Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter (original) (raw)
Related papers
Functional analysis of the M2D helix of the TRK1 potassium transporter of Saccharomyces cerevisiae
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2003
Eukaryotic KcsA-related K + transporters mediate physiologically relevant K + and Na + fluxes in fungi and plants. ScTRK1 is a characteristic member of the group, and here we report a mutational analysis of the unique M2 D helix of this transporter. Our results support the theoretical models placing this helix in a relevant position in the pore and interacting with P segments. Most single mutations eliminating positively charged or introducing negatively charged residues reduced the V max of Rb + influx to a half, several together showed an additive effect, and four practically suppressed transport. In contrast, the introduction of only one positively charged residue practically abolished the function of the transporter. Almost all mutations in the M2 D helix affected the two Rb + binding sites of the transporter, mimicking mutations in the selectivity filter.
FEBS Letters, 1998
HKT1 encodes a high affinity Na + coupled K + transporter expressed in the cortical cells of Triticum aestivum roots. To identify regions of the protein involved in the binding and transport of Na + and K + , mutations were introduced into a domain of HKT1 containing 16 amino acids that are highly conserved across a range of putative K + transport proteins from different phyla. Two mutations had a significant effect on the functional characteristics of the transporter. A yeast growth assay showed that concentrations of NaCl between 2.5 to 50 mM stimulated the growth of yeast expressing HKT1 containing the E464Q substitution, but not the growth of yeast expressing HKT1. Kinetic analysis confirmed that the E464Q mutation lowered the affinity of HKT1 for Na + but did not affect its affinity for K + . A second mutation in the same region F463L was created that also lowered the affinity of the transporter for Na + . The importance of these highly conserved amino acid residues is highlighted by the fact that they have remained conserved through evolution. The results of this mutational analysis suggest that this domain in HKT1 plays a role in the binding and transport of Na + .
Journal of Bacteriology, 2010
The Na + -dependent K + uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the K + -translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated K + uptake and in modulating Na + dependency. Expression of KtrB alone in a K + uptake-deficient Escherichia coli strain conferred low K + uptake activity that was not stimulated by Na + . Coexpression of both KtrA and KtrE with KtrB increased the K + transport activity in a Na + -dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis . Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent K m for K + in all cases. However, none of the mutations el...
Molecular insights into the structure and function of plant K+ transport mechanisms
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2000
Our understanding of plant potassium transport has increased in the past decade through the application of molecular biological techniques. In this review, recent work on inward and outward rectifying K channels as well as high affinity K transporters is described. Through the work on inward rectifying K channels, we now have precise details on how the structure of these proteins determines functional characteristics such as ion conduction, pH sensitivity, selectivity and voltage sensing. The physiological function of inward rectifying K channels in plants has been clarified through the analysis of expression patterns and mutational analysis. Two classes of outward rectifying K channels have now been cloned from plants and their initial characterisation is reviewed. The physiological role of one class of outward rectifying K channel has been demonstrated to be involved in long distance transport of K from roots to shoots. The molecular structure and function of two classes of energised K transporters are also reviewed. The first class is energised by Na and shares structural similarities with K transport mechanisms in bacteria and fungi. Structure-function studies suggest that it should be possible to increase the K and Na selectivity of these transporters, which will enhance the salt tolerance of higher plants. The second class of K transporter is comprised of a large gene family and appears to have a dual affinity for K. A suite of molecular techniques, including gene cloning, oocyte expression, RNA localisation and gene inactivation, is now being used to fully characterise the biophysical and physiological function of plants K transport mechanisms.
Amino Acids, 2008
A nitrogen-related signal transduction pathway, consisting of the three phosphotransfer proteins EI Ntr , NPr, and IIA Ntr , was discovered recently to regulate the uptake of K + in Escherichia coli. In particular, dephosphorylated IIA Ntr inhibits the activity of the K + transporter TrkA. Since the phosphorylation state of IIA Ntr is partially determined by its reversible phosphorylation by NPr, we have determined the three-dimensional structure of NPr by solution NMR spectroscopy. In total, we obtained 973 NOE-derived distance restraints, 112 chemical shiftderived backbone angle restraints, and 35 hydrogen-bond restraints derived from temperature coefficients (wave). We propose that temperature wave is useful for identifying exposed beta-strands and assists in establishing protein folds based on chemical shifts. The deduced structure of NPr contains three a-helices and four b-strands with the three helices all packed on the same face of the b-sheet. The active site residue His16 of NPr for phosphoryl transfer was found to be neutral and in the Ne2-H tautomeric state. There appears to be increased motion in the active site region of NPr compared to HPr, a homologous protein involved in the uptake and regulation of carbohydrate utilization.
Frontiers in Plant Science, 2018
Potassium (K+) is the most abundant cation in plants, and its uptake and transport are key to growth, development and responses to the environment. Here, we report that Arabidopsis thaliana K+ uptake permease 5 (AtKUP5) contains an adenylate cyclase (AC) catalytic center embedded in its N-terminal cytosolic domain. The purified recombinant AC domain generates cAMP in vitro; and when expressed in Escherichia coli, increases cAMP levels in vivo. Both the AC domain and full length AtKUP5 rescue an AC-deficient E. coli mutant, cyaA, and together these data provide evidence that AtKUP5 functions as an AC. Furthermore, full length AtKUP5 complements the Saccharomyces cerevisiae K+ transport impaired mutant, trk1 trk2, demonstrating its function as a K+ transporter. Surprisingly, a point mutation in the AC center that impairs AC activity, also abolishes complementation of trk1 trk2, suggesting that a functional catalytic AC domain is essential for K+ uptake. AtKUP5-mediated K + uptake is not affected by cAMP, the catalytic product of the AC, but, interestingly, causes cytosolic cAMP accumulation. These findings are consistent with a role for AtKUP5 as K+ flux sensor, where the flux-dependent cAMP increases modulate downstream components essential for K+ homeostasis, such as cyclic nucleotide gated channels.
Pflügers Archiv - European Journal of Physiology, 2011
Patch clamp studies of the potassium-transport proteins TRK1,2 in Saccharomyces cerevisiae have revealed large chloride efflux currents: at clamp voltages negative to -100 mV, and intracellular chloride concentrations >10 mM (J. Membr. Biol. 198:177, 2004). Stationary-state current-voltage analysis led to an in-series two-barrier model for chloride activation: the lower barrier (α) being 10-13 kcal/mol located ∼30% into the membrane from the cytoplasmic surface; and the higher one (β) being 12-16 kcal/mol located at the outer surface. Measurements carried out with lyotrophic anions and osmoprotective solutes have now demonstrated the following new properties: (1) selectivity for highly permeant anions changes with extracellular pH; at pH o =5.5: I − ≈Br − >Cl − >SCN − >NO 3 − , and at pH o 7.5: I − ≈Br − >SCN − >NO 3 − >Cl − . (2) NO 2 − acts like "superchoride", possibly enhancing the channel's intrinsic permeability to Cl − . (3) SCN − and NO 3 − block chloride permeability. (4) The order of selectivity for several slightly permeant anions (at pH o =5.5 only) is formate > gluconate > acetate ≫ phosphate −1 . (5) All anion conductances are modulated (choked) by osmoprotective solutes. (6) The data and descriptive two-barrier model evoke a hypothetical structure ( Biophys. J. 77:789, 1999) consisting of an intramembrane homotetramer of fungal TRK molecules, arrayed radially around a central cluster of four single helices (TM7) from each monomer. That tetrameric cluster would resemble the hydrophobic core of (pentameric) ligand-gated ion channels, and would suggest voltage-modulated hydrophobic gating to underlie anion permeation.
Evidence for potassium transport activity of Arabidopsis KEA1-KEA6
Scientific Reports
Arabidopsis thaliana contains the putative K + efflux transporters KEA1-KEA6, similar to KefB and KefC of Escherichia coli. KEA1-KEA3 are involved in the regulation of photosynthetic electron transport and chloroplast development. KEA4-KEA6 mediate pH regulation of the endomembrane network during salinity stress. However, the ion transport activities of KEA1-KEA6 have not been directly characterized. In this study, we used an E. coli expression system to examine KEA activity. KEA1-KEA3 and KEA5 showed bi-directional K + transport activity, whereas KEA4 and KEA6 functioned as a K + uptake system. The thylakoid membrane-localized Na + /H + antiporter NhaS3 from the model cyanobacterium Synechocystis is the closest homolog of KEA3. Changing the putative Na + /H + selective site of KEA3 (Gln-Asp) to that of NhaS3 (Asp-Asp) did not alter the ion selectivity without loss of K + transport activity. The first residue in the conserved motif was not a determinant for K + or Na + selectivity. Deletion of the possible nucleotide-binding KTN domain from KEA3 lowered K + transport activity, indicating that the KTN domain was important for this function. The KEA3-G422R mutation discovered in the Arabidopsis dpgr mutant increased K + transport activity, consistent with the mutant phenotype. These results indicate that Arabidopsis KEA1-KEA6 act as K + transport systems, and support the interpretation that KEA3 promotes dissipation of ΔpH in the thylakoid membrane. Tight control of intracellular pH and ion concentrations through the activity of membrane transport systems is crucial for maintaining intracellular ion homeostasis during changes in the environment and for allowing the formation of an electrochemical potential across the membrane. In chloroplasts and cyanobacteria, the flux of protons and other ions across the thylakoid membrane is highly correlated with light energy conversion in the photosynthetic electron transport system. Light reactions generate proton (H +) motive force (pmf), which is the sum of a pH gradient component (ΔpH) and an electric potential component (∆Ψ) across the thylakoid membrane. Since the potassium ion (K +) is the major essential cation in most living cells, including plant cells and cyanobacteria, K + concentration and K + flux across the membrane conducted by K + transport systems are a main contributor to the electric potential component ∆Ψ 1-3 and to the intracellular osmotic homeostasis in response to external osmotic changes. Several K + transporters are present in chloroplasts and in cyanobacteria. In Synechocystis sp. PCC 6803, two types of K + uptake transporters, KtrB and KdpA 4,5 and K + channels such as SynK 6,7 and SynCaK 8 have been reported. Using antibodies, SynK has been shown to be present both in fractions of thylakoid membrane and plasma membrane. In chloroplasts of Arabidopsis thaliana, the two-pore K + channel TPK3 was detected in the thylakoid membrane fraction as well 6. Upon further examination TPK3 was found to reside in the thylakoid stromal lamellae 9. Recent studies also revealed that putative K + /H + antiporters, KEA1-KEA3, are expressed in Arabidopsis chloroplasts 10. Arabidopsis has six genes encoding KEAs 11,12. KEAs belong to the monovalent cation/proton antiporter (CPA) super family. The CPA family consists of two subfamilies, CPA1 and CPA2; the Na +-H + exchangers (NHX) belong to the CPA1 family, and the K + efflux antiporters (KEA) and cation-H + exchangers (CHX) belong to the CPA2 family. The Arabidopsis NHXs have been studied in great detail; NHX1-NHX6 reside in the endosome membrane, and NHX7(SOS1)-NHX8 function in the plasma membrane 13. NHXs play a critical role in salt tolerance and pH homeostasis. Characterization of KEA1-KEA6 has recently been reported by several groups but detailed information on the function of these transporters remains elusive 10,14-20 .
Helical jackknives control the gates of the double-pore K(+) uptake system KtrAB
eLife, 2017
Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K(+) channels, RCK domains regulate ion fluxes. Until now, a single regulatory mechanism has been proposed for all RCK-regulated channels, involving signal transduction from the RCK domain to the gating area. Here, we present an inactive ADP-bound structure of KtrAB from Vibrio alginolyticus, determined by cryo-electron microscopy, which, combined with EPR spectroscopy and molecular dynamics simulations, uncovers a novel regulatory mechanism for ligand-induced action at a distance. Exchange of activating ATP to inactivating ADP triggers short helical segments in the K(+)-translocating KtrB dimer to organize into two long helices that penetrate deeply into the regulatory RCK domains, thus connecting nucleotide-binding sites and ion gates. As KtrAB and its homolog TrkAH have been implicated as bacterial pathogenicity factors, the discovery of this func...