Alternating Hemiplegia of Childhood mutations have a differential effect on Na+,K+-ATPase activity and ouabain binding

De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na⁺,K⁺-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na⁺,K⁺-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K⁺/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na⁺,K⁺-ATPase. Functional impairment of Na⁺,K⁺-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.     

Na+, K+-ATPase Isozyme Diversity; Comparative Biochemistry and Physiological Implications of Novel Functional Interactions

Na⁺, K⁺-ATPase is ubiquitously expressed in the plasma membrane ofall animal cells where it serves as the principal regulator of intracellularion homeostasis. Na⁺, K⁺-ATPase is responsible for generating andmaintaining transmembrane ionic gradients that are of vital importance forcellular function and subservient activities such as volume regulation, pHmaintenance, and generation of action potentials and secondary activetransport. The diversity of Na⁺, K⁺-ATPase subunit isoforms andtheir complex spatial and temporal patterns of cellular expression suggestthat Na⁺, K⁺-ATPase isozymes perform specialized physiologicalfunctions. Recent studies have shown that the subunit isoformspossess considerably different kinetic properties and modes of regulationand the subunit isoforms modulate the activity, expression and plasmamembrane targeting of Na⁺, K⁺-ATPase isozymes. This review focuseson recent developments in Na⁺, K⁺-ATPase research, and in particular reportsof expression of isoforms in various tissues and experiments aimed atelucidating the intrinsic structural features of isoforms important forNa⁺, K⁺-ATPase function.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

A study on Na+-coupled oxidative phosphorylation: ATP formation supported by artificially imposed ΔpNa and ΔpK inVibrio alginolyticus cells

Addition of Na⁺ to the K⁺-loadedVibrio alginolyticus cells, creating a 250-fold Na⁺ gradient, is shown to induce a transient increase in the intracellular ATP concentration, which is abolished by the Na⁺/H⁺ antiporter, monensin. The pNa-supported ATP synthesis requires an additional driving force supplied by endogenous respiration or, alternatively, by a K⁺ gradient (high [K⁺] inside). In the former case, ATP formation is resistant to the protonophorous uncoupler. Dicyclohexylcarbodiimide and diethylstilbestrol, but not vanadate, completely inhibit Na⁺ pulse-induced ATP formation. The data agree with the assumption that Na⁺-ATP-synthase is involved in oxidative phosphorylation inV. alginolyticus. Interrelation of H⁺ and Na⁺ cycles in bacteria is discussed.Abbreviations and electrochemical gradients of H⁺ and Na⁺, respectively - transmembrane electric potential difference - pH, pNa, and pK concentration gradients of H⁺, Na⁺, and K⁺, respectively - CCCP carbonyl cyanidem-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diesthylstilbestrol - HQNO 2-heptyl-4-hydroxyquinolineN-oxide - Tricine N[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Open-channel block of Na+ channels by intracellular Mg2+

1. Macroscopic and single-channel currents through several types of cloned rat brain Na⁺ channels, expressed in Xenopus oocytes, were measured using the patch-clamp technique. 2. For all cloned channel types and for endogenous Na⁺ channels in chromaffin cells, intracellular Mg²⁺ blocks outward currents in a voltage-dependent manner similar to that in rat brain type II Na⁺ channel (Pusch et al. 1989). 3. A sodium-channel mutant (cZ-2) with long single-channel open times was used to examine the voltage-dependent reduction of single-channel outward current amplitudes by intracellular Mg²⁺. This reduction could be described by a simple blocking mechanism with half-maximal blockage at 0 mV in 1.8 mM intracellular Mg²⁺ and a voltage-dependence of e-fold per 39 mV (in 125 mM [Na] _i ); this corresponds to a binding-site at an electrical distance of 0.32 from the inside of the membrane. 4. At low Mg²⁺ concentrations and high voltages, the open-channel current variance is significantly elevated with respect to zero [Mg] _i . This indicates that Mg²⁺ acts as a fast blocker rather than gradually decreasing current, e.g. by screening of surface charges. Analysis of the open-channel variance yielded estimates of the block and unblock rate constants, which are of the order of 2 · 10⁸ M^–1 s^–1 and 3.6 · 10⁵ s^–1 at 0 mV for the mutant cZ-2. 5. A quantitative analysis of tail-currents of wild-type 11 channels showed that the apparent affinity for intracellular Mg²⁺ strongly depends on [Na] _i . This effect could be explained in terms of a multi-ion pore model. 6. Simulated action potentials, calculated on the basis of the Hodgkin-Huxley theory, are significantly reduced in their amplitude and delayed in their onset by postulating Mg²⁺ block at physiological levels of [Mg] _i .abbreviations [Na]_i intracellular Na⁺ concentration - [K] _i intracellular K⁺ concentration - [Mg] _i intracellular Mg²⁺ concentration - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis-[\-amino-ethyl ether] N,N-tetra acetic acid - TEA tetraethylammonium     

A stable Na+/H+ antiporter of thermophilic bacterium PS3

As a first step in the isolation of a stable Na⁺/H⁺ antiporter, its reaction in sonicated membrane vesicles of thermophilic bacterium PS3 has been characterized. The sonicated vesicles showed quenching of quinacrine fluorescence in either NADH oxidation or ATP hydrolysis. The quenching was reversed by the addition of Na⁺, Li⁺, Mn²⁺, Cd²⁺, and Co²⁺, but not of choline⁺ or Ca²⁺, regardless of their counter anions.²²Na⁺ was taken up into the vesicles by NADH oxidation, and the²²Na⁺ uptake was inhibited by the addition of an uncoupler. H⁺ release was observed on addition of Na⁺ to sonicated vesicles. The magnitude of the pH difference across the membrane induced by NADH oxidation was constant at pH 7.0 to 9.1, but the Na⁺/H⁺ antiport was affected by the pH of the medium (optimum pH=8.5). TheK _m 's of the antiporter for Na⁺ and Li⁺ were 2.5 and 0.1 mM, respectively, but theV _max values for the two ions were the same at pH 8.0. In the presence of Li⁺, no further decrease of fluorescence quenching was observed on addition of Na⁺ andvice versa. The Na⁺/H⁺ antiporter activity in PS3 was stable at 70°C, and the optimum temperature for activity was 55–60°C. In contrast to mesophilic cation/H⁺ antiporters, this antiporter was not inhibited by a thiol reagent.Abbreviations Tricine N-tris(hydroxymethyl)methylglycine - MOPS morpholinopropane sulfonic acid - TMAHO tetramethylammonium hydroxide - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - H⁺ — ATPase proton-translocating adenosine triphosphatase - electrochemical proton gradient across membrane - electrochemical Na⁺ gradient across membrane - pH pH difference across membrane     

Cloning,sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis

Na⁺/H⁺ antiporter activity is wide-spread and plays essential physiological roles. We found that several Enterobacteriaceae share conserved sequences with nhaA, the gene coding for an E. coli antiporter. A nhaA strain which is sensitive to Na⁺ and Li⁺, was used to clone by complementation a DNA fragment from Salmonella enteritidis which confers resistance to the ions. The cloned fragment increased Na⁺/H⁺ antiport activity in membranes isolated from strains carrying the respective hybrid plasmid. DNA sequence analysis of the insert revealed two open reading frames. Both encode putative polypeptides which are closely homologous to the nhaA and nhaR gene products from Escherichia coli. The antiporter activity displays properties very similar to that of the E. coli NhaA, namely, it is activiated by alkaline pH and recognizes Li⁺ with high affinity.Abbreviations _H ⁺ Proton electrochemical potential - pH transmembrane pH gradient - _Na ⁺ Sodium electrochemical potential - SDS Sodium dodecyl sulfate - CIP Calf intestine alkaline phosphates - ORF open reading frame     

Sodium Blocking Induced by a Point Mutation at the C-Terminal End of the Pore Helix of the KAT1 Channel

A plant hyperpolarization-activating K⁺ channel, KAT1, is highly selective for K⁺ over Na⁺ and is little affected by external Na⁺, which is crucial to take up K⁺ effectively in a Na⁺-containing environment. It has been shown that a mutation at the location (Thr256) preceding the selectivity signature sequence dramatically enhanced the sensitivity of the KAT1 channel to external Na⁺. We report here electrophysiological experiments for the mechanism of action of external Na⁺ on KAT1 channels. The Thr256 residue was substituted with either glutamine (Q) or glutamate (E). The wild-type channel was insensitive to external Na⁺. However, the activity of both mutant channels was significantly depressed by Na⁺ with apparent dissociation constants of 6.7 mm and 11.3 mm for T256Q and T256E, respectively. The instantaneous current-voltage relationships revealed distinct blocking mechanisms for these mutants. For T256Q a typical voltage-dependent fast blocking was shown. On the other hand, the blocking for the T256E mutant was voltage-independent at low Na⁺ concentrations and became voltage-dependent at higher concentrations. At extreme hyperpolarization the blocking was relieved significantly. These data strongly suggest that the mutation at the end of the pore helix rearranged the selectivity filter and allows Na⁺ to penetrate into the pore. Received: 16 October 2000/Revised: 20 February 2001     

Divalent cations as probes for structure-function relationships of cloned voltage-dependent sodium channels

1. Several cloned sodium channels were expressed in oocytes and compared with respect to their sensitivity to internal Mg²⁺ concerning the open-channel block and to external Ca²⁺ concerning open-channel block and shifts in steady-state activation. 2. A quantitative comparison between wild-type II channels and a mutant with a positive charge in the S4 segment of repeat I neutralized (K226Q) revealed no significant differences in the Mg²⁺ block. 3. The blocking effect of extracellular Ca²⁺ ions on single-channel inward currents was studied for type II, mutant K226Q and type III. A quantitative comparison showed that all three channel types differ significantly in their Ca⁺ sensitivity. 4. The influence of extracellular Ca²⁺ on the voltage dependence of steady-state activation of macroscopic currents was compared for type II and K226Q channels. Extracellular Ca⁺ increases the voltage of half-maximal activation, V_1/2, more for K226Q than for wild-type II channels; a plot of V _1/2 against [Ca] _o , is twice as steep for the mutant K226Q as for the wild-type on a logarithmic concentration scale. 5. The differential effects of extracellular Ca⁺ and intracellular Mg²⁺ on wild-type II and K226Q channels are discussed in terms of structural models of the Na⁺ channel protein.Abbreviations [Na] _i intracellular Na⁺ concentration - [Mg] intracellular Mg²⁺ concentration - [Ca] _o extracellular Ca²⁺ concentration     

Different modes of electrogenic Na+ absorption in the coprodeum of the chicken embryo: role of extracellular Ca2+

Summary Transepithelial electrogenic Na⁺ transport (I_Na) was investigated in the coprodeum of 20-days-old chicken embryos in Ussing chambers. Short circuit current (I_sc) and transepithelial resistance (R_t) were 14.7±4.8 A · cm^-2 (n=12) and 0.53±0.09 k · cm^-2 (n=12), respectively. I_Na was calculated from changes in I_sc by substitution of mucosal Na⁺ by (N-methyl-d-glucamine) (NMDG). I_sc inversed during Na⁺ removal, and I_Na was found to be 27.8±4.7 A · cm^-2 (n=12). Amiloride (100 mol · l^-1) inhibited only about 60% of I_Na. Analysis of I_sc fluctuations revealed a Lorentzian component in the power density spectrum with a corner frequency of about 57 Hz. This component was not correlated to I_Na, and its origin is still unclear. Removal of mucosal Ca²⁺ increased I_Na about 2.5-fold due to an increase of the amiloride-insensitive component of I_Na in additionally investigated adult tissues. The results clearly show that this is due to a non-selective cation channel with an apparent order of selectivity Cs⁺>Na⁺=K⁺>Rb⁺>Li⁺. The Ca²⁺ concentration required to block 50% of the I_sc was about 18 mol · l^-1. The I _sc ^Ca could also be supressed by other divalent cations such as Mg²⁺ and Ba²⁺. Additionally, an I_Na-linked Lorentzian component occurred which dominated the control spectrum with a significantly higher corner frequency (about 88 Hz). The results indicate that Na⁺ absorption in the coprodeum of the chicken embryo is more complex than in adult hens. However, the Ca²⁺ sensitivity of I_Na is similar to comparable effects described for other epithelia. This possibly reflects the existence of two types of amiloride-insensitive apical cation channels as pathways for Na⁺ absorption, which may be involved to differing degrees in ontogenetic developments of nonselective channels to Na⁺-specific ion channels.Abbreviations DPL direct-linear-plot method - slope of the back-ground noise component - EGTA ethylene glycol-bi(2-amino-ethylether)-N,N,N,N-tetraacetic acid - f frequency - f _c corner frequency of the Lorentzian noise component - G _t transepithelial conductance - HEPES N-hydroxyethylpiperazine-N-ethanesulfonic acid - I _sc short-circuit current - I _Na transepithelial sodium current - I _sc ^Ca Ca²⁺-sensitive short-circuit current - K _m ^Ca Michaelis-Menten constant for Ca²⁺ - K _B power density of the background noise component at f=1Hz - m mucosal - NMDG N-methyl-D-glucamine - R _t transepithelial resistance - s serosal - SEM standard error of mean - S(f) power density of the Lorentzian noise component - S _o plateau value of the Lorentzian noise component     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Identification and Characterization of the Na+/H+ Antiporter Nhas3 from the Thylakoid Membrane of Synechocystis sp. PCC 6803

Na⁺/H⁺ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na⁺/H⁺ antiporters, nhaS1–5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na⁺/H⁺ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na⁺ concentrations. NhaS3 had no K⁺/H⁺ exchange activity itself but enhanced K⁺ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO₂ to high CO₂ conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO₂ consumption caused by the ceasing of O₂-evolving photosynthesis. This is the first report of a Na⁺/H⁺ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H⁺, Na⁺, and K⁺ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.Na⁺/H⁺ antiporters are integral membrane proteins that transport Na⁺ and H⁺ in opposite directions across the membrane and that occur in virtually all cell types. These transporters play an important role in the regulation of cytosolic pH and Na⁺ concentrations and influence proton or sodium motive force across the membrane (1, 2). In Escherichia coli, three Na⁺/H⁺ antiporters (NhaA, NhaB, and ChaA) have been described in detail. Of these, NhaA is the functionally best characterized transporter. The crystal structure of NhaA has been resolved (3). In addition, mutants of nhaA, nhaB, and chaA as well as the triple mutant have been generated (4). The triple mutant was shown to be hypersensitive to extracellular Na⁺. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains six genes encoding Na⁺/H⁺ antiporters (NhaS1–5 and sll0556). NhaS1 (slr1727) has also been designated SynNhaP (5, 6). Null mutants of nhaS1, nhaS2, nhaS4, and nhaS5 have been generated; however, a null mutant of nhaS3 could not be obtained, indicating that it is an essential gene (6–8). By heterologous expression in E. coli, Na⁺/H⁺ exchange activities could be shown for NhaS1–5 (5, 6). Inactivation of nhaS1 and nhaS2 results in retardation of growth of Synechocystis (5, 6). It has been reported that in these mutants the concentration of Na⁺ in cytosol and intrathylakoid space (lumen) increases and impairs the photosynthetic and/or respiratory activity of the cell (9, 10). Therefore the Na⁺ extrusion by Synechocystis Na⁺/H⁺ antiporters similar to E. coli NhaA, NhaB, and ChaA is essential for the adaptation to salinity stress.In contrast to the case in E. coli, Na⁺ is an essential element for the growth of some cyanobacteria (11, 12). Interestingly, the Na⁺/H⁺ antiporter homolog NhaS4 was identified as an uptake system for Na⁺ from the medium during a screen for mutations in Synechocystis that result in lack of growth at low Na⁺ concentrations (7). The requirement of a Na⁺ uptake antiporter for cell growth is consistent with the physiology of Synechocystis. Specifically, photoautotrophic bacteria like cyanobacteria share some components (plastoquinone, cytochrome b₆f, and c₆) of the thylakoid membrane for electron transport for both photophosphorylation and respiratory oxidative phosphorylation. Na⁺/H⁺ antiporters therefore may coordinate both H⁺ and Na⁺ gradients across the plasma and thylakoid membranes to adapt to daily environmental changes (11). It remains to be determined whether the six Na⁺/H⁺ antiporters are localized to the plasma membrane or to the thylakoid membrane in Synechocystis. Information on the membrane localization will also provide information on the physiological role in Synechocystis. In this study, we explored the membrane localization of NhaS3, the role of specific amino acid residues for its function, and the effect of CO₂ concentration and circadian rhythms on the expression pattern of nhaS3 to gain insight into the physiological role of NhaS3 in Synechocystis.     

Different Ionic Specificities of ATP Synthesis in Extremely Alkaliphilic Sulfate-Reducing and Acetogenic Bacteria

Ionic specificity of oxidative phosphorylation was studied in Natroniella acetigenaand Desulfonatronum lacustre, which are new alkaliphilic anaerobes that were isolated from soda lakes and have a pH growth optimum of 9.5–9.7. The ability of their cells to synthesize ATP in response to the imposition of artificial pH⁺and pNa⁺gradients was studied. As distinct from other marine and freshwater sulfate reducers and extremely alkaliphilic anaerobes, D. lacustreuses a Na⁺-translocating ATPase for ATP synthesis. The alkaliphilic acetogen N. acetigena, which develops at a much higher Na⁺concentration in the medium, generated primary pH⁺for ATP synthesis. Thus, the high Na⁺concentrations and alkaline pH values typical of soda lakes do not predetermine the type of bioenergetics of their inhabitants.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.     

Amiloride-sensitive Na+/H+ exchange stimulated by cellular acidification or shrinkage in red blood cells of the frog,Rana temporaria

Simultaneous net uptake of Na⁺ and net extrusion of H⁺, both inhibited by amiloride, could be stimulated in red blood cells of the frog, Rana temporaria, either by intracellular acidification or cellular shrinkage. Net transports of Na⁺ and H⁺ were transient, dying out after 10–20 min (20°C) when stimulated by intracellular acidification but developing more slowly and proceeding for more than 60 min (20°C) when stimulated by cellular shrinkage. Evidence is presented suggesting a coupling between the transports of Na⁺ and H⁺ with an exchange ratio of 1:1 Na⁺/H⁺ exchange, stimulated by intracellular acidification, was able to readjust intracellular pH also when operating in parallel to a fully working anion exchanger in CO₂/HCO ₃ ^- -buffered media. Inhibition of anion exchange resulted in reduced cellular net uptake of Na⁺.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonate - DMSO dimethylsulphoxide - IU international unit - pH _e extracellular pH - pH _i intracellular pH - RBC red blood cell     

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels

The members of the RCK family of cloned voltage-dependent K⁺ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K⁺ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg²⁺ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K⁺ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg²⁺ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K⁺ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K⁺ and thereby indirectly affects internal Mg²⁺ block and channel closing.Abbreviations TEA tetraethylammonium - EGTA Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - 2S3B model 2-site 3-barrier model Correspondence to: S. H. Heinemann     

Sodium channels contribute to action potential generation in canine and human pancreatic islet B cells

Summary Pancreatic islet B cells depolarize and display trains of action potentials in response to stimulatory concentrations of glucose. Based on data from rodent islets these action potentials are considered to be predominantly Ca²⁺ dependent. Here we describe Na⁺-dependent action potentials and Na⁺ currents recorded from canine and human pancreatic islet B cells. Current-clamp recording using the nystatin perforated-patch technique demonstrates that B cells from both species display tetrodotoxin-sensitive Na⁺ action potentials in response to modest glucose-induced depolarization. In companion whole-cell voltage-clamp experiments on canine B cells, the underlying Na⁺ current displays steep voltage-dependent activation and inactivation over the range of –50 to –40 mV. The Na⁺ current is sensitive to tetrodotoxin block with aK ₁=3.2nm and has a reversal potential which changes with [Na⁺] _o as predicted by the Nernst equation. These results suggest that a voltage-dependent Na⁺ current may contribute significantly to action potential generation in some species outside the rodent family.     

Bacterial sodium ion-coupled energetics

For many bacteria Na⁺ bioenergetics is important as a link between exergonic and endergonic reactions in the membrane. This article focusses on two primary Na⁺ pumps in bacteria, the Na⁺-translocating oxaloacetate decarboxylase ofKlebsiella pneumoniae and the Na⁺-translocating F₁F₀ ATPase ofPropionigenium modestum. Oxaloacetate decarboxylase is an essential enzyme of the citrate fermentation pathway and has the additional function to conserve the free energy of decarboxylation by conversion into a Na⁺ gradient. Oxaloacetate decarboxylase is composed of three different subunits and the related methylmalonyl-CoA decarboxylase consists of five different subunits. The genes encoding these enzymes have been cloned and sequenced. Remarkable are large areas of complete sequence identity in the integral membrane-bound -subunits including two conserved aspartates that may be important for Na⁺ translocation. The coupling ratio of the decarboxylase Na⁺ pumps depended on and decreased from two to zero Na⁺ uptake per decarboxylation event as increased from zero to the steady state level.InP. modestum, is generated in the course of succinate fermentation to propionate and CO₂. This is used by a unique Na⁺-translocating F₁F₀ ATPase for ATP synthesis. The enzyme is related to H⁺-translocating F₁F₀ ATPases. The F₀ part is entirely responsible for the coupling of ion specificity. A hybrid ATPase formed by in vivo complementation of anEscherichia coli deletion mutant was completely functional as a Na⁺-ATP synthase conferring theE. coli strain the ability of Na⁺-dependent growth on succinate. The hybrid consisted of subunits a, c, b, and part of fromP. modestum and of the remaining subunits fromE. coli. Studies on Na⁺ translocation through the F₀ part of theP. modestum ATPase revealed typical transporter-like properties. Sodium ions specifically protected the ATPase from the modification of glutamate-65 in subunit c by dicyclohexylcarbodiimide in a pH-dependent manner indicating that the Na⁺ binding site is at this highly conserved acidic amino acid residue of subunit c within the middle of the membrane.     

Sodium-transport NADH-quinone reductase of a marineVibrio alginolyticus

The respiratory chain of a marine bacterium,Vibrio alginolyticus, required Na⁺ for maximum activity, and the site of Na⁺-dependent activation was localized on the NADH-quinone reductase segment. The Na⁺-dependent NADH-quinone reductase extruded Na⁺ as a direct result of redox reaction. It was composed of three subunits, , , and , with apparentMr of 52, 46, and 32 KDa, respectively. The reduction of ubiquinone-1 to ubiquinol proceeded via ubisemiquinone radicals. The former reaction was catalyzed by the FAD-containing subunit. This reaction showed no specific requirement for Na⁺. For the formation of ubiquinol, the presence of the subunit and the FMN-containing subunit was essential. The latter reaction specifically required Na⁺ for activity and was strongly inhibited by 2-n-heptyl-4-hydroxyquinolineN-oxide. It was assigned to the coupling site for Na⁺ transport. The mode of energy coupling of redox-driven Na⁺ pump was compared with those of decarboxylase- and ATP-driven Na⁺ pumps found in other bacteria.