Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants

Mäser, Pascal; Hosoo, Yoshihiro; Goshima, Shinobu; Horie, Tomoaki; Eckelman, Brendan P.; Yamada, K.; Yoshida, Kazuya; Bakker, Evert P.; Shinmyo, Atsuhiko; Oiki, Shigetoshi; Schroeder, Julian I.; Uozumi, Nobuyuki

doi:10.1073/pnas.082123799

Cited by 254 publications

(285 citation statements)

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“…1B). Mutation of these conserved glycines in either the KtrB or HKT proteins has been shown to cause changes in the transport properties as expected if these residues form part of the transport pathway (2,10,11). Although the evidence described above is compelling, to our knowledge there have been no published biochemical or biophysical studies that directly probe the three-dimensional properties of these proteins, a gap that has limited our understanding of this superfamily.…”

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confidence: 91%

Probing the Structure of the Dimeric KtrB Membrane Protein

Albright

Joh

Morais-Cabral

2007

Journal of Biological Chemistry

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The KtrAB ion transporter is a complex of two proteins, KtrB and KtrA. The integral membrane protein KtrB is expected to adopt the structural architecture typified by the pore domain of potassium channels. Here we show that homo-dimerization of KtrB proteins is most likely a general property of this family of transporters. Using cysteine mutants and bifunctional cross-linkers we define regions of the Bacillus subtilis KtrB molecule that are close to the molecular 2-fold axis and to the dimer interface. Fitting of the cross-linking data to a potassium channel-like model suggests structural similarities between potassium channels and KtrB proteins in the extracellular half of the molecule and differences in the cytoplasmic regions.Regulation of intracellular concentrations of sodium and potassium ions is a characteristic of all living organisms and is achieved by a battery of macromolecules that include ion transporter membrane proteins. The KtrAB transporter family plays a role in these processes by mediating Na ϩ -dependent potassium ion uptake in many prokaryotes (1-3). At the genetic level this transporter is usually arranged as a two gene operon (3) encoding the KtrB membrane protein and the cytoplasmic KtrA protein. KtrB proteins are 400 -600 residues long and predicted to have 8 transmembrane helices (TMs) 2 (1-3). KtrA proteins are ϳ220 amino acids long and contain RCK domains that form octameric rings and bind adenine-ribose-phosphatecontaining compounds (4, 5). RCK domains are commonly found associated with membrane proteins, including potassium channels, and function as activity regulators.The KtrAB transporters are part of a large superfamily of ion transport membrane proteins that also includes the plant HKT, the yeast Trk1,2, and the prokaryotic TrK (1, 3). Some of the members of the superfamily, including KtrAB, are thought to be either Na , 3, 6). Sequence homology and hydrophobicity profiles have led to the proposal that all membrane proteins in this superfamily adopt the architecture of the ion pore of potassium channels (1, 2). One KtrB polypeptide is thought to contain four repeats of the TM-P loop -TM structural motif observed in potassium channels, such as KcsA (7, 8). The repeats, labeled A to D in Fig. 1A, do not have the same sequence, except for a few conserved positions (Fig. 1B), and are connected by relatively large cytoplasmic loops (15-36 residues), which show no similarity to one another. The experimentally determined transmembrane topology of HKT1 from Arabidopsis thaliana revealed 8 TMs that agree well with a KcsA-like architecture (9). Moreover, members of the superfamily tend to have one or more conserved glycine residues in the regions equivalent to the potassium channel selectivity filter sequence GYG (glycine-tyrosineglycine) (1, 2) (Fig. 1B). Mutation of these conserved glycines in either the KtrB or HKT proteins has been shown to cause changes in the transport properties as expected if these residues form part of the transport pathway (2, 10, 11). Although the evidence desc...

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confidence: 91%

Probing the Structure of the Dimeric KtrB Membrane Protein

Albright

Joh

Morais-Cabral

2007

Journal of Biological Chemistry

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“…B, Alignment of expected selectivity filter sequences (Cao et al, 2011) in the four pore domains (P A , P B , P C , P D ) of different Na + -K + cotransporters and K + transporters from the HKT/Trk/Ktr superfamily (Corratgé-Faillie et al, 2010;Cao et al, 2011). The star above the last residue of the putative selectivity filter in P A domain indicates the position of a reported determinant of K + permeability: A Gly is present at this position in transporters highly permeable to K + , while a Ser is present in transporters weakly or not permeable to this cation (Mäser et al, 2002). Accession numbers of the transporter sequences: BsKtrB NP_390988; HcTrk1 CAL36606.1; OsHKT2;1 Q0D9S3.1; OsHKT2;2 BAB61791.1; OsHKT2;4 Q8L4K5.1; ScTrk1 NP_012406.1; VaKtrB ZP_01261970.1.…”

Section: Oshkt2;4 Is Selective For K + Among Monovalent Cationsmentioning

confidence: 99%

The Rice Monovalent Cation Transporter OsHKT2;4: Revisited Ionic Selectivity

et al. 2012

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The family of plant membrane transporters named HKT (for high-affinity K+ transporters) can be subdivided into subfamilies 1 and 2, which, respectively, comprise Na+-selective transporters and transporters able to function as Na+-K+ symporters, at least when expressed in yeast (Saccharomyces cerevisiae) or Xenopus oocytes. Surprisingly, a subfamily 2 member from rice (Oryza sativa), OsHKT2;4, has been proposed to form cation/K+ channels or transporters permeable to Ca2+ when expressed in Xenopus oocytes. Here, OsHKT2;4 functional properties were reassessed in Xenopus oocytes. A Ca2+ permeability through OsHKT2;4 was not detected, even at very low external K+ concentration, as shown by highly negative OsHKT2;4 zero-current potential in high Ca2+ conditions and lack of sensitivity of OsHKT2;4 zero-current potential and conductance to external Ca2+. The Ca2+ permeability previously attributed to OsHKT2;4 probably resulted from activation of an endogenous oocyte conductance. OsHKT2;4 displayed a high permeability to K+ compared with that to Na+ (permeability sequence: K+ > Rb+ ≈ Cs+ > Na+ ≈ Li+ ≈ NH4 +). Examination of OsHKT2;4 current sensitivity to external pH suggested that H+ is not significantly permeant through OsHKT2;4 in most physiological ionic conditions. Further analyses in media containing both Na+ and K+ indicated that OsHKT2;4 functions as K+-selective transporter at low external Na+, but transports also Na+ at high (>10 mm) Na+ concentrations. These data identify OsHKT2;4 as a new functional type in the K+ and Na+-permeable HKT transporter subfamily. Furthermore, the high permeability to K+ in OsHKT2;4 supports the hypothesis that this system is dedicated to K+ transport in the plant.

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“…A. thaliana wild-type HKT1 appears to be an Na ϩ -Na ϩ transporter (47), whereas T. aestivum wild-type HKT1 seems to couple potassium transport with sodium influx (9,12). But most importantly, the S68G switch in A. thaliana HKT1 and the G91S switch in T. aestivum HKT1 completely interchange the two phenotypes (48). In yeast Trk1p, extensive mutagenesis of the proposed eighth transmembrane helix, which should halfbracket the fourth putative P-loop, has demonstrated impressive changes in transport affinity due to modifications of charge along the helix, particularly at native Lys 1147 , Lys 1158 , and adjacent neutral residues (49).…”

Section: Discussionmentioning

confidence: 99%