Evidence That Ser775 in the α Subunit of the Na,K-ATPase Is a Residue in the Cation Binding Pocket

Blostein, Rhoda; Wilczynska, Ania; Karlish, Steven J. D.; Argüello, José M.; Lingrel, Jerry B.

doi:10.1074/jbc.272.40.24987

Cited by 49 publications

(47 citation statements)

References 33 publications

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“…DISCUSSION We have taken advantage of the functional differences and the high degree of sequence homology between the H,K-and Na,K-ATPases to attempt to identify the determinant of the electrogenicity of cation transport by the group IIc P-ATPases. In the fifth transmembrane segment, several amino acid residues have been shown to play a role in cation binding in both the H,K-and Na,K-ATPases (22,23,(25)(26)(27)(28)(29)(30)(31) and also in SERCA (32). The middle of the fifth transmembrane segment region is highly similar between the H,K-and Na,K-ATPases (Fig.…”

Section: Electrogenic Transport By the Lys 800 Mutants Of The Hkatpamentioning

confidence: 83%

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

Burnay

Crambert

Kharoubi‐Hess

et al. 2003

Journal of Biological Chemistry

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Section: Electrogenic Transport By the Lys 800 Mutants Of The Hkatpamentioning

confidence: 83%

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

Burnay

Crambert

Kharoubi‐Hess

et al. 2003

Journal of Biological Chemistry

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“…Here, we show that Q783A has normal Ca 2ϩ transport but a 60-fold reduction in the apparent affinity for Mn 2ϩ . Only one such mutation with a similar dramatic effect on ion selectivity has been reported in studies on other ion pumps; substitution of Ser 775 in M5 with alanine in the Na ϩ /K ϩ -ATPase causes a 30-fold decrease in only K ϩ but not Na ϩ affinity (25,26). It is worth noting that only two of five residues required for Ca 2ϩ binding in SERCA were essential in Pmr1.…”

Section: Discussionmentioning

confidence: 98%

Phenotypic Screening of Mutations in Pmr1, the Yeast Secretory Pathway Ca2+/Mn2+-ATPase, Reveals Residues Critical for Ion Selectivity and Transport

Wei

Chen

Rosas

et al. 2000

Journal of Biological Chemistry

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Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca 2؉ and Mn 2؉ ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca 2؉ pumps and screened for loss of Ca 2؉ and Mn 2؉ transport on the basis of hypersensitivity to 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) and Mn 2؉ toxicity, respectively. Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn 2؉ toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including 45 Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable 45 Ca transport; of these, a subset also showed defects in trafficking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn 774 and Asp 778 in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site. Class 3 mutants appear to have altered selectivity for Ca 2؉ and Mn 2؉ ions, as exemplified by mutant Q783A in M6. These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps.

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“…Only ATP9A and ATP9B show a semiconservative replacement of the lysine with arginine. The Na þ ,K þ -ATPase residue in the corresponding position is a serine, which contributes its side-chain oxygen to K þ binding (2,3,16,18). In addition, the asparagine (corresponding to Asn 874 ) next to the serine as well as a threonine and a glutamate in M5 also contribute to K þ binding in Na þ ,K þ -ATPase (2,3,17,18).…”

Section: Discussionmentioning

confidence: 99%

Critical role of a transmembrane lysine in aminophospholipid transport by mammalian photoreceptor P ₄ -ATPase ATP8A2

Coleman¹,

Vestergaard

Molday

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

172

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ATP8A2 is a P 4 -ATPase ("flippase") located in membranes of retinal photoreceptors, brain cells, and testis, where it mediates transport of aminophospholipids toward the cytoplasmic leaflet. It has long been an enigma whether the mechanism of P 4 -ATPases resembles that of the well-characterized cation-transporting P-type ATPases, and it is unknown whether the flippases interact directly with the lipid and with counterions. Our results demonstrate that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate (Asp 416 ) in the P-type ATPase signature sequence and exists in E 1 P and E 2 P forms similar to the archetypical P-type ATPases. Using the properties of the phosphoenzyme, the partial reaction steps of the transport cycle were examined, and the roles of conserved residues Asp 196 , Glu 198 , Lys 873 , and Asn 874 in the transport mechanism were elucidated. The former two residues in the A-domain T/D-G-E-S/T motif are involved in catalysis of E 2 P dephosphorylation, the glutamate being essential. Transported aminophospholipids activate the dephosphorylation similar to K þ activation of dephosphorylation in Na þ ,K þ -ATPase. Lys 873 mutants (particularly K873A and K873E) display a markedly reduced sensitivity to aminophospholipids. Hence, Lys 873 , located in transmembrane segment M5 at a "hot spot" for cation binding in Ca 2þ -ATPase and Na þ ,K þ -ATPase, appears to participate directly in aminophospholipid binding or to mediate a crucial interaction within the ATP8A2-CDC50 complex. By contrast, Lys 865 is unimportant for aminophospholipid sensitivity.to the E 1 form as a counterion is not required for activation of phosphorylation from ATP. Therefore, phospholipids could be the only substrate transported by ATP8A2.lipid transport mechanism | mutagenesis | phospholipid flippase | phosphatidylserine | membrane asymmetry P -type ATPases are a large family of membrane pumps believed to be transiently phosphorylated at the conserved aspartate residue of the DKTGT motif during the catalytic cycle. An important challenge is to understand how these proteins couple the utilization of ATP with transport of substances across the membrane. Among the most well-characterized P-type ATPases are the sarcoplasmic reticulum Ca 2þ -ATPase and the Na þ ,K þ -ATPase. During the reaction cycle of these ATPases, the intermediates E 1 , E 1 P, E 2 P, and E 2 (P representing phosphorylation) are formed sequentially, involving large movements of the three cytoplasmic domains, N (nucleotide binding), P (phosphorylation), and A ("actuator"), that are coupled through a linker region with the ion translocation occurring in the membrane domain M (1-3). For many other P-type ATPases, much less is known regarding the transport mechanism due to lack of structural and biochemical data, and even the existence of a phosphorylated intermediate is a conjecture based on sequence homology. P 4 -ATPases constitute a subfamily of P-type ATPases implicated in the transport of aminophospholipids (4, 5). Using ATP as an energy source, ...

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Evidence That Ser775 in the α Subunit of the Na,K-ATPase Is a Residue in the Cation Binding Pocket

Cited by 49 publications

References 33 publications

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

Phenotypic Screening of Mutations in Pmr1, the Yeast Secretory Pathway Ca2+/Mn2+-ATPase, Reveals Residues Critical for Ion Selectivity and Transport

Critical role of a transmembrane lysine in aminophospholipid transport by mammalian photoreceptor P ₄ -ATPase ATP8A2

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Evidence That Ser775 in the α Subunit of the Na,K-ATPase Is a Residue in the Cation Binding Pocket

Cited by 49 publications

References 33 publications

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

Phenotypic Screening of Mutations in Pmr1, the Yeast Secretory Pathway Ca2+/Mn2+-ATPase, Reveals Residues Critical for Ion Selectivity and Transport

Critical role of a transmembrane lysine in aminophospholipid transport by mammalian photoreceptor P 4 -ATPase ATP8A2

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Critical role of a transmembrane lysine in aminophospholipid transport by mammalian photoreceptor P ₄ -ATPase ATP8A2