Cysteine‐scanning mutagenesis study of the sixth transmembrane segment of the Na,K‐ATPase α subunit

Guennoun, Saïda; Horisberger, J. D.

doi:10.1016/s0014-5793(02)02323-2

Cited by 31 publications

(35 citation statements)

References 21 publications

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“…5C), argues that PTX neither permanently nor grossly distorts the Na ϩ ͞K ϩ pump structure. Also, in PTX-treated Na ϩ ͞K ϩ pumps, cysteine scanning mutagenesis of two membrane-spanning helices (22,23) that, on the basis of point mutation studies (e.g., ref. 15) and comparisons with the Ca 2ϩ -ATPase crystal structure (16,17), are believed to contribute to the lining of the Na ϩ ͞K ϩ pump's normal ion translocation pathway, suggests that the pore of the PTX-induced channels includes at least part of that pathway.…”

Section: Resultsmentioning

confidence: 99%

“…To begin addressing these questions in the Na ϩ ͞K ϩ pump, we have exploited the lethal toxin, palytoxin (PTX), isolated from marine coelenterates of the genus Palythoa (18). PTX has been shown to bind to the Na ϩ ͞K ϩ pump (18)(19)(20), and to somehow cause appearance of nonselective cation channels (21) that likely usurp at least some portion of the pump's intrinsic ion translocation pathway (22,23). We examined, in excised patches, changes in the gating of PTX-induced channels in the Na ϩ ͞K ϩ pump in response to addition or removal of ATP, or of Na ϩ and K ϩ ions, at the cytoplasmic or external membrane surface, respectively.…”

Section: ϫ6mentioning

confidence: 99%

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Na ⁺ /K ⁺ -pump ligands modulate gating of palytoxin-induced ion channels

Artigas

Gadsby

2002

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

138

174

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The Na ؉ ͞K ؉ pump is a ubiquitous P-type ATPase that binds three cytoplasmic Na ؉ ions deep within its core where they are temporarily occluded before being released to the extracellular surface. The 3Na ؉ ͞2K ؉ -exchange transport cycle is completed when two extracellular K ؉ ions bind and become temporarily occluded within the protein and subsequently released to the cytoplasm. Coupling of Na ؉ -ion occlusion to phosphorylation of the pump by ATP and of K ؉ -ion occlusion to its dephosphorylation ensure the vectorial nature of net transport. The occluded-ion conformations, with binding sites inaccessible from either side, represent intermediate states in these alternating-access descriptions of transport. They afford protection against potentially catastrophic effects of inadvertently allowing simultaneous access from both membrane sides. The marine toxin, palytoxin, converts Na ؉ ͞K ؉ pumps into nonselective cation channels, possibly by disrupting the normal strict coupling between opening of one access pathway in the Na ؉ ͞K ؉ ATPase and closing of the other. We show here that gating of the channels in palytoxin-bound Na ؉ ͞K ؉ pumps in excised membrane patches is modulated by the pump's physiological ligands: cytoplasmic application of ATP promotes opening of the channels, and extracellular replacement of Na ؉ ions by K ؉ ions promotes closing of the channels. This suggests that, despite the presence of bound palytoxin, certain partial reactions of the normal Na ؉ ͞K ؉ -transport cycle persist and remain capable of effecting the conformational changes that control access to the pump's cation-binding sites. These findings affirm the alternatingaccess model of ion pumps and offer the possibility of examining ion occlusion͞deocclusion reactions in single pump molecules.

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Section: Resultsmentioning

confidence: 99%

Section: ϫ6mentioning

confidence: 99%

Na ⁺ /K ⁺ -pump ligands modulate gating of palytoxin-induced ion channels

Artigas

Gadsby

2002

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

138

174

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“…The Na ϩ -K ϩ -ATPase is an archetypal active transporter that catalyzes the net efflux of three Na ϩ ions in exchange for the net accumulation of two K ϩ ion per reaction cycle. Recent analyses of the interactions of the Na ϩ -K ϩ -ATPase with PTX, a marine coral poison, indicate that this prototypical carrier-type transporter may continue to provide paradigmatic insights regarding the fundamental function and structure of such transport proteins (3,4,15,16,20,22). Significantly, these PTX studies suggest that the basic functional and structural features that define ion transporters vs. ion channels may be much more similar than expected.…”

Section: Ion Transport Proteins As Channels Vs Transporters: Not As mentioning

confidence: 99%

Ion homeostasis, channels, and transporters: an update on cellular mechanisms

Dubyak

2004

Advances in Physiology Education

112

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The steady-state maintenance of highly asymmetric concentrations of the major inorganic cations and anions is a major function of both plasma membranes and the membranes of intracellular organelles. Homeostatic regulation of these ionic gradients is critical for most functions. Due to their charge, the movements of ions across biological membranes necessarily involves facilitation by intrinsic membrane transport proteins. The functional characterization and categorization of membrane transport proteins was a major focus of cell physiological research from the 1950s through the 1980s. On the basis of these functional analyses, ion transport proteins were broadly divided into two classes: channels and carrier-type transporters (which include exchangers, cotransporters, and ATP-driven ion pumps). Beginning in the mid-1980s, these functional analyses of ion transport and homeostasis were complemented by the cloning of genes encoding many ion channels and transporter proteins. Comparison of the predicted primary amino acid sequences and structures of functionally similar ion transport proteins facilitated their grouping within families and superfamilies of structurally related membrane proteins. Postgenomics research in ion transport biology increasingly involves two powerful approaches. One involves elucidation of the molecular structures, at the atomic level in some cases, of model ion transport proteins. The second uses the tools of cell biology to explore the cell-specific function or subcellular localization of ion transport proteins. This review will describe how these approaches have provided new, and sometimes surprising, insights regarding four major questions in current ion transporter research. 1) What are the fundamental differences between ion channels and ion transporters? 2) How does the interaction of an ion transport protein with so-called adapter proteins affect its subcellular localization or regulation by various intracellular signal transduction pathways? 3) How does the specific lipid composition of the local membrane microenvironment modulate the function of an ion transport protein? 4) How can the basic functional properties of a ubiquitously expressed ion transport protein vary depending on the cell type in which it is expressed?

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“…We have recently published the results of studies in which we have examined the accessibility of the residues of the fifth and sixth TM segments by systematic mutagenesis of all the residues of these domains into cysteine (8,9) and then probing the accessibility of the introduced thiol groups with sulfhydryl reagents. These studies have allowed us to define the aspects of the corresponding TM helices that form the pathway for cations across the membrane.…”

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confidence: 99%

The Fourth Transmembrane Segment of the Na,K-ATPase α Subunit

Horisberger

Kharoubi‐Hess

Guennoun³

et al. 2004

Journal of Biological Chemistry

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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

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Cysteine‐scanning mutagenesis study of the sixth transmembrane segment of the Na,K‐ATPase α subunit

Cited by 31 publications

References 21 publications

Na ⁺ /K ⁺ -pump ligands modulate gating of palytoxin-induced ion channels

Na ⁺ /K ⁺ -pump ligands modulate gating of palytoxin-induced ion channels

Ion homeostasis, channels, and transporters: an update on cellular mechanisms

The Fourth Transmembrane Segment of the Na,K-ATPase α Subunit

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Cysteine‐scanning mutagenesis study of the sixth transmembrane segment of the Na,K‐ATPase α subunit

Cited by 31 publications

References 21 publications

Na + /K + -pump ligands modulate gating of palytoxin-induced ion channels

Na + /K + -pump ligands modulate gating of palytoxin-induced ion channels

Ion homeostasis, channels, and transporters: an update on cellular mechanisms

The Fourth Transmembrane Segment of the Na,K-ATPase α Subunit

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Product

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Na ⁺ /K ⁺ -pump ligands modulate gating of palytoxin-induced ion channels

Na ⁺ /K ⁺ -pump ligands modulate gating of palytoxin-induced ion channels