Expression and functional characterization of missense mutations in ATP8A2 linked to severe neurological disorders.

ATP8A2 is a P4-ATPase (adenosine triphosphate) that actively flips phosphatidylserine and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. Mutations in the ATP8A2 gene have been reported to cause severe autosomal recessive neurological diseases in humans characterized by intellectual disability, hypotonia, chorea, and hyperkinetic movement disorders with or without optic and cerebellar atrophy. To determine the effect of disease-associated missense mutations on ATP8A2, we expressed six variants with the accessory subunit CDC50A in HEK293T cells. The level of expression, cellular localization, and functional activity were analyzed by western blot analysis, immunofluorescence microscopy, and ATPase activity assays. Two variants (p.Ile376Met and p.Lys429Met) expressed at normal ATP8A2 levels and preferentially localized to the Golgi-recycling endosomes, but were devoid of ATPase activity. Four variants (p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg) expressed poorly, localized to the endoplasmic reticulum, and lacked ATPase activity. The expression of these variants was increased twofold by the addition of the proteasome inhibitor MG132. We conclude that the p.Ile376Met and p.Lys429Met variants fold in a native-like conformation, but lack key amino acid residues required for ATP-dependent lipid transport. In contrast, the p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg variants are highly misfolded and undergo rapid proteosomal degradation.

Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase.

The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na+,K+-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na+ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ∼30-fold reduction of apparent Na+ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E1P and E2P. The apparent affinity for K+ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na+-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E1-E2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K+ than the WT enzyme, which together with the reduced Na+ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na+ interaction at site III.

Proteomic Analysis and Functional Characterization of P4-ATPase Phospholipid Flippases from Murine Tissues.

P4-ATPases are a subfamily of P-type ATPases that flip phospholipids across membranes to generate lipid asymmetry, a property vital to many cellular processes. Mutations in several P4-ATPases have been linked to severe neurodegenerative and metabolic disorders. Most P4-ATPases associate with one of three accessory subunit isoforms known as CDC50A (TMEM30A), CDC50B (TMEM30B), and CDC50C (TMEM30C). To identify P4-ATPases that associate with CDC50A, in vivo, and determine their tissue distribution, we isolated P4-ATPases-CDC50A complexes from retina, brain, liver, testes, and kidney on a CDC50A immunoaffinity column and identified and quantified P4-ATPases from their tryptic peptides by mass spectrometry. Of the 12 P4-ATPase that associate with CDC50 subunits, 10 P4-ATPases were detected. Four P4-ATPases (ATP8A1, ATP11A, ATP11B, ATP11C) were present in all five tissues. ATP10D was found in low amounts in liver, brain, testes, and kidney, and ATP8A2 was present in significant amounts in retina, brain, and testes. ATP8B1 was detected only in liver, ATP8B3 and ATP10A only in testes, and ATP8B2 primarily in brain. We also show that ATP11A, ATP11B and ATP11C, like ATP8A1 and ATP8A2, selectively flip phosphatidylserine and phosphatidylethanolamine across membranes. These studies provide new insight into the tissue distribution, relative abundance, subunit interactions and substrate specificity of P4-ATPase-CDC50A complexes.

Dynamics of P-type ATPase transport revealed by single-molecule FRET.

Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.

Disease mutations reveal residues critical to the interaction of P4-ATPases with lipid substrates.

Phospholipid flippases (P4-ATPases) translocate specific phospholipids from the exoplasmic to the cytoplasmic leaflet of membranes. While there is good evidence that the overall molecular structure of flippases is similar to that of P-type ATPase ion-pumps, the transport pathway for the "giant" lipid substrate has not been determined. ATP8A2 is a flippase with selectivity toward phosphatidylserine (PS), possessing a net negatively charged head group, whereas ATP8B1 exhibits selectivity toward the electrically neutral phosphatidylcholine (PC). Setting out to elucidate the functional consequences of flippase disease mutations, we have identified residues of ATP8A2 that are critical to the interaction with the lipid substrate during the translocation process. Among the residues pinpointed are I91 and L308, which are positioned near proposed translocation routes through the protein. In addition we pinpoint two juxtaposed oppositely charged residues, E897 and R898, in the exoplasmic loop between transmembrane helices 5 and 6. The glutamate is conserved between PS and PC flippases, whereas the arginine is replaced by a negatively charged aspartate in ATP8B1. Our mutational analysis suggests that the glutamate repels the PS head group, whereas the arginine minimizes this repulsion in ATP8A2, thereby contributing to control the entry of the phospholipid substrate into the translocation pathway.

Arginine substitution of a cysteine in transmembrane helix M8 converts Na+,K+-ATPase to an electroneutral pump similar to H+,K+-ATPase.

Na+,K+-ATPase and H+,K+-ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H+,K+-ATPase avoids binding of Na+ at the site corresponding to the Na+-specific site of the Na+,K+-ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na+,K+-ATPase with arginine, present in the H+,K+-ATPase at the corresponding position, converted the normal 3Na+:2K+:1ATP stoichiometry of the Na+,K+-ATPase to electroneutral 2Na+:2K+:1ATP stoichiometry similar to the electroneutral transport mode of the H+,K+-ATPase. The electroneutral C932R mutant of the Na+,K+-ATPase retained a wild-type-like enzyme turnover rate for ATP hydrolysis and rate of cellular K+ uptake. Only a relatively minor reduction of apparent Na+ affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na+ affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine+ group of the M8 arginine replaces Na+ at the third site, thus preventing Na+ binding there, although allowing Na+ to bind at the two other sites and become transported. Hence, in the H+,K+-ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

Neurological disease mutations of α3 Na+,K+-ATPase: Structural and functional perspectives and rescue of compromised function.

Na+,K+-ATPase creates transmembrane ion gradients crucial to the function of the central nervous system. The α-subunit of Na+,K+-ATPase exists as four isoforms (α1-α4). Several neurological phenotypes derive from α3 mutations. The effects of some of these mutations on Na+,K+-ATPase function have been studied in vitro. Here we discuss the α3 disease mutations as well as information derived from studies of corresponding mutations of α1 in the light of the high-resolution crystal structures of the Na+,K+-ATPase. A high proportion of the α3 disease mutations occur in the transmembrane sector and nearby regions essential to Na+ and K+ binding. In several cases the compromised function can be traced to disturbance of the Na+ specific binding site III. Recently, a secondary mutation was found to rescue the defective Na+ binding caused by a disease mutation. A perspective is that it may be possible to develop an efficient pharmaceutical mimicking the rescuing effect.

P4-ATPases as Phospholipid Flippases-Structure, Function, and Enigmas.

P4-ATPases comprise a family of P-type ATPases that actively transport or flip phospholipids across cell membranes. This generates and maintains membrane lipid asymmetry, a property essential for a wide variety of cellular processes such as vesicle budding and trafficking, cell signaling, blood coagulation, apoptosis, bile and cholesterol homeostasis, and neuronal cell survival. Some P4-ATPases transport phosphatidylserine and phosphatidylethanolamine across the plasma membrane or intracellular membranes whereas other P4-ATPases are specific for phosphatidylcholine. The importance of P4-ATPases is highlighted by the finding that genetic defects in two P4-ATPases ATP8A2 and ATP8B1 are associated with severe human disorders. Recent studies have provided insight into how P4-ATPases translocate phospholipids across membranes. P4-ATPases form a phosphorylated intermediate at the aspartate of the P-type ATPase signature sequence, and dephosphorylation is activated by the lipid substrate being flipped from the exoplasmic to the cytoplasmic leaflet similar to the activation of dephosphorylation of Na(+)/K(+)-ATPase by exoplasmic K(+). How the phospholipid is translocated can be understood in terms of a peripheral hydrophobic gate pathway between transmembrane helices M1, M3, M4, and M6. This pathway, which partially overlaps with the suggested pathway for migration of Ca(2+) in the opposite direction in the Ca(2+)-ATPase, is wider than the latter, thereby accommodating the phospholipid head group. The head group is propelled along against its concentration gradient with the hydrocarbon chains projecting out into the lipid phase by movement of an isoleucine located at the position corresponding to an ion binding glutamate in the Ca(2+)- and Na(+)/K(+)-ATPases. Hence, the P4-ATPase mechanism is quite similar to the mechanism of these ion pumps, where the glutamate translocates the ions by moving like a pump rod. The accessory subunit CDC50 may be located in close association with the exoplasmic entrance of the suggested pathway, and possibly promotes the binding of the lipid substrate. This review focuses on properties of mammalian and yeast P4-ATPases for which most mechanistic insight is available. However, the structure, function and enigmas associated with mammalian and yeast P4-ATPases most likely extend to P4-ATPases of plants and other organisms.

Rescue of Na+ affinity in aspartate 928 mutants of Na+,K+-ATPase by secondary mutation of glutamate 314.

The Na(+),K(+)-ATPase binds Na(+) at three transport sites denoted I, II, and III, of which site III is Na(+)-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na(+) affinity in the α1-, α2-, and α3-isoforms of Na(+),K(+)-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na(+)-coordinating residues in site III. Remarkably, the Na(+) affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na(+) binding and does not affect the E1-E2 conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na(+) affinity is likely intrinsic to the Na(+) binding pocket, and the underlying mechanism could be a tightening of Na(+) binding at Na(+) site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na(+),K(+) pump function in intact cells. Rescue of Na(+) affinity and Na(+) and K(+) transport by second-site mutation is unique in the history of Na(+),K(+)-ATPase and points to new possibilities for treatment of neurological patients carrying Na(+),K(+)-ATPase mutations.

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

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.

Searching PubMed for studies on bacteremia, bloodstream infection, septicemia, or whatever the best term is: a note of caution.

BACKGROUND: There is inconsistency in the terminology used to describe bacteremia. To demonstrate the impact on information retrieval, we compared the yield of articles from PubMed MEDLINE using the terms "bacteremia," "bloodstream infection," and "septicemia." METHODS: We searched for articles published between 1966 and 2009, and depicted the relationships among queries graphically. To examine the content of the retrieved articles, we extracted all Medical Subject Headings (MeSH) terms and compared topic similarity using a cosine measure. RESULTS: The recovered articles differed greatly by term, and only 53 articles were captured by all terms. Of the articles retrieved by the "bacteremia" query, 21,438 (84.1%) were not captured when searching for "bloodstream infection" or "septicemia." Likewise, only 2,243 of the 11,796 articles recovered by free-text query for "bloodstream infection" were retrieved by the "bacteremia" query (19%). Entering "bloodstream infection" as a phrase, 46.1% of the records overlapped with the "bacteremia" query. Similarity measures ranged from 0.52 to 0.78 and were lowest for "bloodstream infection" as a phrase compared with "septicemia." CONCLUSION: Inconsistent terminology has a major impact on the yield of queries. Agreement on terminology should be sought and promoted by scientific journals. An immediate solution is to add "bloodstream infection" as entry term for bacteremia in the MeSH vocabulary.

Ca2+-ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum Ca2+-ATPase 2 isoform (SERCA2c).

We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676-684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166+/-26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.