Change to alanine of one out of four selectivity filter glycines in KtrB causes a two orders of magnitude decrease in the affinities for both K+ and Na+ of the Na+ dependent K+ uptake system KtrAB from Vibrio alginolyticus

Tholema, Nancy; Bakker, Evert P.; Suzuki, Ayako; Nakamura, Tatsunosuke

doi:10.1016/s0014-5793(99)00504-9

Cited by 83 publications

(92 citation statements)

References 29 publications

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“…For proton motive force tests in E. coli, the strain LF12 (⌬(kdpDE)54, nagA, malT, ⌬hemA) was constructed as described by Futatsugi et al (doctoral thesis, 2001) 1 based on the strain TK2642(⌬(kdpDE)54, nagA, malT), a kind gift of Wolf Epstein. Transformants of the E. coli mutant were cultured in a synthetic medium supplemented with the required antibiotics and indicated cations as described previously (27,40,41). Gene expression was induced by the addition of 0.25 mM IPTG 2 to the medium.…”

Section: Methodsmentioning

confidence: 99%

“…PCC 6803-K ϩ influx was essentially measured as described by Tholema et al (27). E. coli LB2003 cells were cultured in a synthetic medium in the presence of 30 mM KCl, 0.25 mM IPTG, 25 g/ml of ampicillin, and/or 15 g/ml of chloramphenicol at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…10 mM glucose was added to the suspension 10 min prior to the start of the K ϩ uptake measurement. The net uptake of K ϩ was measured by the silicone filtration technique (21,27,45), the K ϩ contents of the cell pellets being determined by flame photometry (45).…”

Section: Methodsmentioning

confidence: 99%

“…The K ϩ transport activity of both wheat HKT1 and V. alginolyticus KtrAB is enhanced by Na ϩ (27,29). To test whether this was also the case for Synechocystis KtrABE, the LB2003 cells were suspended in low Na ϩ buffer (200 mM HEPEStriethanolamine buffer; 1.9 M Na ϩ and 0.36 M K ϩ , as measured by flame photometry).…”

Section: Three Synechocystis Ktr Gene Products Are Required For K ϩmentioning

confidence: 99%

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Na+-dependent K+ Uptake Ktr System from the Cyanobacterium Synechocystis sp. PCC 6803 and Its Role in the Early Phases of Cell Adaptation to Hyperosmotic Shock

Matsuda

Kobayashi

Katoh

et al. 2004

Journal of Biological Chemistry

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107

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Transmembrane ion transport processes play a key role in the adaptation of cells to hyperosmotic conditions. Previous work has shown that the disruption of a ktrB/ntpJ-like putative Na ؉ /K ؉ transporter gene in the cyanobacterium Synechocystis sp. PCC 6803 confers increased Na ؉ sensitivity, and inhibits HCO 3 ؊ uptake. Here, we report on the mechanistic basis of this effect. Heterologous expression experiments in Escherichia coli show that three Synechocystis genes are required for K ؉ transport activity. They encode an NAD ؉ -binding peripheral membrane protein (ktrA; sll0493), an integral membrane protein, belonging to a superfamily of K ؉ transporters (ktrB; formerly ntpJ; slr1509), and a novel type of ktr gene product, not previously found in Ktr systems (ktrE; slr1508). In E. coli, Synechocystis KtrABEmediated K ؉ uptake occurred with a moderately high affinity (K m of about 60 M), and depended on both Na ؉ and a high membrane potential, but not on ATP. KtrABE neither mediated Na ؉ uptake nor Na ؉ efflux. In Synechocystis sp. PCC 6803, KtrB-mediated K ؉ uptake required Na ؉ and was inhibited by protonophore. A ⌬ktrB strain was sensitive to long term hyperosmotic stress elicited by either NaCl or sorbitol. Hyperosmotic shock led initially to loss of net K ؉ from the cells. The ⌬ktrB cells shocked with sorbitol failed to reaccumulate K ؉ up to its original level. These data indicate that in strain PCC 6803 K ؉ uptake via KtrABE plays a crucial role in the early phase of cell turgor regulation after hyperosmotic shock.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Three Synechocystis Ktr Gene Products Are Required For K ϩmentioning

confidence: 99%

See 2 more Smart Citations

Na+-dependent K+ Uptake Ktr System from the Cyanobacterium Synechocystis sp. PCC 6803 and Its Role in the Early Phases of Cell Adaptation to Hyperosmotic Shock

Matsuda

Kobayashi

Katoh

et al. 2004

Journal of Biological Chemistry

Self Cite

107

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“…The Ktr ion transporters are composed of two essential components 3,16 : a membrane protein (KtrB or KtrD) and a cytosolic regulatory protein (KtrA or KtrC). Studies with cells show that Ktr proteins 8,19 transport K + but are also permeable to Na + , and that K + transport is ATP 20 and Na + dependent 5,8,18 . Furthermore, it has been shown that truncated KtrA forms octameric rings 21,22 and that KtrB assembles as homodimers 6,21 .…”

mentioning

confidence: 99%

The structure of the KtrAB potassium transporter

Vieira-Pires

Szöllősi

Morais-Cabral

2013

Nature

106

163

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In bacteria, archaea, fungi and plants the Trk, Ktr and HKT ion transporters are key components of osmotic regulation, pH homeostasis and resistance to drought and high salinity. These ion transporters are functionally diverse: they can function as Na + or K + channels and possibly as cation/K + symporters. They are closely related to potassium channels both at the level of the membrane protein and at the level of the cytosolic regulatory domains. Here we describe the crystal structure of a Ktr K + transporter, the KtrAB complex from Bacillus subtilis. The structure shows the dimeric membrane protein KtrB assembled with a cytosolic octameric KtrA ring bound to ATP, an activating ligand. A comparison between the structure of KtrAB-ATP and the structures of the isolated fulllength KtrA protein with ATP or ADP reveals a ligand-dependent conformational change in the octameric ring, raising new ideas about the mechanism of activation in these transporters.The Trk/Ktr/HKT superfamily of ion transporters includes the Trk, Ktr and HKT transporter families 1 . These transporters are closely related to the superfamily of tetrameric cation channels 2-4 , which includes potassium, sodium and calcium channels. The membrane proteins in both superfamilies share the same architecture 2,5-9 : four subunits or repeats, each with the TM-P loop-TM (where TM indicates transmembrane helix) structural motif first seen in the KcsA potassium channel structure 10 , assemble to form an ion pore with a 'selectivity filter' along its central axis. In addition, the cytosolic regulatory proteins of the Trk and Ktr transporters and of the MthK 11,12 and large-conductance Ca 2+ -gated (BK) K + channels [13][14][15] are RCK (regulate conductance of K+) domains.Ktr ion transporters are crucial K + transport systems in some bacteria 16,17 , with a role in resistance to osmotic stress and high salinity by mediating the early uptake of K + (refs 16, 18). The Ktr ion transporters are composed of two essential components 3,16 : a membrane protein (KtrB or KtrD) and a cytosolic regulatory protein (KtrA or KtrC). Studies with cells show that Ktr proteins 8,19 transport K + but are also permeable to Na + , and that K + transport is ATP 20 and Na + dependent 5,8,18 . Furthermore, it has been shown that truncated KtrA forms octameric rings 21,22 and that KtrB assembles as homodimers 6,21 . The structures described here clarify the molecular basis of some of these properties as well as the structural relationship between these transporters and ion channels.

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Adaptation to Life at High Salt Concentrations in Archaea, Bacteria, and Eukarya

Gunde‐Cimerman¹,

Oren²,

Plemenitaš³

2005

Cellular Origin, Life in Extreme Habitats and Astrobiology

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Change to alanine of one out of four selectivity filter glycines in KtrB causes a two orders of magnitude decrease in the affinities for both K⁺ and Na⁺ of the Na⁺ dependent K⁺ uptake system KtrAB from Vibrio alginolyticus

Cited by 83 publications

References 29 publications

Na+-dependent K+ Uptake Ktr System from the Cyanobacterium Synechocystis sp. PCC 6803 and Its Role in the Early Phases of Cell Adaptation to Hyperosmotic Shock

Na+-dependent K+ Uptake Ktr System from the Cyanobacterium Synechocystis sp. PCC 6803 and Its Role in the Early Phases of Cell Adaptation to Hyperosmotic Shock

The structure of the KtrAB potassium transporter

Adaptation to Life at High Salt Concentrations in Archaea, Bacteria, and Eukarya

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