Structural Requirements for the Interaction between the Na+/K+-ATPase α- and β-Subunits

Lemas, M. Victor; Hamrick, M.; Emerick, Mark C.; Hwang, B.; Kostich, Mitch; Fambrough, Douglas M.

doi:10.1007/978-3-642-72511-1_46

Cited by 37 publications

(55 citation statements)

References 9 publications

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“…There is strong evidence (23)(24)(25) that specific interaction between the ␣-and ␤-subunit occurs in this region of the ␣-subunit. Of the five chimeras prepared in this study, only two (HN34 and HN34͞56) showed considerable K ϩ -activated ATPase activity.…”

Section: Discussionmentioning

confidence: 99%

High-affinity ouabain binding by a chimeric gastric H ⁺ ,K ⁺ -ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α ₁ -subunit of rat Na ⁺ ,K ⁺ -ATPase

Koenderink

Hermsen²,

Swarts³

et al. 2000

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

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Na ؉ ,K ؉ -ATPase and gastric H ؉ ,K ؉ -ATPase are two related enzymes that are responsible for active cation transport. Na ؉ ,K ؉ -ATPase activity is inhibited specifically by ouabain, whereas H ؉ ,K ؉ -ATPase is insensitive to this drug. Because it is not known which parts of the catalytic subunit of Na ؉ ,K ؉ -ATPase are responsible for ouabain binding, we prepared chimeras in which small parts of the ␣-subunit of H ؉ ,K ؉ -ATPase were replaced by their counterparts of the ␣1-subunit of rat Na ؉ ,K ؉ -ATPase. A chimeric enzyme in which transmembrane segments 5 and 6 of H ؉ ,K ؉ -ATPase were replaced by those of Na ؉ ,K ؉ -ATPase could form a phosphorylated intermediate, but hardly showed a K ؉ -stimulated dephosphorylation reaction. When transmembrane segments 3 and 4 of Na ؉ ,K ؉ -ATPase were also included in this chimeric ATPase, K ؉ -stimulated dephosphorylation became apparent. This suggests that there is a direct interaction between the hairpins M3-M4 and M5-M6. Remarkably, this chimeric enzyme, HN34͞56, had obtained a high-affinity ouabain-binding site, whereas the rat Na ؉ ,K ؉ -ATPase, from which the hairpins originate, has a low affinity for ouabain. The low affinity of the rat Na ؉ ,K ؉ -ATPase previously had been attributed to the presence of two charged amino acids in the extracellular domain between M1 and M2. In the HN34͞56 chimera, the M1͞M2 loop, however, originates from H ؉ ,K ؉ -ATPase, which has two polar uncharged amino acids on this position. Placement of two charged amino acids in the M1͞M2 loop of chimera HN34͞56 results in a decreased ouabain affinity. This indicates that although the M1͞M2 loop affects the ouabain affinity, binding occurs when the M3͞M4 and M5͞M6 hairpins of Na ؉ ,K ؉ -ATPase are present.

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Section: Discussionmentioning

confidence: 99%

High-affinity ouabain binding by a chimeric gastric H ⁺ ,K ⁺ -ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α ₁ -subunit of rat Na ⁺ ,K ⁺ -ATPase

Koenderink

Hermsen²,

Swarts³

et al. 2000

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

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“…Lemas et al (35) have shown that a 26-sequence peptide (Asn 889 -Ala 914 ) in the predicted M7M8 extracellular loop interacts directly with an external fragment of the ␤-subunit. It should be pointed out that their study utilized a co-immunoprecipitation protocol, and the functional importance of the association was not determined.…”

Section: Figmentioning

confidence: 99%

Site-directed Chemical Labeling of Extracellular Loops in a Membrane Protein

Kaplan

2000

Journal of Biological Chemistry

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We have mapped the membrane topology of the renal Na,K-ATPase ␣-subunit by using a combination of introduced cysteine mutants and surface labeling with a membrane impermeable Cys-directed reagent, N-biotinylaminoethyl methanethiosulfonate. To begin our investigation, two cysteine residues (Cys 911 and Cys 964 ) in the wild-type ␣-subunit were substituted to create a background mutant devoid of exposed cysteines (Lutsenko, S., Daoud, S., and Kaplan, J. H. (1997) J. Biol. Chem. 272, 5249 -5255). Into this background construct were then introduced single cysteines in each of the five putative extracellular loops (P118C, T309C, L793C, L876C, and M973C) and the resulting ␣-subunit mutants were co-expressed with the ␤-subunit in baculovirusinfected insect cells. All of our expressed Na,K-ATPase mutants were functionally active. Their ATPase, phosphorylation, and ouabain binding activities were measured, and the turnover of the phosphoenzyme intermediate was close to the wild-type enzyme, suggesting that they are folded properly in the infected cells. Incubation of the insect cells with the cysteine-selective reagent revealed essentially no labeling of the ␣-subunit of the background construct and labeling of all five mutants with single cysteine residues in putative extracellular loops. Two additional mutants, V969C and L976C, were created to further define the M9M10 loop. The lack of labeling for these two mutants showed that although Met 973 is apparently exposed, Val 969 and Leu 976 are not, demonstrating that this method may also be utilized to define membrane aqueous boundaries of membrane proteins. Our labeling studies are consistent with a specific 10-transmembrane segment model of the Na,KATPase ␣-subunit. This strategy utilized only functional Na,K-ATPase mutants to establish the membrane topology of the entire ␣-subunit, in contrast to most previously applied methods. Na,K-ATPase (sodium pump) is an integral membrane protein that is present in most animal cells. The enzyme consists of two subunits, a large catalytic ␣-subunit (about 110 kDa) and a glycosylated ␤-subunit (ϳ55 kDa); both subunits are required for enzymatic activity (1-3). Na,K-ATPase utilizes the energy derived from ATP hydrolysis to transport Na ϩ and K ϩ ions across plasma membranes against their electrochemical gradients and is a member of the P 2 -ATPase family (4). The ion gradients generated by the sodium pump are important for regulating a variety of physiological functions such as cell excitability, contractility, and secondary active transport. Several isoforms of the ␣-and ␤-subunits have been cloned from various species, and the primary sequences have been described (5). The secondary structural information on the protein, on the other hand, remains controversial despite extensive investigation; such information is essential for understanding structure/function relationships of the Na,K-ATPase. Based on hydropathy analysis (6), protease accessibility (7, 8), and immunochemical studies (9, 10), the amino-terminal third of the ␣-subun...

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“…Na ϩ pumps are expressed as ␣␤ dimers. The catalytic subunit, ␣, contains the Na ϩ , K ϩ , ATP, and ouabain binding sites; the smaller ␤-subunit may chaperone ␣ to the PM (17). There are four ␣-subunit isoforms (18).…”

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confidence: 99%

Local subplasma membrane Ca ²⁺ signals detected by a tethered Ca ²⁺ sensor

Lee¹,

Song²,

Nakai

et al. 2006

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

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Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent ''junctional'' sarcoplasmic͞endo-plasmic reticulum (jS͞ER) constitute specialized Ca 2؉ signaling complexes in many cell types. We examined the possibility that some Ca 2؉ signals arising in the junctional space between the PM and jS͞ER may represent cross-talk between the PM and jS͞ER. The Ca 2؉ sensor protein, GCaMP2, was targeted to different PM domains by constructing genes for fusion proteins with either the ␣1 or ␣2 isoform of the Na ؉ pump catalytic (␣) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that ␣2(f)GCaMP2, like native Na ؉ pumps with ␣2-subunits, sorted to PM domains that colocalized with subjacent S͞ER; ␣1(f)GCaMP2, like Na ؉ pumps with ␣1-subunits, was more uniformly distributed. The GCaMP2 moieties in both constructs were tethered just beneath the PM. Both constructs detected global Ca 2؉ signals evoked by serotonin (in arterial smooth muscle cells) and ATP, and by store-operated Ca 2؉ channel-mediated Ca 2؉ entry after S͞ER unloading with cyclopiazonic acid (in Ca 2؉ -free medium). When cytosolic Ca 2؉ diffusion was markedly restricted with EGTA, however, only ␣2(f)GCaMP2 detected the local, storeoperated Ca 2؉ channel-mediated Ca 2؉ entry signal. Thus, ␣1 Na ؉ pumps are apparently excluded from the PM microdomains occupied by ␣2 Na 2؉ pumps. The jS͞ER and adjacent PM may communicate by Ca 2؉ signals that are confined to the tiny junctional space between the two membranes. Similar methods may be useful for studying localized Ca 2؉ signals in other subPM microdomains and signals associated with other organelles.arterial smooth muscle ͉ astrocytes ͉ Na ϩ pump ͉ PLasmERosome ͉ store-operated Ca 2ϩ entry

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Structural Requirements for the Interaction between the Na+/K+-ATPase α- and β-Subunits

Cited by 37 publications

References 9 publications

High-affinity ouabain binding by a chimeric gastric H ⁺ ,K ⁺ -ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α ₁ -subunit of rat Na ⁺ ,K ⁺ -ATPase

High-affinity ouabain binding by a chimeric gastric H ⁺ ,K ⁺ -ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α ₁ -subunit of rat Na ⁺ ,K ⁺ -ATPase

Site-directed Chemical Labeling of Extracellular Loops in a Membrane Protein

Local subplasma membrane Ca ²⁺ signals detected by a tethered Ca ²⁺ sensor

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Structural Requirements for the Interaction between the Na+/K+-ATPase α- and β-Subunits

Cited by 37 publications

References 9 publications

High-affinity ouabain binding by a chimeric gastric H + ,K + -ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α 1 -subunit of rat Na + ,K + -ATPase

High-affinity ouabain binding by a chimeric gastric H + ,K + -ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α 1 -subunit of rat Na + ,K + -ATPase

Site-directed Chemical Labeling of Extracellular Loops in a Membrane Protein

Local subplasma membrane Ca 2+ signals detected by a tethered Ca 2+ sensor

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Product

Resources

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High-affinity ouabain binding by a chimeric gastric H ⁺ ,K ⁺ -ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α ₁ -subunit of rat Na ⁺ ,K ⁺ -ATPase

High-affinity ouabain binding by a chimeric gastric H ⁺ ,K ⁺ -ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α ₁ -subunit of rat Na ⁺ ,K ⁺ -ATPase

Local subplasma membrane Ca ²⁺ signals detected by a tethered Ca ²⁺ sensor