A Na+-translocating pyrophosphatase in the acetogenic bacterium Acetobacterium woodii

The anaerobic acetogenic bacterium Acetobacterium woodii employs a novel type of Na(+)-motive anaerobic respiration, caffeate respiration. However, this respiration is at the thermodynamic limit of energy conservation, and even worse, in the first step, caffeate is activated by caffeyl-CoA synthetase, which hydrolyzes ATP to AMP and pyrophosphate. Here, we have addressed whether or not the energy stored in the anhydride bond of pyrophosphate is conserved by A. woodii. Inverted membrane vesicles of A. woodii have a membrane-bound pyrophosphatase that catalyzes pyrophosphate hydrolysis at a rate of 70-120 milliunits/mg of protein. Pyrophosphatase activity was dependent on the divalent cation Mg(2+). In addition, activity was strictly dependent on Na(+) with a K(m) of 1.1 mM. Hydrolysis of pyrophosphate was accompanied by (22)Na(+) transport into the lumen of the inverted membrane vesicles. Inhibitor studies revealed that (22)Na(+) transport was primary and electrogenic. Next to the Na(+)-motive ferredoxin:NAD(+) oxidoreductase (Fno or Rnf), the Na(+)-pyrophosphatase is the second primary Na(+)-translocating enzyme in A. woodii.     

Functional characterization of a NapA Na(+)/H(+) antiporter from Thermus thermophilus

Na(+)/H(+) antiporters are ubiquitous membrane proteins and play an important role in cell homeostasis. We amplified a gene encoding a member of the monovalent cation:proton antiporter-2 (CPA2) family (TC 2.A.37) from the Thermus thermophilus genome and expressed it in Escherichia coli. The gene product was identified as a member of the NapA subfamily and was found to be an active Na(+)(Li(+))/H(+) antiporter as it conferred resistance to the Na(+) and Li(+) sensitive strain E. coli EP432 (DeltanhaA, DeltanhaB) upon exposure to high concentration of these salts in the growth medium. Fluorescence measurements using the pH sensitive dye 9-amino-6-chloro-2-methoxyacridine in everted membrane vesicles of complemented E. coli EP432 showed high Li(+)/H(+) exchange activity at pH 6, but marginal Na(+)/H(+) antiport activity. Towards more alkaline conditions, Na(+)/H(+) exchange activity increased to a relative maximum at pH 8, where by contrast the Li(+)/H(+) exchange activity reached its relative minimum. Substitution of conserved residues D156 and D157 (located in the putative transmembrane helix 6) with Ala resulted in the complete loss of Na(+)/H(+) activity. Mutation of K305 (putative transmembrane helix 10) to Ala resulted in a compromised phenotype characterized by an increase in apparent K(m) for Na(+) (36 vs. 7.6 mM for the wildtype) and Li(+) (17 vs. 0.22 mM), In summary, the Na(+)/H(+) antiport activity profile of the NapA type transporter of T. thermophilus resembles that of NhaA from E. coli, whereas in contrast to NhaA the T. thermophilus NapA antiporter is characterized by high Li(+)/H(+) antiport activity at acidic pH.     

Carboxyl-terminal hydrophilic tail of a NhaP type Na+/H+ antiporter from cyanobacteria is involved in the apparent affinity for Na+ and pH sensitivity

Little information is available on the C-terminal hydrophilic tails of prokaryotic Na(+)/H(+) antiporters. To address functional properties of the C-terminal tail, truncation mutants in this domain were constructed. Truncation of C-terminal amino acid residues of NhaP1 type antiporter from Synechocystis PCC6803 (SynNhaP1) did not change the V(max) values, but increased the K(m) values for Na(+) and Li(+) about 3 to 15-fold. Truncation of C-terminal tail of a halotolerant cyanobacterium Aphanothece halophytica (ApNhaP1) significantly decreased the V(max) although it did not alter the K(m) values for Na(+). The C-terminal part of SynNhaP1 was expressed in E. coli and purified as a 16kDa soluble protein. Addition of purified polypeptide to the membrane vesicles expressing the C-terminal truncated SynNhaP1 increased the exchange activities. Change of Glu519 and Glu521 to Lys in C-terminal tail altered the pH dependence of Na(+)/H(+) and Li(+)/H(+) exchange activities. These results indicate that the specific acidic amino acid residues at C-terminal domain play important roles for the K(m) and the pH dependence of the exchange activity.     

Na+-pyrophosphatase: a novel primary sodium pump

Membrane-bound pyrophosphatase (PPase) is commonly believed to couple pyrophosphate (PPi) hydrolysis to H+ transport across the membrane. Here, we demonstrate that two newly isolated bacterial membrane PPases from the mesophile Methanosarcina mazei (Mm-PPase) and the moderate thermophile Moorella thermoacetica and a previously described PPase from the hyperthermophilic bacterium Thermotoga maritima catalyze Na+ rather than H+ transport into Escherichia coli inner membrane vesicles (IMV). When assayed in uncoupled IMV, the three PPases exhibit an absolute requirement for Na+ but display the highest hydrolyzing activity in the presence of both Na+ and K+. Steady-state kinetic analysis of PPi hydrolysis by Mm-PPase revealed two Na+ binding sites. One of these sites can also bind K+, resulting in a 10-fold increase in the affinity of the other site for Na+ and a 2-fold increase in maximal velocity. PPi-driven 22Na+ transport into IMV containing Mm-PPase was unaffected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, inhibited by the Na+ ionophore monensin, and activated by the K+ ionophore valinomycin. The Na+ transport was accompanied by the generation of a positive inside membrane potential as reported by Oxonol VI. These findings define Na+-dependent PPases as electrogenic Na+ pumps. Phylogenetic analysis suggests that ancient gene duplication preceded the split of Na+- and H+-PPases.     

Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity

Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.     

H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl

H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.     

Importance for absorption of Na+ from freshwater of lysine, valine and serine substitutions in the alpha1a-isoform of Na,K-ATPase in the gills of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

In the gills of rainbow trout and Atlantic salmon, the alpha1a- and alpha1b-isoforms of Na,K-ATPase are expressed reciprocally during salt acclimation. The alpha1a-isoform is important for Na(+) uptake in freshwater, but the molecular basis for the functional differences between the two isoforms is not known. Here, three amino acid substitutions are identified in transmembrane segment 5 (TM5), TM8 and TM9 of the alpha1a-isoform compared to the alpha1b-isoform, and the functional consequences are examined by mutagenesis and molecular modeling on the crystal structures of Ca-ATPase or porcine kidney Na,K-ATPase. In TM5 of the alpha1a-isoform, a lysine substitution, Asn783 --> Lys, inserts the epsilon-amino group in cation site 1 in the E(1) form to reduce the Na(+)/ATP ratio. In the E(2) form the epsilon-amino group approaches cation site 2 to force ejection of Na(+) to the blood phase and to interfere with binding of K(+). In TM8, a Asp933 --> Val substitution further reduces K(+) binding, while a Glu961 --> Ser substitution in TM9 can prevent interaction of FXYD peptides with TM9 and alter Na(+) or K(+) affinities. Together, the three substitutions in the alpha1a-isoform of Na,K-ATPase act to promote binding of Na(+) over K(+) from the cytoplasm, to reduce the Na(+)/ATP ratio and the work done in one Na,K pump cycle of active Na(+) transport against the steep gradient from freshwater (10-100 microM: Na(+)) to blood (160 mM: Na(+)) and to inhibit binding of K(+) to allow Na(+)/H(+) rather than Na(+)/K(+) exchange.     

A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans

The H(+) proton-translocating inorganic pyrophosphatase (H(+)-PPase) family is composed of two phylogenetically distinct types of enzymes: K(+)-dependent and K(+)-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H(+)-PPase from the thermophilic bacterium Carboxydothermus hydrogenoformans. Both PP(i)-hydrolyzing and PP(i)-energized H(+) translocation activities of the recombinant enzyme in Escherichia coli inner membrane vesicles are strictly K(+)-dependent. Here we deduce the K(+) requirement of all available H(+)-PPase sequences based on the K(+) dependence of C. hydrogenoformans H(+)-PPase in conjunction with phylogenetic analyses. Our data reveal that K(+)-independent H(+)-PPases possess conserved Lys and Thr that are absent in K(+)-dependent H(+)-PPases. We further demonstrate that a A460K substitution in C. hydrogenoformans H(+)-PPase is sufficient to confer K(+) independence to both PP(i) hydrolysis and PP(i)-energized H(+) translocation. In contrast, a A463T mutation does not affect the K(+) dependence of H(+)-PPase.     

A structural determinant for the control of PIP2 sensitivity in G protein-gated inward rectifier K+ channels

Inward rectifier K(+) (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP(2)), but G protein-gated Kir (K(G)) channels further require either G protein βγ subunits (Gβγ) or intracellular Na(+) for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of K(G) channel subunit Kir3.2 obtained in the presence and the absence of Na(+). The Na(+)-free Kir3.2, but not the Na(+)-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na(+)-dependent activation, lowered PIP(2) sensitivity. The conservation of these residues within the K(G) channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP(2) sensitivity.     

Transport mechanism and pH regulation of the Na+/H+ antiporter NhaA from Escherichia coli: an electrophysiological study

Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na(+) or H(+) gradients as the driving force. Under symmetrical pH conditions with a Na(+) gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the K(m) for Na(+) at acidic pH. Under symmetrical Na(+) concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na(+) and H(+) binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na(+) induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na(+) translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.     

Chemical reactivities of cysteine substitutions in subunit a of ATP synthase define residues gating H+ transport from each side of the membrane

Subunit a plays a key role in coupling H(+) transport to rotations of the subunit c-ring in F(1)F(o) ATP synthase. In Escherichia coli, H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F(o) subunit c. Based upon the Ag(+) sensitivity of Cys substituted into subunit a, H(+) are thought to reach Asp-61 via aqueous pathways mapping to surfaces of TMH 2-5. In this study we have extended characterization of the most Ag(+)-sensitive residues in subunit a with cysteine reactive methanethiosulfonate (MTS) reagents and Cd(2+). The effect of these reagents on ATPase-coupled H(+) transport was measured using inside-out membrane vesicles. Cd(2+) inhibited the activity of all Ag(+)-sensitive Cys on the cytoplasmic side of the TMHs, and three of these substitutions were also sensitive to inhibition by MTS reagents. On the other hand, Cd(2+) did not inhibit the activities of substitutions at residues 119 and 120 on the periplasmic side of TMH2, and residues 214 and 215 in TMH4 and 252 in TMH5 at the center of the membrane. When inside-out membrane vesicles from each of these substitutions were sonicated during Cd(2+) treatment to expose the periplasmic surface, the ATPase-coupled H(+) transport activity was strongly inhibited. The periplasmic access to N214C and Q252C, and their positioning in the protein at the a-c interface, is consistent with previous proposals that these residues may be involved in gating H(+) access from the periplasmic half-channel to Asp-61 during the protonation step.     

Involvement in K+ access of Leu318 at the extracellular domain flanking M3 and M4 of the Na+,K+-ATPase alpha-subunit

The effect of point mutation in the sequence 316TWLE319, which occurs in the extracellular loop flanking the third (M3) and the fourth (M4) transmembrane segment (L3/4) of the Na+,K+-ATPase alpha-subunit, was examined. Mutation of Glu319 to Asp yielded an enzyme with full activity, whereas substituting Glu319 to Ala resulted in a severe loss of activity. A negative charge was introduced along the sequence, one residue at a time, from Thr316 to Leu318 (by E-scanning) in the mutant construct with Glu319 already mutated to Gln. The activity that had been reduced to 60% by the mutation of Glu319 to Gln was restored upon the introduction of a negative charge by E-scanning. When Leu318 was replaced by Glu in a series of scanning experiments, the K+ sensitivity of the ATPase activity was lowered. The lowering of K+ sensitivity was further demonstrated when a mutation of Leu318 to Glu was introduced into the wild-type enzyme. Furthermore, mutants with Leu318 to Gln, Arg, and Phe displayed lower K+ sensitivity similar to that of Leu318 to Glu mutant. Leu318 may be in access path for K+, and any substitution at this position may interfere with access of K+ from outside the cell.     

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.     

Quaternary organic amines inhibit Na,K pump current in a voltage-dependent manner: direct evidence of an extracellular access channel in the Na,K-ATPase

The effects of organic quaternary amines, tetraethylammonium (TEA) chloride and benzyltriethylammonium (BTEA) chloride, on Na,K pump current were examined in rat cardiac myocytes superfused in extracellular Na(+)-free solutions and whole-cell voltage-clamped with patch electrodes containing a high Na(+)-salt solution. Extracellular application of these quaternary amines competitively inhibited extracellular K(+) (K(+)(o)) activation of Na,K pump current; however, the concentration for half maximal inhibition of Na,K pump current at 0 mV (K(0)(Q)) by BTEA, 4.0 +/- 0.3 mM, was much lower than the K(0)(Q) for TEA, 26.6 +/- 0.7 mM. Even so, the fraction of the membrane electric field dissipated during K(+)(o) activation of Na,K pump current (lambda(K)), 39 +/- 1%, was similar to lambda(K) determined in the presence of TEA (37 +/- 2%) and BTEA (35 +/- 2%), an indication that the membrane potential (V(M)) dependence for K(+)(o) activation of the Na,K pump current was unaffected by TEA and BTEA. TEA was found to inhibit the Na,K pump current in a V(M)-independent manner, i.e., inhibition of current dissipated 4 +/- 2% of the membrane electric field. In contrast, BTEA dissipated 40 +/- 5% of the membrane electric field during inhibition of Na,K pump current. Thus, BTEA inhibition of the Na,K-ATPase is V(M)-dependent. The competitive nature of inhibition as well as the similar fractions of the membrane electric field dissipated during K(+)(o)-dependent activation and BTEA-dependent inhibition of Na,K pump current suggest that BTEA inhibits the Na,K-ATPase at or very near the enzyme's K(+)(o) binding site(s) located in the membrane electric field. Given previous findings that organic quaternary amines are not occluded by the Na,K-ATPase, these data clearly demonstrate that an ion channel-like structure provides access to K(+)(o) binding sites in the enzyme.     

Molecular mechanism of the ATP synthase's F(o) motor probed by mutational analyses of subunit a

The most prominent residue of subunit a of the F(1)F(o) ATP synthase is a universally conserved arginine (aR227 in Propionigenium modestum), which was reported to permit no substitution with retention of ATP synthesis or H(+)-coupled ATP hydrolysis activity. We show here that ATP synthases with R227K or R227H mutations in the P.modestum a subunit catalyse ATP-driven Na(+) transport above or below pH 8.0, respectively. Reconstituted F(o) with either mutation catalysed 22Na(+)(out)/Na(+)(in) exchange with similar pH profiles as found in ATP-driven Na(+) transport. ATP synthase with an aR227A substitution catalysed Na(+)-dependent ATP hydrolysis, which was completely inhibited by dicyclohexylcarbodiimide, but not coupled to Na(+) transport. This suggests that in the mutant the dissociation of Na(+) becomes more difficult and that the alkali ions remain therefore permanently bound to the c subunit sites. The reconstituted mutant enzyme was also able to synthesise ATP in the presence of a membrane potential, which stopped at elevated external Na(+) concentrations. These observations reinforce the importance of aR227 to facilitate the dissociation of Na(+) from approaching rotor sites. This task of aR227 was corroborated by other results with the aR227A mutant: (i) after reconstitution into liposomes, F(o) with the aR227A mutation did not catalyse 22Na(+)(out)/Na(+)(in) exchange at high internal sodium concentrations, and (ii) at a constant (Delta)pNa(+), 22Na(+) uptake was inhibited at elevated internal Na(+) concentrations. Hence, in mutant aR227A, sodium ions can only dissociate from their rotor sites into a reservoir of low sodium ion concentration, whereas in the wild-type the positively charged aR227 allows the dissociation of Na(+) even into compartments of high Na(+) concentration.     

H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na(+) interaction with the pore of Shaker B K(+) channels: zero and low K(+) conditions.

The Shaker B K(+) conductance (G(K)) collapses (in a reversible manner) if the membrane is depolarized and then repolarized in, 0 K(+), Na(+)-containing solutions (Gómez-Lagunas, F. 1997. J. Physiol. 499:3-15; Gómez-Lagunas, F. 1999. Biophys. J. 77:2988-2998). In this work, the role of Na(+) ions in the collapse of G(K) in 0-K(+) solutions, and in the behavior of the channels in low K(+) was studied. The main findings are as follows. First, in 0-K(+) solutions, the presence of Na(+) ions is an important factor that speeds the collapse of G(K). Second, external Na(+) fosters the drop of G(K) by binding to a site with a K(d) = 3.3 mM. External K(+) competes, in a mutually exclusive manner, with Na(o)(+) for binding to this site, with an estimated K(d) = 80 microM. Third, NMG and choline are relatively inert regarding the stability of G(K); fourth, with [K(o)(+)] = 0, the energy required to relieve Na(i)(+) block of Shaker (French, R.J., and J.B. Wells. 1977. J. Gen. Physiol. 70:707-724; Starkus, J.G., L. Kuschel, M. Rayner, and S. Heinemann. 2000. J. Gen. Physiol. 110:539-550) decreases with the molar fraction of Na(i)(+) (X(Na,i)), in an extent not accounted for by the change in Delta(mu)(Na). Finally, when X(Na,i) = 1, G(K) collapses by the binding of Na(i)(+) to two sites, with apparent K(d)s of 2 and 14.3 mM.     

Rapid induction of P/C-type inactivation is the mechanism for acid-induced K+ current inhibition

Extracellular acidification is known to decrease the conductance of many voltage-gated potassium channels. In the present study, we investigated the mechanism of H(+)(o)-induced current inhibition by taking advantage of Na(+) permeation through inactivated channels. In hKv1.5, H(+)(o) inhibited open-state Na(+) current with a similar potency to K(+) current, but had little effect on the amplitude of inactivated-state Na(+) current. In support of inactivation as the mechanism for the current reduction, Na(+) current through noninactivating hKv1.5-R487V channels was not affected by [H(+)(o)]. At pH 6.4, channels were maximally inactivated as soon as sufficient time was given to allow activation, which suggested two possibilities for the mechanism of action of H(+)(o). These were that inactivation of channels in early closed states occurred while hyperpolarized during exposure to acid pH (closed-state inactivation) and/or inactivation from the open state was greatly accelerated at low pH. The absence of outward Na(+) currents but the maintained presence of slow Na(+) tail currents, combined with changes in the Na(+) tail current time course at pH 6.4, led us to favor the hypothesis that a reduction in the activation energy for the inactivation transition from the open state underlies the inhibition of hKv1.5 Na(+) current at low pH.     

Decoupling the Na+-K+-ATPase in vivo: a possible new role in the gills of freshwater fishes

The literature suggests that when Na(+)-K(+)-ATPase has reduced access to its glycosphingolipid cofactor sulfogalactosyl ceramide (SGC), it is converted to a Na(+) uniporter. We recently showed that such segregation can occur within a single membrane when Na(+)-K(+)-ATPase is excluded from membrane microdomains or 'lipid rafts' enriched in SGC (D. Lingwood, G. Harauz, J.S. Ballantyne, J. Biol. Chem. 280, 36545-36550). Specifically we demonstrated that Na(+)-K(+)-ATPase localizes to SGC-enriched rafts in the gill basolateral membrane (BLM) of rainbow trout exposed to seawater (SW) but not freshwater (FW). We therefore proposed that since the freshwater gill Na(+)-K(+)-ATPase was separated from BLM SGC it should also transport Na(+) only, suggesting a new role for the pump in this epithelium. In this paper we discuss the biochemical evidence for SGC-based modulation of transport stoichiometry and highlight how a unique asparagine-lysine substitution in the FW pump isoform and FW gill transport energetics gear the Na(+)-K(+)-ATPase to perform Na(+) uniport.     

Extracellular determinants of anion discrimination of the Cl-/H+ antiporter protein CLC-5

Mammalian CLC proteins comprise both Cl- channels and Cl-/H+ antiporters that carry out fundamental physiological tasks by transporting Cl- across plasma membrane and intracellular compartments. The NO3- over Cl- preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl-/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3- over Cl- preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl-. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext.