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

Burnay, Muriel; Crambert, Gilles; Kharoubi‐Hess, Solange; Geering, Käthi; Horisberger, J. D.

doi:10.1074/jbc.m300946200

Cited by 51 publications

(40 citation statements)

References 37 publications

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“…The lysine of transmembrane helix M5 replacing S777 of Na + , K + -ATPase in the H + ,K + -ATPases has previously been attributed a role as an internal cation similar to the present proposal for the arginine of M8 (7,9). In favor of this function of the M5 lysine is the finding that alanine substitution confers electrogenicity to the nongastric H + ,K + -ATPase, thus raising the number of cations transported toward the extracellular side per cycle (7).…”

Section: Discussionmentioning

confidence: 60%

“…1). The occupation of site I by this lysine with its potential positive charge might account, in part, for the difference in transport stoichiometry between H + ,K + -ATPase and Na + ,K + -ATPase (7, 9), but consistent results for gain of electrogenicity upon mutation of this lysine in the gastric and nongastric H + ,K + -ATPases have not been obtained (7,10). It remains to be explained how the H + ,K + -ATPases avoid binding Na + (or H + ) at the site corresponding to Significance This study explores the structural basis for the difference between the closely related Na + ,K + -ATPase and H + ,K + -ATPase with respect to ion transport stoichiometry, electrogenicity, and cation selectivity, which has long been an enigma.…”

mentioning

confidence: 94%

“…1H + :1K + :1ATP at low luminal pH and 2H + :2K + :1ATP at neutral pH (6)(7)(8). A low-resolution crystal structure of the H + ,K + -ATPase obtained at acidic pH in the presence of the K + congener Rb + shows that one bound Rb + ion occupies a site equivalent to site II of the Na + ,K + -ATPase, whereas the occupancy of site I is very low (8).…”

mentioning

confidence: 99%

“…Crystal structures of the Na + ,K + -ATPase show that two Na + ions bind at sites that overlap substantially with the K + sites (so-called sites I and II), whereas the third Na + ion is bound at a distinct Na + -specific site (site III) (1,4,5). Two isoforms of H + ,K + -ATPase, with different expression profiles, perform electroneutral H + ,K + -exchange by a mechanism basically similar to the Na + ,K + -ATPase mechanism (6,7). The nongastric H + ,K + -ATPase participates in K + reabsorption in the kidney and colon, whereas the better characterized gastric H + ,K + -ATPase is responsible for acid secretion into the lumen of the stomach and has been proposed to have two different stoichiometries:…”

mentioning

confidence: 99%

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Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Holm

Khandelwal

Einholm

et al. 2016

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

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Na+,K+-ATPase and H+,K+-ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H+,K+-ATPase avoids binding of Na+ at the site corresponding to the Na+-specific site of the Na+,K+-ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na+,K+-ATPase with arginine, present in the H+,K+-ATPase at the corresponding position, converted the normal 3Na+:2K+:1ATP stoichiometry of the Na+,K+-ATPase to electroneutral 2Na+:2K+:1ATP stoichiometry similar to the electroneutral transport mode of the H+,K+-ATPase. The electroneutral C932R mutant of the Na+,K+-ATPase retained a wild-type–like enzyme turnover rate for ATP hydrolysis and rate of cellular K+ uptake. Only a relatively minor reduction of apparent Na+ affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na+ affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine+ group of the M8 arginine replaces Na+ at the third site, thus preventing Na+ binding there, although allowing Na+ to bind at the two other sites and become transported. Hence, in the H+,K+-ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

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Section: Discussionmentioning

confidence: 60%

mentioning

confidence: 94%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Holm

Khandelwal

Einholm

et al. 2016

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

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“…Thr-140 may be crucial for alkaline cation binding, as the K m of the T140C mutant NhaA was higher than that of D141C NhaA. The importance of threonine and serine hydroxylation for cation transport has also been reported for the Na ϩ , K ϩ -ATPase, and K ϩ channels and other types of Na ϩ /H ϩ antiporters such as NhaDs (33)(34)(35). It is noteworthy that cysteine substitution of Phe-144 induced significant defects in antiporter activity and that this residue is located on the same side of the TM4 ␣-helix as Thr-140 (Figs.…”

Section: Alteration Of [ 14 C]nem Labeling In the Presence Of LImentioning

confidence: 96%

The Fourth Transmembrane Domain of the Helicobacter pylori Na+/H+ Antiporter NhaA Faces a Water-filled Channel Required for Ion Transport

Kuwabara

Inoue

Tsuboi

et al. 2004

Journal of Biological Chemistry

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Cysteine-scanning mutagenesis was performed from Ser-130 to Leu-160 in the fourth transmembrane domain (TM4) of the Na ؉ /H ؉ antiporter NhaA from Helicobacter pylori to determine the topology of each residue and to identify functionally important residues. All of the mutants were based on cysteine-less NhaA (Cys-less NhaA), which functions very similarly to the wild-type protein, and were expressed at a level similar to Cys-less NhaA. Discontinuity of [14 C]N-ethylmaleimide (NEM)-reactive residues suggested that TM4 comprises residues Gly-135 to Val-156. Even within TM4, NEM reactivity was high for I136C, D141C to A143C, L146C, M150C, and G153C to R155C. These residues are thought to be located on one side of the ␣-helical structure of TM4 and to face a putative water-filled channel.

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Cation Uptake Studies with Atomic Absorption Spectrophotometry (AAS)

Friedrich

2015

Pumps, Channels, and Transporters

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

Cited by 51 publications

References 37 publications

Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

The Fourth Transmembrane Domain of the Helicobacter pylori Na+/H+ Antiporter NhaA Faces a Water-filled Channel Required for Ion Transport

Cation Uptake Studies with Atomic Absorption Spectrophotometry (AAS)

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

Cited by 51 publications

References 37 publications

Arginine substitution of a cysteine in transmembrane helix M8 converts Na + ,K + -ATPase to an electroneutral pump similar to H + ,K + -ATPase

Arginine substitution of a cysteine in transmembrane helix M8 converts Na + ,K + -ATPase to an electroneutral pump similar to H + ,K + -ATPase

The Fourth Transmembrane Domain of the Helicobacter pylori Na+/H+ Antiporter NhaA Faces a Water-filled Channel Required for Ion Transport

Cation Uptake Studies with Atomic Absorption Spectrophotometry (AAS)

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Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase