Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

Palasis, Maria; Kuntzweiler, Theresa A.; Argüello, José M.; Lingrel, Jerry B.

doi:10.1074/jbc.271.24.14176

Cited by 101 publications

(81 citation statements)

References 43 publications

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“…It has been proposed that binding of ouabain to this hairpin inhibits cation transport by immobilizing these transmembrane domains (20). Our results support this hypothesis and further demonstrate that when in H ϩ ,K ϩ -ATPase M5-M6 is replaced by the similar region of Na ϩ ,K ϩ -ATPase, the resulting chimera (HN56) hardly showed a K ϩ -stimulated dephosphorylation activity.…”

Section: Discussionsupporting

confidence: 85%

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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

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: Discussionsupporting

confidence: 85%

“…The K ϩ -ouabain antagonism suggests that the K ϩ -binding site, which probably includes M4, M5, and M6, also may be involved in ouabain binding (20). In the present study we have investigated the role of transmembrane hairpins M1-M2, M3-M4, and M5-M6 in the binding of ouabain.…”

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confidence: 96%

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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“…Although evidence for direct interaction of these residues with cardiotonic steroids is lacking, the consensus interpretation pictures the steroid docking on the Na͞K pump's external surface, interacting with many of the identified amino acids (14,(38)(39)(40). Identification of the conserved Thr atop TM6 (34) and neighboring residues in the TM5-TM6 hairpin loop (35) as key determinants of ouabain apparent affinity led to the suggestion that cardiotonic steroids might act by preventing motion of the TM5-TM6 hairpin loop, thereby impeding ion passage to or from the deeper binding sites between TM4, TM5, and TM6 (35). In accord with such a mechanism, binding of ouabain traps two Na ions inside the phosphorylated Na͞K pump (41), and cardiotonic steroid can bind to, and shut, PTX-bound Na͞K pump-channels (see Fig.…”

Section: Discussionmentioning

confidence: 99%

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Artigas

Gadsby

2006

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

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The Na͞K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na͞K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na͞K pump and the site of its interaction with ouabain. Palytoxin uncouples the pump's gates, which normally open strictly alternately, thus allowing both gates to sometimes be open, so transforming the pump into an ion channel. Palytoxin therefore permits electrophysiological analysis of even a single Na͞K pump. We used outside-out patch recording of Xenopus ␣1␤3 Na͞K pumps, which were made ouabain-resistant by point mutation, after expressing them in Xenopus oocytes. Endogenous, ouabain-sensitive, Xenopus ␣1␤3 Na͞K pumps were silenced by continuous exposure to ouabain. We found that side-chain charge of two residues at either end of the ␣ subunit's first extracellular loop, known to make a major contribution to ouabain affinity, strongly influenced conductance of single palytoxin-bound pumpchannels by an electrostatic mechanism. The effects were mimicked by modification of cysteines introduced at those two positions with variously charged methanethiosulfonate reagents. The consequences of these modifications demonstrate that both residues lie in a wide vestibule near the mouth of the pump's ion pathway. Bound ouabain protects the site with the strongest influence on conductance from methanethiosulfonate modification, while leaving the site with the weaker influence unprotected. The results suggest a method for mapping the footprint of bound cardiotonic steroid on the extracellular surface of the Na͞K pump.Na,K-ATPase ͉ palytoxin ͉ single-channel conductance ͉ cysteine modification ͉ electrostatic mechanism

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“…It is also known that the carboxyl-selective chemical reagent 4-(diazomethyl)-7-(diethylamino)coumarin (DEAC) binds to Glu 77g in the H5 transmembrane region and inactivates the enzyme via disruption of Na+ and Kf binding [28], and indeed amino acid substitutions at this site decrease the apparent affinities for both cations [17,18]. Furthermore, the H5-H6 loop contains Phe786, Leu7g3 and Thr7g7, which have been shown to be important determinants of ouabain sensitivity [13,38,39]. Finally, the H5-H6 loop may also be important in energy transduction, since phosphorylation from ATP appears to alter the insertion of this loop into the plasma membrane, as demonstrated by the increased labeling of G~u~'~ by DEAC when the enzyme is phosphorylated [28].…”

Section: Discussionmentioning

confidence: 99%

Critical effects on catalytic function produced by amino acid substitutions at Asp⁸⁰⁴ and Asp⁸⁰⁸ of the al isoform of Na,K‐ATPase

1996

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At two intramembrane carboxyl-containing amino acids of the sheep al isoform of Na,K-ATPase (Aspso and Aspass), both charge-conserving (Asp to Glu) and charge-deleting (Asp to Asn, Leu and Ala) replacements were made and the altered enzymes studied. Nucleotide changes encoding the amino acid substitutions were placed in a cDNA encoding a ouabainresistant enzyme (sheep al RD) and the encoded enzymes were expressed in ouabain-sensitive HeLa cells. Transfections with cDNAs carrying all As so4 L carrying AspsesAla, Asp substitutions, along with those Asn, and AspSOSLeu replacements failed to confer ouabain resistance to the cells, indicating critical roles for Aspse4 and AspSo*. Only the expression of the Aspso8Glu enzyme produced ouabain-resistant HeLa cells, demonstrating that the altered protein was functional. When the inactive proteins Aspsa4Ala and AspsasAla were expressed using an alternative selection system (the protein carrying the amino acid substitution was the ouabain-sensitive wild-type sheep al Na,K-ATPase, which was expressed in ouabain-resistant 3T3 cells), intact cells were able to bind extracellular ouabain with high affinity (& = l-30 nM), indicating that the inactive proteins were synthesized and folded properly in the plasma membrane. The results demonstrate that carboxyl side chains at positions 8oJ and 808 are critical for enzyme catalytic function.

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Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

Cited by 101 publications

References 43 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

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Critical effects on catalytic function produced by amino acid substitutions at Asp⁸⁰⁴ and Asp⁸⁰⁸ of the al isoform of Na,K‐ATPase

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Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

Cited by 101 publications

References 43 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

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Critical effects on catalytic function produced by amino acid substitutions at Asp804 and Asp808 of the al isoform of Na,K‐ATPase

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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

Critical effects on catalytic function produced by amino acid substitutions at Asp⁸⁰⁴ and Asp⁸⁰⁸ of the al isoform of Na,K‐ATPase