated a drastic reduction in Na(+) affinity in the mutant, which can be understood by structural modelling of the E1 and E2 conformations of the wild-type and mutant enzymes on the basis of the strategic location of the C terminus in relation to the third Na(+) binding site. The dramatic clinical presentation, together with the biochemical findings, provides both in vivo and in vitro evidence for a crucial role of the C terminus of the alpha-subunit in the function of the Na(+)/K(+)-ATPase and a key impact of Na(+) affinity in the pathophysiology of RDP.

The Rapid-onset Dystonia Parkinsonism Mutation D923N of the Na+,K+-ATPase α3 Isoform Disrupts Na+ Interaction at the Third Na+ Site

Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific α3-isoform of Na+,K+-ATPase. Here, we present the functional consequences of RDP mutation D923N. Relative to the wild type, the mutant exhibits a remarkable ∼200-fold reduction of Na+ affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E1 form with intracellular Na+. This is the largest effect on Na+ affinity reported so far for any Na+,K+-ATPase mutant. D923N also affects the interaction with extracellular Na+ normally driving the E1P to E2P conformational transition backward. However, no impairment of K+ binding was observed for D923N, leading to the conclusion that Asp923 is specifically associated with the third Na+ site that is selective toward Na+. The crystal structure of the Na+,K+-ATPase in E2 form shows that Asp923 is located in the cytoplasmic half of transmembrane helix M8 inside a putative transport channel, which is lined by residues from the transmembrane helices M5, M7, M8, and M10 and capped by the C terminus, recently found involved in recognition of the third Na+ ion. Structural modeling of the E1 form of Na+,K+-ATPase based on the Ca2+-ATPase crystal structure is consistent with the hypothesis that Asp923 contributes to a site binding the third Na+ ion. These results in conjunction with our previous findings with other RDP mutants suggest that a selective defect in the handling of Na+ may be a general feature of the RDP disorder.

Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1.

XR5118 [(3 Z,6 Z )-6-benzylidine-3-(5-(2-dimethylaminoethyl-thio-))-2-(thienyl)methylene-2,5-dipiperazinedione hydrochloride] can inactivate the anti-proteolytic activity of the serpin plasminogen activator inhibitor-1 (PAI-1), a potential therapeutic target in cancer and cardiovascular diseases. Serpins inhibit their target proteases by the P(1) residue of their reactive centre loop (RCL) forming an ester bond with the active-site serine residue of the protease, followed by insertion of the RCL into the serpins large central beta-sheet A. In the present study, we show that the RCL of XR5118-inactivated PAI-1 is inert to reaction with its target proteases and has a decreased susceptibility to non-target proteases, in spite of a generally increased proteolytic susceptibility of specific peptide bonds elsewhere in PAI-1. The properties of XR5118-inactivated PAI-1 were different from those of the so-called latent form of PAI-1. Alanine substitution of several individual residues decreased the susceptibility of PAI-1 to XR5118. The localization of these residues in the three-dimensional structure of PAI-1 suggested that the XR5118-induced inactivating conformational change requires mobility of alpha-helix F, situated above beta-sheet A, and is in agreement with the hypothesis that XR5118 binds laterally to beta-sheet A. These results improve our understanding of the unique conformational flexibility of serpins and the biochemical basis for using PAI-1 as a therapeutic target.

Importance of Leu99 in Transmembrane Segment M1 of the Na+,K+-ATPase in the Binding and Occlusion of K+

Twenty-six point mutations were introduced into the N-terminal and middle parts of transmembrane segment M1 of the Na+,K+-ATPase and its cytosolic extension. None of the alterations to charged and polar residues in the N-terminal part of M1 and its cytosolic extension had any major effect on the cation binding properties, thus rejecting the hypothesis that these residues are involved in cation selectivity. By contrast, specific residues in the middle part of M1, particularly Leu99, were found critical to K+ interaction of the enzyme. Hence, mutation L99A reduced the affinity for K+ activation of E2P dephosphorylation 17-fold, and L99F reduced the equilibrium level of the K+-occluded intermediate [K2]E2 and increased the rate of K+ deocclusion 39-fold, i.e. more than seen for mutation E329Q of the cation-binding glutamate in M4. L99Q affected K+ interaction in yet another way, the equilibrium level of [K2]E2 being slightly increased despite an increased rate of K+ deocclusion, suggesting that the K+ ions leave and enter the occlusion pocket more frequently than in the wild type. L99Q furthermore affected the ability to discriminate between Na+ and K+ on the extracellular side. Our findings can be explained by a structural model in which Leu99 and Glu329 interact and cooperate in K+ binding and gating of the K+ sites. The disturbance of K+ interaction in mutants with alteration to Leu91, Phe95, Ser96, or Leu98 could be a consequence of the roles of these residues in positioning the M1 helix optimally for the interaction between Leu99 and Glu329. Phe95 may serve to stabilize the pivot for movement of M1 through interaction with Ile287 in M3.

Plasminogen activator inhibitor-1 polymers, induced by inactivating amphipathic organochemical ligands.

Negatively charged organochemical inactivators of the anti-proteolytic activity of plasminogen activator inhibitor-1 (PAI-1) convert it to inactive polymers. As investigated by native gel electrophoresis, the size of the PAI-1 polymers ranged from dimers to multimers of more than 20 units. As compared with native PAI-1, the polymers exhibited an increased resistance to temperature-induced unfolding. Polymerization was associated with specific changes in patterns of digestion with non-target proteases. During incubation with urokinase-type plasminogen activator, the polymers were slowly converted to reactive centre-cleaved monomers, indicating substrate behaviour of the terminal PAI-1 molecules in the polymers. A quadruple mutant of PAI-1 with a retarded rate of latency transition also had a retarded rate of polymerization. Studying a number of serpins by native gel electrophoresis, ligand-induced polymerization was observed only with PAI-1 and heparin cofactor II, which were also able to copolymerize. On the basis of these results, we suggest that the binding of ligands in a specific region of PAI-1 leads to so-called loop-sheet polymerization, in which the reactive centre loop of one molecule binds to beta-sheet A in another molecule. Induction of serpin polymerization by small organochemical ligands is a novel finding and is of protein chemical interest in relation to pathological protein polymerization in general.

Relationship between intracellular Na+ concentration and reduced Na+ affinity in Na+,K+-ATPase mutants causing neurological disease

Background: Na+,K+-ATPase mutations extending the C terminus cause neurological disease. Results: C-terminal extension reduces Na+ affinity. Analysis of several mutants establishes a relationship between change in Na+ affinity and change of intracellular Na+ and K+ concentrations. Conclusion: The Na+ affinity of the Na+,K+-ATPase is a major in vivo determinant of the intracellular Na+ concentration. Significance: Insight in pathophysiology and regulation of the Na+,K+-ATPase is obtained. The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na+,K+-ATPase α2 and α3 isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na+,K+-ATPase mutations, extending the C terminus by either 28 residues (“+28” mutation) or an extra tyrosine (“+Y”), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific α isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na+ affinity without disturbance of K+ binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of α2 despite a high expression level. A significant rise of [Na+]i and reduction of [K+]i was detected in cells expressing mutants with reduced Na+ affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na+ affinity were found to reduce [Na+]i. It is concluded that the Na+ affinity of the Na+,K+-ATPase is an important determinant of [Na+]i.

Molecular Cloning and Characterization of Porcine Na+/K+-ATPase Isoforms α1, α2, α3 and the ATP1A3 Promoter

Na+/K+-ATPase maintains electrochemical gradients of Na+ and K+ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na+/K+-ATPase exists in four different isoforms (α1–α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na+/K+-ATPase mutations, we have determined the porcine coding sequences of the α1–α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na+ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na+ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.

Mutagenesis of Residues Involved in Control of the Ca2+ Entry Pathway and Conformational Changes Associated with Ca2+ Binding in the SR Ca2+-ATPase

Abstract: Rapid kinetic measurements were used to study the rate of Ca2+ dissociation from the high‐affinity Ca2+ sites of the dephosphoenzyme (i.e., from the E1Ca2 form toward the cytoplasmic side) as well as the rate of Ca2+ binding with associated conformational changes (E2→E1Ca2 transition) in the wild type and mutants of the sarcoplasmic reticulum Ca2+‐ATPase expressed in mammalian cells. Cluster mutations as well as single mutations in transmembrane segment M3 resulted in conspicuous effects on the rate of Ca2+ migration. Furthermore, mutation of Asp59 in transmembrane segment M1 to arginine exerted a profound effect on Ca2+ interaction. The data demonstrate an important role for M3 residues in control of the Ca2+ entry pathway and provide functional evidence in support of a close relationship between this pathway and the water‐accessible channel leading between transmembrane segments M1 and M3 in the thapsigargin stabilized E2 structure. In addition, rapid kinetic measurements demonstrated that the hydrogen bond network involving Asp813 of loop L6‐7 and Lys758 of M5 is important for the E2→E1Ca2 transition.

Importance of a potential protein kinase A phosphorylation site of Na+,K+-ATPase and its interaction network for Na+ binding

The molecular mechanism underlying PKA-mediated regulation of Na+,K+-ATPase was explored in mutagenesis studies of the potential PKA site at Ser-938 and surrounding charged residues. The phosphomimetic mutations S938D/E interfered with Na+ binding from the intracellular side of the membrane, whereas Na+ binding from the extracellular side was unaffected. The reduction of Na+ affinity is within the range expected for physiological regulation of the intracellular Na+ concentration, thus supporting the hypothesis that PKA-mediated phosphorylation of Ser-938 regulates Na+,K+-ATPase activity in vivo. Ser-938 is located in the intracellular loop between transmembrane segments M8 and M9. An extended bonding network connects this loop with M10, the C terminus, and the Na+ binding region. Charged residues Asp-997, Glu-998, Arg-1000, and Lys-1001 in M10, participating in this bonding network, are crucial to Na+ interaction. Replacement of Arg-1005, also located in the vicinity of Ser-938, with alanine, lysine, methionine, or serine resulted in wild type-like Na+ and K+ affinities and catalytic turnover rate. However, when combined with the phosphomimetic mutation S938E only lysine substitution of Arg-1005 was compatible with Na+,K+-ATPase function, and the Na+ affinity of this double mutant was reduced even more than in single mutant S938E. This result indicates that the positive side chain of Arg-1005 or the lysine substituent plays a mechanistic role as interaction partner of phosphorylated Ser-938, transducing the phosphorylation signal into a reduced affinity of Na+ site III. Electrostatic interaction of Glu-998 is of minor importance for the reduction of Na+ affinity by phosphomimetic S938E as revealed by combining S938E with E998A.