Gain-of-function mutations indicate that Escherichia coli Kch forms a functional K+ conduit in vivo

Although Kch of Escherichia coli is thought to be a K(+) channel by sequence homology, there is little evidence that it actually conducts K(+) ions in vitro or in vivo. We isolated gain-of-function (GOF) Kch mutations that render bacteria specifically sensitive to K(+) ions. Millimolar added K(+), but not Na(+) or sorbitol, blocks the initiation or continuation of mutant growth in liquid media. The mutations are mapped at the RCK (or KTN) domain, which is considered to be the cytoplasmic sensor controlling the gate. Additional mutations directed to the K(+)-filter sequence rescue the GOF mutant. The apparent K(+)-specific conduction through the 'loose-cannon' mutant channel suggests that the wild-type Kch channel also conducts, albeit in a regulated manner. Changing the internal ATG does not erase the GOF toxicity, but removes kch's short second product, suggesting that it is not required for channel function in vivo. The mutant phenotypes are better explained by a perturbation of membrane potential instead of internal K(+) concentration. Possible implications on the normal function of Kch are discussed.     

Potassium/proton antiport system of Escherichia coli

The intracellular level of potassium (K(+)) in Escherichia coli is regulated through multiple K(+) transport systems. Recent data indicate that not all K(+) extrusion system(s) have been identified (15). Here we report that the E. coli Na(+) (Ca(2+))/H(+) antiporter ChaA functions as a K(+) extrusion system. Cells expressing ChaA mediated K(+) efflux against a K(+) concentration gradient. E. coli strains lacking the chaA gene were unable to extrude K(+) under conditions in which wild-type cells extruded K(+). The K(+)/H(+) antiporter activity of ChaA was detected by using inverted membrane vesicles produced using a French press. Physiological growth studies indicated that E. coli uses ChaA to discard excessive K(+), which is toxic for these cells. These results suggest that ChaA K(+)/H(+) antiporter activity enables E. coli to adapt to K(+) salinity stress and to maintain K(+) homeostasis.     

GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na(+)-sensitive phenotype of an Escherichia coli mutant that is deficient in Na(+)/H(+) antiport activity (strain KNabc). GerN also reduced the concentration of K(+) required to support growth of an E. coli mutant deficient in K(+) uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na(+)/H(+) antiport activity, with a K(m) for Na(+) estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li(+), but not K(+), served as a substrate. GerN-mediated Na(+)/H(+) antiport was further demonstrated in everted vesicles as energy-dependent accumulation of (22)Na(+). GerN also used K(+) as a coupling ion without completely replacing H(+), as indicated by partial inhibition by K(+) of H(+) uptake into right-side-out vesicles loaded with Na(+). K(+) translocation as part of the antiport was supported by the stimulatory effect of intravesicular K(+) on (22)Na(+) uptake by everted vesicles and the dependence of GerN-mediated (86)Rb(+) efflux on the presence of Na(+) in trans. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na(+)/H(+)-K(+) antiport. GerN-mediated Na(+)/H(+)-K(+) antiport was much more rapid than GerN-mediated Na(+)/H(+) antiport.     

Analysis of a putative voltage-gated prokaryotic potassium channel.

Most of the completely sequenced prokaryotic genomes contain genes of potassium channel homologues, but there is still not much known about the role of these proteins in prokaryotes. Here we describe the large-scale overproduction and purification of a prokaryotic voltage-gated potassium channel homologue, Kch, from Escherichia coli. After successful overproduction of the protein, a specific increase in the potassium permeability of the cells was found. Kch could be purified in large amounts using classical purification methods to prevent aggregation of the protein. The physiological state of the protein was revealed to be a homotetramer and the protein was shown to be localized to the cytoplasmic membrane of the cells. In the course of the localization studies, we found a specific increase in the density of the cytoplasmic membrane on Kch production. This was linked to the observed increase in the protein to lipid ratio in the membranes. Another observed change in the membrane composition was an increase in the cardiolipin to phosphatidylglycerol ratio, which may indicate a specific cardiolipin requirement of Kch. On the basis of some of our results, we discuss a function for Kch in the maintenance of the membrane potential in E. coli.     

Separable gating mechanisms in a Mammalian pacemaker channel

Despite permeability to both K(+) and Na(+), hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels contain the K(+) channel signature sequence, GYG, within the selectivity filter of the pore. Here, we show that this region is involved in regulating gating in a mouse isoform of the pacemaker channel (mHCN2). A mutation in the GYG sequence of the selectivity filter (G404S) had different effects on the two components of the wild-type current; it eliminated the slowly activating current (I(f)) but, surprisingly, did not affect the instantaneous current (I(inst)). Confocal imaging and immunocytochemistry showed G404S protein on the periphery of the cells, consistent with the presence of channels on the plasma membrane. Experiments with the wild-type channel showed that the rate of I(f) deactivation and I(f) amplitude had a parallel dependence on the ratio of K(+)/Na(+) driving forces. In addition, the amplitude of fully activated I(f), unlike I(inst), was not well predicted by equal and independent flow of K(+) and Na(+). The data are consistent with two separable gating mechanisms associated with pacemaker channels: one (I(f)) that is sensitive to voltage, to a mutation in the selectivity filter, and to driving forces for permeating cations and another (I(inst)) that is insensitive to these influences.     

Nitrophenylphosphate as a tool to characterize gill Na, K-ATPase activity in hyperregulating Crustacea

The kinetic properties of a gill Na(+), K(+)-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na(+), K(+)-ATPase hydrolyzed PNPP (K(+)-phosphatase activity) obeying Michaelis-Menten kinetics with K(M)=1.72+/-0.06 mmol l(-1) and V(max)=259.1+/-11.6 U mg(-1). ATP was a competitive inhibitor of K(+)-phosphatase activity with a K(i)=50.1+/-2.5 micromol l(-1). A cooperative effect for the stimulation of the enzyme by potassium (K(0.5)=3.62+/-0.18 mmol l(-1); n(H)=1.5) and magnesium ions (K(0.5)=0.61+/-0.02 mmol l(-1), n(H)=1.3) was found. Sodium ions had no effect on K(+)-phosphatase activity up to 1.0 mmol l(-1), but above 80 mmol l(-1) inhibited the original activity by approximately 75%. In the range of 0-10 mmol l(-1), sodium ions did not affect stimulation of the K(+)-phosphatase activity by potassium ions. Ouabain (K(i)=762.4+/-26.7 micromol l(-1)) and orthovanadate (K(i)=0.25+/-0.01 micromol l(-1)) completely inhibited the K(+)-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K(+)-phosphatase activity of the Na(+), K(+)-ATPase alone and suggest that the use of PNPP as a substrate to characterize K(+)-phosphatase activity may be a useful technique in comparative osmoregulatory studies of Na(+), K(+)-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme.     

Regulation of Kir channels by intracellular pH and extracellular K(+): mechanisms of coupling

ROMK channels are regulated by internal pH (pH(i)) and extracellular K(+) (K(+)(o)). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H(+), increasing the apparent pKa for inhibition by approximately 0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K(+)(o), consistent with coupling between the responses to pH(i) and K(+)(o). The ion selectivity sequence of the activation of the channel by cations was K(+) congruent with Rb(+) > NH(4)(+) > Na(+), similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K(+) from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K(+)(o). We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K(+)(o) between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K(+) to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba(2+) block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H(+). We conclude that decreasing pH(I) increases the sensitivity of ROMK2 channels to K(+)(o) by altering the properties of the selectivity filter.     

Requirement for sodium in the anaerobic growth of Aerobacter aerogenes on citrate

Anaerobic growth of Aerobacter aerogenes on citrate as a carbon source required the presence of Na(+). The growth rate increased with increasing Na(+) concentration and was optimal at 0.10 m Na(+). The requirement was specific for Na(+), which could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). K(+) was required for growth in the presence of Na(+), the optimal K(+) concentration being 0.15 mm. Enzyme profiles were determined on cells grown in three different media: (i) intermediate Na(+), high K(+) concentration, (ii) high Na(+), high K(+) concentration, and (c) high Na(+), low K(+) concentration. All cells contained the enzymes of the citrate fermentation pathway, namely, citritase and the Na(+)-requiring oxalacetate (OAA) decarboxylase. All of the enzymes of the citric acid cycle were present, except alpha-ketoglutarate dehydrogenase which could not be detected. The incomplete citric acid cycle was, in effect, converted into two biosynthetic pathways leading to glutamate and succinate, respectively. The specific activities of citritase and OAA decarboxylase were lowest in medium (i), and under these conditions the activity of OAA decarboxylase appeared to be limited in vivo by the availability of Na(+). Failure of A. aerogenes to grow anaerobically on citrate in the absence of Na(+) can be explained at the enzymatic level by the Na(+) requirement of the OAA decarboxylase step of the citrate fermentation pathway and by the absence of an alternate pathway of citrate catabolism.     

Role of sodium in determining alternate pathways of aerobic citrate catabolism in Aerobacter aerogenes

In contrast to the absolute Na(+) requirement for anaerobic growth of Aerobacter aerogenes on citrate as sole carbon source, aerobic growth of this microorganism did not require the presence of Na(+). However, Na(+) (optimal concentration, 10 mm) did increase the maximal amount of aerobic growth by 60%, even though it did not change the rate of growth. This increase in growth was specifically affected by Na(+), which could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). Enzyme profiles were determined in A. aerogenes cells grown aerobically on citrate in media of varying cationic composition. Cells grown in Na(+)-free medium possessed all the enzymes of the citric acid cycle including alpha-ketoglutarate dehydrogenase, which is repressed by anaerobic conditions of growth. The enzymes of the anaerobic citrate fermentation pathway, citritase and oxalacetate decarboxylase, were also present in these cells, but this pathway of citrate catabolism was effectively blocked by the absence of Na(+), which is essential for the activation of the oxalacetate decarboxylase step. Thus, in Na(+)-free medium, aerobic citrate catabolism proceeded solely via the citric acid cycle. Addition of 10 mm Na(+) to the aerobic citrate medium resulted in the activation of oxalacetate decarboxylase and the repression of alpha-ketoglutarate dehydrogenase, thereby diverting citrate catabolism from the (aerobic) citric acid cycle mechanism to the fermentation mechanism characteristic of anaerobic growth. The further addition of 2% potassium acetate to the medium caused repression of citritase and derepression of alpha-ketoglutarate dehydrogenase, switching citrate catabolism back into the citric acid cycle.     

Ion binding affinity in the cavity of the KcsA potassium channel

The hydrophobic cell membrane interior presents a large energy barrier for ions to permeate. Potassium channels reduce this barrier by creating a water-filled cavity at the middle of their ion conduction pore to allow ion hydration and by directing the C-terminal "end charge" of four alpha-helices toward the water-filled cavity. Here we have studied the interaction of monovalent cations with the cavity of the KcsA K(+) channel using X-ray crystallography. In these studies, Tl(+) was used as an analogue for K(+) and the total ion-stabilization energy for Tl(+) in the cavity was estimated by measuring its binding affinity. Binding affinity for the Na(+) ion was also measured, revealing a weak selectivity ( approximately 7-fold) favoring Tl(+) over Na(+). The structures of the cavity containing Na(+), K(+), Tl(+), Rb(+), and Cs(+) are compared. These results are consistent with a fairly large (more negative than -100 mV) electrostatic potential inside the cavity, and they also imply the presence of a weak nonelectrostatic component to a cation's interaction with the cavity.     

Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.     

Sodium and Other Inorganic Growth Requirements of Bacteroides amylophilus

Bacteroides amylophilus has growth requirements for Na(+), PO(4) (3-), K(+), and small quantities of Mg(2+). No requirement could be shown for Ca(2+) in media previously found growth-yield-limiting for Bacteroides succinogenes. Deletion of Co(2+), Mn(2+), Cl(-), or SO(4) (2-) did not affect growth. Quantitative studies indicate that Na(+), K(+), and PO(4) (3-) have differing effects on the growth of B. amylophilus. A concentration of sodium and potassium ions affects both growth rate and growth yield, whereas a phosphate concentration markedly affects growth yield, but affects growth rate only slightly, if at all. The sodium requirement of B. amylophilus is absolute. It cannot be replaced by K(+), Li(+), Rb(+), or Cs(+). The latter three monovalent cations are toxic to B. amylophilus if supplied to the organism at Na(+)-replacing concentrations. K(+) is inactive at similar concentrations. The K(+) requirement of B. amylophilus may be satisfied by Rb(+). The concentration of Na(+) required by B. amylophilus for abundant growth suggests that B. amylophilus should be considered a slightly halophilic organism. The results suggest that Na(+) may be a more frequent requirement among terrestial bacteria obtained from relatively low-salt environments than has been previously believed.     

Channel induction by palytoxin in yeast cells expressing Na+,K+-ATPase or its chimera with sarco/endoplasmic reticulum Ca2+-ATPase

Palytoxin (PTX) induces a cation channel through interaction with Na(+),K(+)-ATPase. It is unclear how this action relates to the enzyme catalytic activity. We examined whether the action of PTX depends on the catalytic domain specific for Na(+),K(+)-ATPase. Wild-type Na(+),K(+)-ATPase alpha-subunit (NNN) or its chimera (NCN), in which the catalytic domain was replaced with that of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, was co-expressed with beta-subunit in the yeast Saccharomyces cerevisiae. PTX (0.1-100 nM) increased K(+) efflux in NNN- or NCN-transfected cells to a similar degree but not in non-transfected cells. When ouabain-resistant NNN and NCN were expressed, PTX also increased K(+) efflux. Ouabain inhibited the effect of PTX in NNN or NCN cells but not in ouabain-resistant cells. These data suggest that the channel-forming action of PTX does not depend on the catalytic domain species.     

Tracing the origin of the RACTK1 K(+) channel

Potassium secretion by the kidney is vital for the maintenance of K(+) homeostasis. RACTK1, a putative inwardly rectifying potassium channel cloned from cultured rabbit collecting duct cells, has been proposed to play a role in this process. However, the lack of homology with any other cloned potassium channel and the inability to reproduce the results across different laboratories has brought into question the existence of RACTK1. Recently, it has been suggested that RACTK1 is a contamination from Escherichia coli. In this work we add conclusive evidence supporting the bacterial origin of RACTK1. Using both genomic PCR and RT-PCR we were unable to detect RACTK1 in a number of mammalian species. In addition sequencing of RACTK1 cDNA confirmed a complete homology between RACTK1 and a region of E. coli genomic DNA. Finally, a hypothesis on how RACTK1 could have been generated from a contamination by E. coli genomic DNA is presented.     

Sodium, an Obligate Growth Requirement for Predominant Rumen Bacteria

Sodium is an obligate growth requirement for most currently recognized predominant species of rumen bacteria. The isoosmotic deletion of Na(+) from a nutritionally adequate defined medium completely eliminated growth of most species. Growth yields and rates were both a function of Na(+) concentration for Na(+)-requiring species, and Na(+) could not be replaced by Rb(+), Li(+), or Cs(+) when these ions were substituted for Na(+) at a concentration equivalent to an Na(+) concentration that supported abundant growth. Li(+), Cs(+), or Rb(+) was toxic at an Na(+)-replacing concentration (15 mM) but not at a K(+)-replacing concentration (0.65 mM). K(+) was also an obligate growth requirement for rumen bacteria in media containing Na(+) and K(+) as major monovalent cations, but K(+) could be replaced, for most species, by Rb(+). The quantities of Na(+) that support rapid and abundant growth of Na(+)-requiring rumen bacteria show that these organisms are slight halophiles. A growth requirement for Na(+) appears more frequent among nonmarine bacteria than has been previously believed.     

A novel current pathway parallel to the central pore in a mutant voltage-gated potassium channel

Voltage-gated potassium channels are proteins composed of four subunits consisting of six membrane-spanning segments S1-S6, with S4 as the voltage sensor. The region between S5 and S6 forms the potassium-selective ion-conducting central α-pore. Recent studies showed that mutations in the voltage sensor of the Shaker channel could disclose another ion permeation pathway through the voltage-sensing domain (S1-S4) of the channel, the ω-pore. In our studies we used the voltage-gated hKv1.3 channel, and the insertion of a cysteine at position V388C (Shaker position 438) generated a current through the α-pore in high potassium outside and an inward current at hyperpolarizing potentials carried by different cations like Na(+), Li(+), Cs(+), and NH(4)(+). The observed inward current looked similar to the ω-current described for the R1C/S Shaker mutant channel and was not affected by some pore blockers like charybdotoxin and tetraethylammonium but was inhibited by a phenylalkylamine blocker (verapamil) that acts from the intracellular side. Therefore, we hypothesize that the hKv1.3_V388C mutation in the P-region generated a channel with two ion-conducting pathways. One, the α-pore allowing K(+) flux in the presence of K(+), and the second pathway, the σ-pore, functionally similar but physically distinct from the ω-pathway. The entry of this new pathway (σ-pore) is presumably located at the backside of Y395 (Shaker position 445), proceeds parallel to the α-pore in the S6-S6 interface gap, ending between S5 and S6 at the intracellular side of one α-subunit, and is blocked by verapamil.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

Chromosomal relocation of prophage-associated bacterial genes

Taylor, M. W. (Stanford University, Stanford, Calif.), and C. Yanofsky. Chromosomal relocation of prophage-associated bacterial genes. J. Bacteriol. 91:1469-1476. 1966.-Two distinguishable colony types, rough-edged and smooth-edged, were observed when tryptophan auxotrophs of Escherichia coli were transformed to tryptophan independence with DNA from the hybrid nondefective transducing phage i(lambda)h(phi80)T(1) (S)tryp A(+)B(+), and with the helper phage lambdai(434). P1kc transduction experiments with cells of the two types of colonies as genetic donors showed that the i(lambda)h(phi80)T(1) (S)tryp A(+)B(+) prophage was located at different regions of the E. coli chromosome. In cells of rough-edged colonies, the prophage was linked to the tryp-cys region, its normal location, whereas in cells of smooth-edged colonies the prophage was associated with the gal region. When transformation experiments were performed with a T(1) (R)tryp(-) deletion mutant as recipient, and phage lambdai(434) as helper, prophage localization was only detected at the gal region. Localization of (lambda)h(phi80)T(1) (S)tryp A(+)B(+) prophage near gal does not appear to be due to the formation of a recombinant phage carrying tryp A(+)B(+), but is due to some type of interaction between the genomes of i(lambda)h(phi80)T(1) (S)tryp A(+)B(+) and the helper phage. When conditions comparable to those used in transformation studies were employed in transduction experiments, including the use of helper phage, two classes of transductants with either cys or gal linkage were also observed. To examine whether the location of the prophage on the E. coli chromosome had any effect on the ability of the prophage-associated tryp A(+) and tryp B(+) genes to function or respond to different repression conditions, specific activities of the A and B subunits of tryptophan synthetase specified by the phage genome were measured. Similar values were obtained regardless of the location of the prophage-associated tryp genes. Furthermore, the prophage-associated tryp genes, free from their normal operator region, permitted enzyme formation which was unaffected by repression or derepression conditions.     

Trk1 and Trk2 define the major K(+) transport system in fission yeast

The trk1(+) gene has been proposed as a component of the K(+) influx system in the fission yeast Schizosaccharomyces pombe. Previous work from our laboratories revealed that trk1 mutants do not show significantly altered content or influx of K(+), although they are more sensitive to Na(+). Genome database searches revealed that S. pombe encodes a putative gene (designated here trk2(+)) that shows significant identity to trk1(+). We have analyzed the characteristics of potassium influx in S. pombe by using trk1 trk2 mutants. Unlike budding yeast, fission yeast displays a biphasic transport kinetics. trk2 mutants do not show altered K(+) transport and exhibit only a slightly reduced Na(+) tolerance. However, trk1 trk2 double mutants fail to grow at low K(+) concentrations and show a dramatic decrease in Rb(+) influx, as a result of loss of the high-affinity transport component. Furthermore, trk1 trk2 cells are very sensitive to Na(+), as would be expected for a strain showing defective potassium transport. When trk1 trk2 cells are maintained in K(+)-free medium, the potassium content remains higher than that of the wild type or trk single mutants. In addition, the trk1 trk2 strain displays increased sensitivity to hygromycin B. These results are consistent with a hyperpolarized state of the plasma membrane. An additional phenotype of cells lacking both Trk components is a failure to grow at acidic pH. In conclusion, the Trk1 and Trk2 proteins define the major K(+) transport system in fission yeast, and in contrast to what is known for budding yeast, the presence of any of these two proteins is sufficient to allow growth at normal potassium levels.     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.