Functional Consequences of Various Leucine Mutations in the M3/M4 Loop of the Na+,K+-ATPase α-Subunit (original) (raw)
Related papers
Biochemistry, 2004
The Na,K-and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K + for cytoplasmic Na + or H + , respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the R subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na + , TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na +-independent hydrolysis of ATP, a 2-fold higher K 0.5 for Na + activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na + over H + for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K + and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the R subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.
Biochemical and Biophysical Research Communications, 2005
The effect of point mutation in the sequence 316 TWLE 319 , which occurs in the extracellular loop flanking the third (M3) and the fourth (M4) transmembrane segment (L3/4) of the Na + ,K +-ATPase a-subunit, was examined. Mutation of Glu 319 to Asp yielded an enzyme with full activity, whereas substituting Glu 319 to Ala resulted in a severe loss of activity. A negative charge was introduced along the sequence, one residue at a time, from Thr 316 to Leu 318 (by E-scanning) in the mutant construct with Glu 319 already mutated to Gln. The activity that had been reduced to 60% by the mutation of Glu 319 to Gln was restored upon the introduction of a negative charge by E-scanning. When Leu 318 was replaced by Glu in a series of scanning experiments, the K + sensitivity of the ATPase activity was lowered. The lowering of K + sensitivity was further demonstrated when a mutation of Leu 318 to Glu was introduced into the wild-type enzyme. Furthermore, mutants with Leu 318 to Gln, Arg, and Phe displayed lower K + sensitivity similar to that of Leu 318 to Glu mutant. Leu 318 may be in access path for K + , and any substitution at this position may interfere with access of K + from outside the cell.
Eight Amino Acids Form the ATP Recognition Site of Na + /K + -ATPase †
Biochemistry, 2003
Point mutations of a part of the H 4 -H 5 loop (Leu 354 -Ile 604 ) of Na + /K + -ATPase have been used to study the ATP and TNP-ATP binding affinities. Besides the previously reported amino acid residues Lys 480 , Lys 501 , Gly 502 , and Cys 549 , we have found four more amino acid residues, viz., Glu 446 , Phe 475 , Gln 482 , and Phe 548 , completing the ATP-binding pocket of Na + /K + -ATPase. Moreover, mutation of Arg 423 has also resulted in a large decrease in the extent of ATP binding. This residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site, probably due to formation of a hydrogen bond with Glu 472 . On the other hand, only some minor effects were caused by mutations of Ile 417 , Asn 422 , Ser 445 , and Glu 505 .
Journal of Biological Chemistry, 1997
During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G 950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and ␣1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and ␣1 have similar apparent affinities for cytoplasmic Na ؉ and extracellular K ؉. In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K ؉ deocclusion pathway of the reaction is rate-limiting. At 1 M ATP K ؉ inhibits Na ؉-ATPase of ␣1, but activates Na ؉-ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E 1) from K ؉-occluded enzyme (E 2 (K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of V max to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than ␣1. These distinct kinetics of E233K suggest a shift in its E 1 /E 2 conformational equilibrium toward E 1. Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu 233 3 Lys mutation.
The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe 323 , Ile 322 , Gly 326 , Ala 330 , Pro 333 , Glu 334 , and Gly 335 . In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ( 333 PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.
Isolation and cloning of the K +-independent, ouabain-insensitive Na +ATPase
Biochimica Et Biophysica Acta-biomembranes, 2011
Primary Na + transport has been essentially attributed to Na + /K + pump. However, there are functional and biochemical evidences that suggest the existence of a K + -independent, ouabain-insensitive Na + pump, associated to a Na + -ATPase with similar characteristics, located at basolateral plasma membrane of epithelial cells. Herein, membrane protein complex associated with this Na + -ATPase was identified. Basolateral membranes from guinea-pig enterocytes were solubilized with polyoxyethylene-9-lauryl ether and Na + -ATPase was purified by concanavalin A affinity and ion exchange chromatographies. Purified enzyme preserves its native biochemical characteristics: Mg 2+ dependence, specific Na + stimulation, K + independence, ouabain insensitivity and inhibition by furosemide (IC 50 : 0.5 mM) and vanadate (IC 50 : 9.1 μM). IgY antibodies against purified Na + -ATPase did not recognize Na + /K + -ATPase and vice versa. Analysis of purified Na + -ATPase by SDS-PAGE and 2D-electrophoresis showed that is constituted by two subunits: 90 (α) and 50 (β) kDa. Tandem mass spectrometry of α-subunit identified three peptides, also present in most Na + /K + -ATPase isoforms, which were used to design primers for cloning both ATPases by PCR from guinea-pig intestinal epithelial cells. A cDNA fragment of 1148 bp (atna) was cloned, in addition to Na + /K + -ATPase α1-isoform cDNA (1283 bp). In MDCK cells, which constitutively express Na + -ATPase, silencing of atna mRNA specifically suppressed Na + -ATPase α-subunit and ouabain-insensitive Na + -ATPase activity, demonstrating that atna transcript is linked to this enzyme. Guinea-pig atna mRNA sequence (2787 bp) was completed using RLM-RACE. It encodes a protein of 811 amino acids (88.9 kDa) with the nine structural motifs of P-type ATPases. It has 64% identity and 72% homology with guinea-pig Na + /K + -ATPase α1-isoform. These structural and biochemical evidences identify the K + -independent, ouabain-insensitive Na + -ATPase as a unique P-type ATPase.
Advances in Na+,K+-ATPase studies: from protein to gene and back to protein
FEBS Letters, 1989
Complete primary stators of both submit of Na',K+-ATPase from various sources have been ~~biisbed by a ~mbination of the methods for molecular cloning and protein chemistry. The gene family homologo~ to the a-subunit cDNA of animal Na+,K*-ATPases has been found in the human genome. Some genes of this family encode the known isoforms (aI and aI1) of the Na+,K+-ATPase catalytic subunit. The proteins coded by other genes can be either new isoforms of the Na+,K+-ATPase catalytic subunit or other ion-transporting ATPases. Expression of the genes of this family proceeds in a tissue-specific manner and changes during the postnatal development and neoplastic transformation. The complete exon-intron structure of one of the genes of this family has been established. This gene codes for the form of the catalytic subunit, the existence of which has been unknown. Apparently, all the genes of the discovered family have a similar intron-exon structure. There is certain correlation between the gene structure and the proposed domain a~angement of the a-subunit. The results obtained have become the basis for the experiments which prove the existence of the earlier unknown afI1 isoform of the Na+,K+-ATPase catalytic subunit and have made possible the study of its function.
Biochemistry, 1999
The phosphorylation capacity of Na + ,K + -ATPase preparations in common use is much less than expected on the basis of the molecular weight of the enzyme deduced from cDNA sequences. This has led to the popularity of half-of-the-sites or flip-flop models for the enzyme reaction mechanism. We have prepared Na + ,K + -ATPase from nasal salt glands of salt-adapted ducks which has a phosphorylation capacity and specific activity near the theoretical maxima. Preparations with specific activities of >60 µmol (mg of protein) -1 min -1 at 37°C had phosphorylation capacities of >60 nmol/mg of protein, and the rate of turnover of the enzyme was 9690 min -1 , within the range reported for the enzyme from other sources. The fraction of the maximal specific activity of the enzyme compared well with the fraction of the protein on SDS-PAGE which was R and chains, especially at the highest specific activity which indicates that all of the R protomers are active. The gels of the most reactive preparations contained only R and chains, but less active preparations contained a number of extraneous proteins. The major contaminant was actin. The preparation did not contain any protein which migrated in the molecular weight range of the γ subunit. The subunit composition of the enzyme was R 1 and 1 only. This is the first report of a pure, homogeneous, fully active preparation of the protein. Reaction models which incorporate a half-of-the-sites or flip-flop mechanism do not apply to this enzyme. Na + ,K + -ATPase 1 (adensoine triphosphatase, EC 3.6.1.3), first identified by Skou , is the biochemical manifestation of the Na + ,K + pump which is responsible for the asymmetric distribution of Na + and K + across animal cell membranes. Early results of ion transport studies with intact cells showed that the movement of Na + out of the cell is coupled to the movement of K + in and both depend on the hydrolysis of ATP, and biochemical studies with enzyme preparations showed that Na + promotes phosphorylation from ATP and K + promotes dephosphorylation (2). The findings formed the basis of the Albers-Post model of the enzyme reaction mechanism which supposes that the enzyme exists in two major conformations, one of which can be phosphorylated by ATP and the other by inorganic phosphate, and that the reaction mechanism is ping-pong with respect to Na + and K + . The model has been remarkably successful in accounting for the physiological, electrophysiological, and biochemical properties of the pump.
K + Congeners That Do Not Compromise Na + Activation of the Na + ,K + -ATPase
Journal of Biological Chemistry, 2014
The Na + , K + -ATPase discriminates between similar and abundant ions. Results: The K + congener acetamidinium interacts with the outward facing sites of the Na + ,K + -ATPase, but does not interact with the inward facing sites. Conclusion: Water in the ion binding cavity regulate ion selectivity of the Na + ,K + -ATPase. Significance: This study identifies new determinants of the ion selectivity of K + -transporting P-type pumps.