Inhibition of Na+,K+-ATPase by ouabain triggers epithelial cell death independently of inversion of the [Na+]i/[K+]i ratio

Treatment with ouabain led to massive death of principal cells from collecting ducts (C7-MDCK), indicated by cell swelling, loss of mitochondrial function, an irregular pattern of DNA degradation, and insensitivity to pan-caspase inhibitor. Equimolar substitution of extracellular Na(+) by K(+) or choline(+) sharply attenuated the effect of ouabain on intracellular Na(+) and K(+) content but did not protect the cells from death in the presence of ouabain. In contrast to ouabain, inhibition of the Na(+)/K(+) pump in K(+)-free medium increased Na(+)(i) content but did not affect cell survival. In control and K(+)-free medium, ouabain triggered half-maximal cell death at concentrations of approximately 0.5 and 0.05 microM, respectively, which was consistent with elevation of Na(+)/K(+) pump sensitivity to ouabain in K(+)-depleted medium. Our results show for the first time that the death of ouabain-treated renal epithelial cells is independent of the inhibition of Na(+)/K(+) pump-mediated ion fluxes and the [Na(+)](i)]/[K(+)](i) ratio.     

Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase

Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct inhibitory effects on Na(+)/Ca(2+) exchange.     

Voltage dependence of the apparent affinity for external Na(+) of the backward-running sodium pump

The steady-state voltage and [Na(+)](o) dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K(+)-free seawater; ATP- and Na(+)-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated (42)K(+) efflux to reverse pump current, I(pump) (both defined by sensitivity to dihydrodigitoxigenin, H(2)DTG), scaled by Faraday's constant, was -1.5 +/- 0.4 (n = 5; expected ratio for 2 K(+)/3 Na(+) stoichiometry is -2.0). Steady-state reverse pump current-voltage (I(pump)-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various V(m) to H(2)DTG or from the difference between membrane I-V relationships obtained by imposing V(m) staircases in the presence or absence of H(2)DTG. With the second method, we also investigated the influence of [Na(+)](o) (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse I(pump)-V relationship. The reverse I(pump)-V relationship is sigmoid, I(pump) saturating at large negative V(m), and each doubling of [Na(+)](o) causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state (22)Na(+) efflux during electroneutral Na(+)/Na(+) exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100-103). This model predicts valence z = nlambda, where n (1.33 +/- 0.05) is the Hill coefficient of Na binding, and lambda (0.61 +/- 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na(+)/Na(+) exchange modes only with additional assumptions that render them less likely.     

Large diameter of palytoxin-induced Na/K pump channels and modulation of palytoxin interaction by Na/K pump ligands

Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pump's ion translocation pathway. We systematically analyzed palytoxin's interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na(+)-containing (K(+)-free) external and internal solutions, lowest for K(+)-containing (Na(+)-free) external and internal solutions, and intermediate for the mixed external Na(+)/internal K(+), and external K(+)/internal Na(+) conditions; with Na(+) solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na(+) solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained approximately 30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the same Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine(+), suggesting that the narrowest section of the pore must be approximately 7.5 A wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle.     

Upregulation of apical NHE3 in renal OK cells overexpressing the rodent alpha(1)-subunit of the Na(+) pump

Vectorial Na(+) reabsorption across the proximal tubule is mediated by apical entry of Na(+), primarily via Na(+)/H(+) exchanger isoform 3 (NHE3), and basolateral extrusion via the Na(+) pump (Na(+)-K(+)-ATPase). We hypothesized that regulation of Na(+) reabsorption should involve not only the activity of the basolateral Na(+)-K(+)-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na(+)-K(+)-ATPase, opossum kidney (OK) cells were transfected with the rodent Na(+)-K(+)-ATPase alpha(1)-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na(+)-K(+)-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na(+)-K(+)-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC(50) values for ouabain: the first value was similar to the IC(50) of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum alpha(1)-isozymes. It is shown that transfection of OK cells with Na(+)-K(+)-ATPase increased Na(+)-K(+)-ATPase and NHE3 activities. This was associated with overexpression of the Na(+)-K(+)-ATPase alpha(1)-subunit and NHE3 in transfected OK cells. The abundance of the Na(+)-K(+)-ATPase beta(1)-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na(+)-K(+)-ATPase in cells transfected with the rodent Na(+) pump alpha(1)-subunit cDNA is expected to stimulate apical Na(+) influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.     

Tyrosine 537 within the Na+,K+-ATPase alpha-subunit is essential for AP-2 binding and clathrin-dependent endocytosis

In renal epithelial cells endocytosis of Na(+),K(+)-ATPase molecules is initiated by phosphorylation of its alpha(1)-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na(+),K(+)-ATPase alpha(1)-subunit. We identified a conserved sequence (Y(537)LEL) within the alpha(1)-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na(+),K(+)-ATPase alpha(1)-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent alpha-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased alpha(1)-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na(+),K(+)-ATPase activity. Because phorbol esters increase Na(+),K(+)-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na(+),K(+)-ATPase endocytosis.     

The role of Na,K-ATPase alpha subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties of the Na,K-pump

The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K-ATPase alpha subunit, in determining the voltage and extracellular K+ (K+(o)) dependence of enzyme-mediated ion transport, were examined in this study. HeLa cells expressing the alpha1 subunit of sheep Na,K-ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+ (37 degrees C). Na,K-pump current produced by the ouabain-resistant control enzyme (RD), containing amino acid substitutions Gln111Arg and Asn122Asp, displayed a membrane potential and K+(o) dependence similar to wild-type Na,K-ATPase during superfusion with 0 and 148 mM Na+-containing salt solutions. Additional substitution of alanine at Ser775 or Glu779 produced 155- and 15-fold increases, respectively, in the K+(o) concentration that half-maximally activated Na,K-pump current at 0 mV in extracellular Na+-free solutions. However, the voltage dependence of Na,K-pump current was unchanged in RD and alanine-substituted enzymes. Thus, large changes in apparent K+(o) affinity could be produced by mutations in the fifth transmembrane segment of the Na,K-ATPase with little effect on voltage-dependent properties of K+ transport. One interpretation of these results is that protein structures responsible for the kinetics of K+(o) binding and/or occlusion may be distinct, at least in part, from those that are responsible for the voltage dependence of K+(o) binding to the Na,K-ATPase.     

Clathrin-mediated endocytosis of Na+,K+-ATPase in response to parathyroid hormone requires ERK-dependent phosphorylation of Ser-11 within the alpha1-subunit

Parathyroid hormone (PTH) inhibits Na(+),K(+)-ATPase activity through protein kinase C- (PKC) and extracellular signal-regulated kinase- (ERK) dependent pathways and increases serine phosphorylation of the alpha(1)-subunit. To determine whether specific serine phosphorylation sites within the Na(+),K(+)-ATPase alpha(1)-subunit are involved in the Na(+),K(+)-ATPase responses to PTH, we examined the effect of PTH in opossum kidney cells stably transfected with wild type rat Na(+),K(+)-ATPase alpha(1)-subunit (WT), serine 11 to alanine mutant alpha(1)-subunit (S11A), or serine 18 to alanine mutant alpha(1)-subunit (S18A). PTH increased phosphorylation and endocytosis of the Na(+),K(+)-ATPase alpha(1)-subunit into clathrin-coated vesicles in cells transfected with WT and S18A rat Na(+),K(+)-ATPase alpha(1)-subunits. PTH did not increase the level of phosphorylation or stimulate translocation of Na(+),K(+)-ATPase alpha(1)-subunits into clathrin-coated vesicles in cells transfected with the S11A mutant. PTH inhibited ouabain-sensitive (86)Rb uptake and Na(+),K(+)-ATPase activity (ouabain-sensitive ATP hydrolysis) in WT- and S18A-transfected opossum kidney cells but not in S11A-transfected cells. Pretreatment of the cells with the PKC inhibitors and ERK inhibitor blocked PTH inhibition of (86)Rb uptake, Na(+),K(+)-ATPase activity, alpha(1)-subunit phosphorylation, and endocytosis in WT and S18A cells. Consistent with the notion that ERK phosphorylates Na(+),K(+)-ATPase alpha(1)-subunit, ERK was shown to be capable of causing phosphorylation of Na(+),K(+)-ATPase alpha(1)-subunit immunoprecipitated from WT and S18A but not from S11A-transfected cells. These results suggest that PTH regulates Na(+),K(+)-ATPase by PKC and ERK-dependent alpha(1)-subunit phosphorylation and that the phosphorylation requires the expression of a serine at the 11 position of the Na(+),K(+)-ATPase alpha(1)-subunit.     

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.     

Extracellular sodium interacts with the HERG channel at an outer pore site

Most voltage-gated K(+) currents are relatively insensitive to extracellular Na(+) (Na(+)(o)), but Na(+)(o) potently inhibits outward human ether-a-go-go-related gene (HERG)-encoded K(+) channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429-30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na(+) (Na(+)(i)) and extracellular Ba(2+) (Ba(2+)(o)), to define a site where Na(+)(o) interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na(+)(o) was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K(+) selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na(+)(o). Quantitatively similar effects of Na(+)(i) to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na(+)(o) sensitivity, this result suggested that Na(+)(o) and Na(+)(i) act at different sites. Extracellular Ba(2+) (Ba(2+)(o)) blocks K(+) channel pores, and thereby serves as a useful probe of K(+) channel structure. HERG channel inactivation promotes relief of Ba(2+) block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265-278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na(+)(o) action. A remote allosteric model predicts that Na(+)(o) will speed relief of Ba(2+)(o) block by promoting inactivation. Instead, Na(+)(o) slowed Ba(2+) egress and Ba(2+) relieved Na(+)(o) inhibition, consistent with Na(+)(o) binding to an outer pore site. The apparent affinities of the outer pore for Na(+)(o) and K(+)(o) as measured by slowing of Ba(2+) egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na(+)(o) inhibition. Na(+)(o) inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.     

Na(+)/K(+) pump expression in the L8 rat myogenic cell line: effects of heterologous alpha subunit transfection

We have characterized the physiological and biochemical properties of the Na(+)/K(+) pump and its molecular expression in L8 rat muscle cells. Pump properties were measured by [(3)H]ouabain binding and (86)Rb uptake. Scatchard plot analysis of specific ouabain binding indicated the presence of a single family of binding sites with a B(max) of approximately 135 fmol/ mg P and a K(D) of 3.3 x 10(-8). (86)Rb uptake due to specific pump activity was found to be 20% of the total in L8 cells. The results indicated lower affinity of L8 cells for ouabain and lower activity of the pump than that reported for chick or rat skeletal muscle in primary culture. Both the alpha(1) and beta(1) protein and mRNA isoforms were expressed in myoblasts and in myotubes, while the alpha(2), alpha(3), and beta(2) isoforms were not detectable. We attempted to overcome low physiological expression of the Na(+)/K(+) pump by employing a vector expressing an avian high affinity alpha subunit. This allowed identification of the transfected subunit separate from that endogenously expressed in L8 cells. Successful transfection into L8 myoblasts and myotubes was recognized by anti-avian alpha subunit monoclonal antibodies. Fusion index, Na(+)/K(+) pump activity, and the level of the transmembrane resting potential were all significantly greater in transfected L8 (tL8) cells than in non-tL8. The total amount of alpha subunit (avian and rat) in tL8 cells was greater than that (only rat) in non-tL8 cells. This relatively high abundance of the Na(+)/K(+) pump in transfected cells may indicate that avian and rat alpha subunits hybridize to form functional pump complexes.     

Catalytic properties of Staphylococcus aureus and Bacillus members of the secondary cation/proton antiporter-3 (Mrp) family are revealed by an optimized assay in an Escherichia coli host

Monovalent cation proton antiporter-3 (Mrp) family antiporters are widely distributed and physiologically important in prokaryotes. Unlike other antiporters, they require six or seven hydrophobic gene products for full activity. Standard fluorescence-based assays of Mrp antiport in membrane vesicles from Escherichia coli transformants have not yielded strong enough signals for characterization of antiport kinetics. Here, an optimized assay protocol for vesicles of antiporter-deficient E. coli EP432 transformants produced higher levels of secondary Na(+)(Li(+))/H(+) antiport than previously reported. Assays were conducted on Mrps from alkaliphilic Bacillus pseudofirmus OF4 and Bacillus subtilis and the homologous antiporter of Staphylococcus aureus (Mnh), all of which exhibited Na(+)(Li(+))/H(+) antiport. A second paralogue of S. aureus (Mnh2) did not. K(+), Ca(2+), and Mg(2+) did not support significant antiport by any of the test antiporters. All three Na(+)(Li(+))/H(+) Mrp antiporters had alkaline pH optima and apparent K(m) values for Na(+) that are among the lowest reported for bacterial Na(+)/H(+) antiporters. Using a fluorescent probe of the transmembrane electrical potential (DeltaPsi), Mrp Na(+)/H(+) antiport was shown to be DeltaPsi consuming, from which it is inferred to be electrogenic. These assays also showed that membranes from E. coli EP432 expressing Mrp antiporters generated higher DeltaPsi levels than control membranes, as did membranes from E. coli EP432 expressing plasmid-borne NhaA, the well-characterized electrogenic E. coli antiporter. Assays of respiratory chain components in membranes from Mrp and control E. coli transformants led to a hypothesis explaining how activity of secondary, DeltaPsi-consuming antiporters can elicit increased capacity for DeltaPsi generation in a bacterial host.     

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.     

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.     

The activity profile of the NhaD-type Na+(Li+)/H+ antiporter from the soda Lake Haloalkaliphile Alkalimonas amylolytica is adaptive for the extreme environment

In extreme alkaliphiles, Na(+)/H(+) antiporters play a central role in the Na(+) cycle that supports pH homeostasis, Na(+) resistance, solute uptake, and motility. Properties of individual antiporters have only been examined in extremely alkaliphilic soil Bacillus spp., whereas the most alkaline natural habitats usually couple high pH with high salinity. Here, studies were conducted on a Na(+)(Li(+))/H(+) antiporter, NhaD, from the soda lake haloalkaliphile Alkalimonas amylolytica. The activity profile of A. amylolytica NhaD at different pH values and Na(+) concentrations reflects its unique natural habitat. In membrane vesicles from antiporter-deficient Escherichia coli EP432 (DeltanhaA DeltanhaB), the pH optimum for NhaD-dependent Na(+)(Li(+))/H(+) antiport was at least 9.5, the highest pH that could be tested; no activity was observed at pH < or =8.5. NhaD supported low Na(+)/H(+) antiport activity at pH 9.5 that was detectable over a range of Na(+) concentrations from 10 mM to at least 800 mM, with a 600 mM optimum. Although A. amylolytica nhaD was isolated by complementing the Li(+) sensitivity of the triple mutant E. coli strain KNabc (DeltanhaA DeltanhaB DeltachaA), sustained propagation of nhaD-bearing plasmids in this strain resulted in a glycine (Gly(327))-->serine mutation in a putative cytoplasmic loop of the mutant transporter. The altered activity profile of NhaD-G327S appears to be adaptive to the E. coli setting: a much higher activity than wild-type NhaD at Na(+) concentrations up to 200 mM but lower activity at 400 to 600 mM Na(+), with a pH optimum and minimal pH for activity lower than those of wild-type NhaD.     

Coexpression with beta(1)-subunit modifies the kinetics and fatty acid block of hH1(alpha) Na(+) channels

Voltage-gated cardiac Na(+) channels are composed of alpha- and beta(1)-subunits. In this study beta(1)-subunit was cotransfected with the alpha-subunit of the human cardiac Na(+) channel (hH1(alpha)) in human embryonic kidney (HEK293t) cells. The effects of this coexpression on the kinetics and fatty acid-induced suppression of Na(+) currents were assessed. Current density was significantly greater in HEK293t cells coexpressing alpha- and beta(1)-subunits (I(Na,alpha beta)) than in HEK293t cells expressing alpha-subunit alone (I(Na,alpha)). Compared with I(Na,alpha), the voltage-dependent inactivation and activation of I(Na,alpha beta) were significantly shifted in the depolarizing direction. In addition, coexpression with beta(1)-subunit prolonged the duration of recovery from inactivation. Eicosapentaenoic acid [EPA, C20:5(n-3)] significantly reduced I(Na,alpha beta) in a concentration-dependent manner and at 5 microM shifted the midpoint voltage of the steady-state inactivation by -22 +/- 1 mV. EPA also significantly accelerated channel transition from the resting state to the inactivated state and prolonged the recovery time from inactivation. Docosahexaenoic acid [C22:6(n-3)], alpha-linolenic acid [C18:3(n-3)], and conjugated linoleic acid [C18:2(n-6)] at 5 microM significantly inhibited both I(Na,alpha beta) and I(Na,alpha.) In contrast, saturated and monounsaturated fatty acids had no effects on I(Na,alpha beta). This finding differs from the results for I(Na,alpha), which was significantly inhibited by both saturated and unsaturated fatty acids. Our data demonstrate that functional association of beta(1)-subunit with hH1(alpha) modifies the kinetics and fatty acid block of the Na(+) channel.     

Energetics of potassium ion transport in Aplysia gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na(+)/K(+) transporter (Na(+)/K(+) pump or Na(+)/K (+) -ATPase). This Na(+)/K(+) pump accounts for both the intracellular Na(+) electrochemical potential (micro) being less than the extracelluar Na(+) micro and the intracellular K(+) micro being more than the extracellular K(+ ) micro. Also, K(+) channel activity resides in both luminal and basolateral membranes of the Aplysia foregut epithelial cells. Increased activity of the Na(+)/K(+) pump, coupled to luminal and basolateral membrane depolarization altered the K(+) transport energetics across the basolateral membrane to a greater extent than the alteration in K(+) transport energetics across the luminal membrane. These results suggest that K(+) transport, either into or out of the Aplysia foregut epithelial cells, is rate-limiting at the basolateral membrane.