Pump-leak sodium fluxes in low salt corn roots

Summary The influx and efflux of sodium from 4-hr washed, low salt corn roots (Zea mays L.) has been studied for characterization of passive and active components. Initial Na⁺ content of the roots is very low, 2.25±0.4 mol/g fresh weight. Na⁺ influx in the presence of 0.2mm Ca²⁺ and 0.002 to 20mm K⁺ is passive (a leak) based upon Goldman-type models, being determined by Na⁺ and cell potential (). Na⁺ was not transported by the K⁺ carrier and influx was unaffected by 50 m dicyclohexylcarbodiimide (DCCD). Permeability of the cells to Na⁺ was of the same order asP _k.Efflux of Na⁺ was by an efficient and rapid active transport system (a pump), thus accounting for the failure of these roots to accumulate high levels of Na⁺. In short-term loading and efflux experiments, internal Na⁺ turnover had a half-time of about 5 min. Sodium efflux was unaffected by DCCD. Net H⁺ flux was zero in the presence of DCCD regardless of sodium efflux, indicating absence of Na⁺/H⁺ antiport. Efflux of Na⁺ was equally rapid into medium containing no Na⁺ and only 0.002mm K⁺. K⁺ influx accounted for less than 4% of Na⁺ efflux, prompting the hypothesis that the Na⁺ (or cation?) efflux pump is the second electrogenic system previously defined based upon electrophysiological measurements.     

The role of ion antiporters in the maintenance of intracellular pH in rat vascular smooth muscle cells

Vascular smooth muscle intracellular pH is maintained by the Na⁺/H⁺ and Cl^–/HCO ₃ ^– antiporters. The Na⁺/H⁺ exchanger is a major route of H⁺ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H^– into the extracellular space in exchange for Na⁺. The Cl^–/HCO ₃ ^– exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na⁺/H⁺ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl^–/HCO ₃ ^– exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in sensing changes in intracellular pH, to which these antiporters respond.Abbreviations CaM Calmodulin - DCCD Dicylohexyl-Carbodiimide - DG Diacylglycerol - DIDS-4 4-Diisthiocyanostilbene-2,2-Disulfonic Acid - IP₃ Inositol Trisphosphate - PKC protein Kinase C - SITS-4 4-Acetamido-4-Isothiocyanstilbene-2,2-Disulfonate - VSMC Vascular Smooth Muscle Cell     

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     

Osmosensitivity of the 2H+−K+-exchange and the H+-ATPase complex F0F1 in anaerobically grownEscherichia coli

Escherichia coli grown anaerobically for osmotic studies upon increased osmolarity in alkaline medium carried out H⁺–K⁺-exchange in two steps, the first of which was DCCD¹ sensitive and osmo-dependent and had the 2H⁺/K⁺ stoichiometry. H⁺-efflux in the presence of protonophore (CCCP) upon increase of osmolarity was shown to be high and inhibited by DCCD, whereas H⁺-efflux induced by a decrease of osmolarity was small and not inhibited by DCCD. The 2H⁺/K⁺-exchange was absent intrkA anduncA mutants. InuncB mutant 2H⁺/K⁺-exchange was not DCCD-and osmosensitive. Competition between DCCD and osmoshock on inhibition of 2H⁺/K⁺-exchange was found. Osmosensitivity of this exchange disappeared in spheroplasts. Osmosensitivity of both 2H⁺/K⁺-exchange and the F₀F₁ and osmoregulation of the F₀F₁ via F₀ and a periplasmic space are postulated.Abbreviations F₀F₁ H⁺-ATPase complex - F₀ H⁺-channel, proteolipid - F₁ H⁺-ATPase - Trk constitutive system for K⁺ uptake - PV periplasmic protein valve - DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - _H or _K transmembrane electrochemical gradient for H⁺ or K⁺ respectively - membrane potential - upshock or downshock increase or decrease of medium osmolarity, respectively - CGSC E. coli Genetic Stock Center, Yale University, USA     

Coupling between H+ transport and anaerobic glycolysis in turtle urinary bladder: Effects of inhibitors of H+ ATPase

Summary The coupling between H⁺ transport (J _H) and anaerobic glycolysis was examinedin vitro in an anaerobic preparation of turtle urinary bladder.J _H was measured as the short-circuit current after Na⁺ transport was abolished with ouabain and by pH stat titration. The media were gassed with N₂ and 1% CO₂ (PO₂<0.5 mm Hg) and contained 10mm glucose. Under these conditions,J _H was not inhibited by 3mm serosal (S) cyanide or by 0.1mm mucosal (M) dinitrophenol. Control anerobic lactate production (J _lac) of 47 bladders was plotted as a function of simultaneously measuredJ _H. The slope ofJ _lac onJ _H was 0.58±0.12 with an intercept forJ _lac atJ _H=0 of 0.55 mol/hr. Values for J _lac/J _H were determined in groups of individual bladders whenJ _H was inhibited by an opposing pH gradient (0.55±0.16), by acetazolamide (0.58±0.19) and by dicyclohexylcarbodiimide, DCCD (0.58±0.14). The constancy of J _lac/J _H indicates a high degree of coupling betweenJ _H andJ _lac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the J _lac/J _H values can be used to estimate the stoichiometry of H⁺ translocation. The movement of slightly less than 2 H⁺ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited byM addition of oligomycin but was inhibited byM addition of DCCD and Dio-9, inhibitors of H⁺ flow in the proteolipid portion of H⁺-translocating ATPases. DCCD inhibited anaerobicJ _H without change in J _lac/J _H or basalJ _lac and, therefore, acted primarily on the H⁺ pump.S addition of vanadate also inhibitedJ _H, but the inhibition was associated with an increase inJ _lac. The site of this apparent uncoupling remains to be defined. The acidification pump of the luminal cell membrane of the turtle bladder has H⁺-ATPase characteristics that differ from mitochondrial ATPase in that H⁺ transport is oligomycin-resistant and vanadate-sensitive. As judged from the flows of H⁺ and lactate, the H⁺/ATP stoichiometry of the pump is about 2.     

Na+-transport modulation induces isoform-specific expression of Na+,K+-ATPase α-subunit isoforms in C2C12 skeletal muscle cell

Changes in demands for Na⁺ transport alter expression of the Na⁺,K⁺-ATPase subunit isoforms. In skeletal muscle, the effects of these changes on expression the 2 isoform, the major isoform expressed in differentiated muscle cell, is not known. Therefore, this study examines regulation of the -subunit isoforms by Na⁺ in the C₂C₁₂ skeletal muscle cell that expresses the 1 and 2 isoforms. Western blot analysis showed that in differentiating C₂C₁₂ muscle cell, but not in undifferentiated myoblast, veratridine, a Na⁺ channel activator, greatly increased expression of the 2 isoform; expression of 1 was unaltered. Because the level of -actinin was unaltered, the data suggest that veratridine treatment did not significantly alter the progression of cell differentiation. Furthermore, a reduction in Na⁺ transport by tetrodotoxin again failed to alter expression of a1. Thus, in C₂C₁₂ skeletal muscle cell, changes in Na⁺ transport alters expression of the 2, but not the 1 isoform. These results differ from those observed previously in muscle cells that express only the 1 isoform. Because mammalian skeletal muscle expresses both the 1- and 2-subunit isoforms, the differential regulation that was observed may be physiologically relevant in these muscle cells in vivo.     

Regulation of pH in the isolated perfused gills of the shore crabCarcinus maenas

Isolated posterior gills (no. 7) of shore crabsCarcinus maenas acclimated to brackish water of a salinity of 10 S were bathed and perfused with 50% sea water (200 mmol·l^-1 Na⁺), and the internal perfusate collected during subsequent periods of 5 min. During a single passage through the gill the pH of the perfusion medium decreased from ca. 8.1 to ca. 7.7, a result implying that the gill possesses structures which recognize unphysiologically high pH values in the haemolymph and regulates them down to physiological values of ca. 7.7. The calculated apparent proton fluxes from the epithelial cells into the haemolymph space amounted to 17.9 mol·g fw^-1·h^-1, a value of only 3.8% of net Na⁺ fluxes observed under comparable conditions. When 0.1 mmol·l^-1 KCN, an inhibitor of mitochondrial cytochrome oxidase, or 5 mmol·l^-1 ouabain, a specific inhibitor of Na⁺/K⁺-ATPase were applied in the internal perfusate, down-regulation of pH was no longer observed and the gill was completely depolarized, i.e. transepithelial potential differences dropped from-7.8 to 0 mV (haemolymph space negative to bath). Regulation of pH was completely inhibited by antagonists of carbonic anhydrase (0.1 mmol·l^-1 acetazolamide or 0.01 mmol·l^-1 ethoxyzolamide) applied in the perfusate. Inhibitors of Na⁺/H⁺ exchange, 0.1 mmol·l^-1 amiloride applied in the external bathing medium or in the internal perfusate, and symmetrical 0.01 mmol·l^-1 5-(N-ethyl-N-isopropyl)amiloride, as well as inhibitors of Cl^-/HCO₃ ^- exchange and Na⁺/HCO₃ ^- cotransport, 0.5 mmol·l^-1 4,4-diisothiocyanatostilbene-2,2-disulphonate or 0.3 mmol·l^-1 4-acetamido-4-isothiocyanatostilbene 2,2-disulphonate applied on both sides of the gill, and inhibitors of H⁺-ATPase, 0.05 mmol·l^-1 N-ethylmaleimide and 0.1 mmol·l^-1 N,N-dicyclohexylcarbodiimide —applied on both sides of the gill — did not alter the acidification of the perfusate observed in controls. Using artificial salines buffered to pH 8.1 with 0.75 mmol·l^-1 tris (hydroxymethyl) aminomethane instead of 2 mmol·l^-1 HCO₃ ^-, apparent proton fluxes were reduced to 11% of controls, a result suggesting that pH regulation by crab gills needs the presence of HCO₃ ^-. The findings obtained suggest that pH regulation by crab gills depends on the oxidative metabolism of the intact branchial epithelium and that carbonic anhydrase plays a central role in this process. Na⁺/H⁺ exchange, anion exchange or cotransport and active proton secretion seem not to be involved. While unimpaired active ion uptake is a prerequisite for pH regulation, ion transport itself is independent of it.Abbreviations acetazolamide (N-[sulphamoyl-1, 3, 4-thiadiazol-2-yl]-acetamide) - amiloride 3,5-diamino-6-chloropyrazinoyl-guanidine - CA carbonic anhydrase - DBI dextrane-bound inhibitor thiadiazolesulphonamide - DCCD N N dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanato-stilbene-2,2-disulphonate - EIPA 5-(N-ethyl-N-isopropyl) amiloride - ethoxyzolamide 6-ethoxy-2-benzothiazole-sulphonamide - fw fresh weight - J _H ⁺ apparent proton flux - NEM N-ethylmaleimide - PD transepithelial potential difference - PEG-STZ polyethylene-glycol-thiadiazolesulphonamide - STTS 4-acetamido-4-isothiocyanatostibene 2,2-disulphonate - SW sea water - TRIS tris(hydroxymethyl)aminomethane     

Structure and function of H+-ATPase

(1) Extensive studies on proton-translocating ATPase (H⁺-ATPase) revealed that H⁺-ATPase is an energy transforming device universally distributed in membranes of almost all kinds of cells. (2) Crystallization of the catalytic portion (F₁) of H⁺-ATPase showed that F₁ is a hexagonal molecule with a central hole. The diameter of F₁ is about 90 Å and its molecular weight is about 380,000. (3) Use of thermophilic F₁ permits the complete reconstitution of F₁ from its five subunits (, , , , and ) and demonstration of the gate function of the -complex, the catalytic function of (supported by and ), and the H⁺-translocating functions of all five subunits. (4) Studies using purified thermostable F₀ showed that F₀ is an H⁺-channel portion of H⁺-ATPase. The direct measurement of H⁺-flux through F₀, sequencing of DCCD-binding protein, and isolation of F₁-binding protein are described. (5) The subunit stoichiometry of F₁ may be ₃₃. (6) Reconstitution of stable H⁺-ATPase-liposomes revealed that ATP is directly synthesized by the flow of H⁺ driven by an electrochemical potential gradient and that H⁺ is translocated by ATP hydrolysis. This rules out functions for all the hypothetical components that do not belong to H⁺-ATPase in H⁺-driven ATP synthesis. The roles of conformation change and other phenomena in ATP synthesis are also discussed.     

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     

Characterization of a Na∶K∶2Cl cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1)

Summary ⁸⁶Rb uptake into LLC-PK₁ cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na⁺ and Cl^– for activity, and a leak component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of⁸⁶Rb,²²Na and³⁶Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na⁺K⁺Cl^– cotransport system. The kinetic parameters for Na⁺ and K⁺ interaction have been analyzed under initial linear zerotrans conditions. The following values were obtained:K _mNa+=0.42±0.05 mmol/liter,V _max=303±24 pmol/mg/6 sec;K _mK+=11.9±1.0 mmol/liter,V _maxK+=307±27 pmol/mg/6 sec. For Cl^– interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na⁺1K⁺2Cl^– can be calculated. It is concluded that the apical membrane of LLC-PK₁ cells contains a Na⁺K⁺2Cl^– cotransport system with properties similar to those described for the thick ascending limb of the loop of Henle.     

Osmoregulation and the control of phloem-sap composition in Ricinus communis L.

Phloem-sap composition was studied in plants of Ricinus communis L. grown on a waterculture medium. The sap possessed a relatively high K⁺:Na⁺ ratio and low levels of Ca²⁺ and free H⁺. Sucrose and K⁺ (together with its associated anions) accounted for 75% of the phloem-sap solute potential (_s). In plants kept in continuous darkness, a decrease in phloem-sap sucrose levels over 24h was accompanied by an increase in K⁺ levels. Measurements of phloem-sap _s and xylem water potential () indicated that this resulted in a partial maintenance of phloem turgor pressure _p. In darkness there was also a marked decrease in the malate content of the leaf tissue, and it is possible that organic carbon from this source was mobilized for export in the phloem. The results support the concept of the phloem sap as a symplastic phase. We interpret the increase in K⁺ levels in the phloem in darkness as an osmoregulatory response to conditions of restricted solute availability. This reponse can be explained on the basis of the sucrose-H⁺ co-transport mechanism of phloem loading.Abbreviations water potential - _s solute potential - _p pressure potential     

Membrane transport ofp-nitrophenyl-α-galactoside by the melibiose carrier ofEscherichia coli

Summary p-Nitrophenyl--galactoside (-pNPG) was found to be a substrate for the melibiose transport system ofEscherichia coli. This sugar enters induced cells via the carrier and is split by -galactosidase to galactose andp-nitrophenol. In mutant cells lacking the -galactosidase [³H]--pNPG accumulated to concentrations 15 times higher than the external medium. The transport of -pNPG is inhibited by both Na⁺ and Li⁺. Na⁺ (10mm) reduced the K_m for -pNPG from 0.45 to 0.18mm and reduced theV _max from 6.7 nmoles/min/mg dry wt to a value of 3.0.     

H2: heterodisulfide oxidoreductase,a second energy-conserving system in the methanogenic strain Gö1

Washed everted vesicles of the methanogenic bacterium strain Gö1 catalyzed an H₂-dependent reduction of the heterodisulfide of HS-CoM (2-mercaptoethanesulfonate) and HS-HTP (7-mercaptoheptanoylthreonine phosphate) (CoM-S-S-HTP). This process was independent of coenzyme F₄₂₀ and was coupled to proton translocation across the cytoplasmic membrane into the lumen of the everted vesicles. The maximal H⁺/CoM-S-S-HTP ratio was 2. The tranmembrane electrochemical gradient thereby generated was shown to induce ATP synthesis from ADP+P_i, exhibiting a stoichiometry of 1 ATP synthesized per 2 CoM-S-S-HTP reduced (H⁺/ATP=4). ATP formation was inhibited by the uncoupler 3,5-di-tert-butyl-4-hydroxy-benzylidene-malononitrile (SF 6847) and by the ATP synthase inhibitor N,N-dicyclohexylcarbodiimide (DCCD). This energy-conserving system showed a stringent coupling. The addition of HS-CoM and HS-HTP at 1 mM each decreased the heterodisulfide reductase activity to 50% of the control. Membranes from Methanolobus tindarius showed F₄₂₀H₂-dependent but no H₂-dependent heterodisulfide oxidoreductase activity. Neither of these activities was detectable in membranes of Methanococcus thermolithotrophicus.Abbreviations _H+ transmembrane electrochemical gradient of H⁺ - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - DCCD N,N-dicyclohexylcarbodiimide - SF 6847 3,5-di-ert-butyl-4-hydroxybenzylidenemalononitrile - Mb. Methanobacterium - Ml. Methanolobus - Mc. Methanococcus - MV methylviologen - BV benzylviologen - MTZ metronidazole     

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     

Na+-translocating NADH-quinone reductase of marine and halophilic bacteria

The respiratory chain of marine and moderately halophilic bacteria requires Na⁺ for maximum activity, and the site of Na⁺-dependent activation is located in the NADH-quinone reductase segment. The Na⁺-dependent NADH-quinone reductase purified from marine bacteriumVibrio alginolyticus is composed of three subunits, , , and , with apparentM _r of 52, 46, and 32kDa, respectively. The FAD-containing -subunit reacts with NADH and reduces ubiquinone-1 (Q-1) by a one-electron transfer pathway to produce ubisemiquinones. In the presence of the FMN-containing -subunit and the -subunit, Q-1 is converted to ubiquinol-1 without the accumulation of free radicals. The reaction catalyzed by the -subunit is strictly dependent on Na⁺ and is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), which is tightly coupled to the electrogenic extrusion of Na⁺. A similar type of Na⁺-translocating NADH-quinone reductase is widely distributed among marine and moderately halophilic bacteria. The respiratory chain ofV. alginolyticus contains another NADH-quinone reductase which is Na⁺ independent and has no energy-transducing capacity. These two types of NADH-quinone reductase are quite different with respect to their mode of quinone reduction and their sensitivity toward NADH preincubation.     

Studies on the kinetics of Na+/H+ exchange in OK cells: Introduction of a new device for the analysis of polarized transport in cultured epithelia

Summary The present study describes a new perfusion technique—based on the use of a routine spectrofluorometer—which enables fluorometric evaluation of polarity, regulation and kinetics of Na⁺/H⁺ exchange at the level of an intact monolayer. Na⁺/ H⁺ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na⁺/H⁺ exchange activity was measured by monitoring changes in intracellular pH (pH _i ) after an acid load, using the pH-sensitive dye 27-bis (carboxyethyl) 5–6-carboxy-fluorescein (BCECF). Initial experiments indicated that OK cells grown on a permeable support had access to apical and basolateral perfusion media. They also demonstrate that OK cells express an apical pH _i , recovery mechanism, which is Na⁺ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pH _i =7.68; pH _o =7.4) where Na⁺/H⁺ exchange is not detectable, transport rate increased as pH _i decreased. A positive cooperativity characterized the interaction of internal H⁺ with the exchanger, and suggests multiple H⁺ binding sites. In contrast, extracellular [Na⁺] increased transport with simple Michaelis-Menten kinetics. The apparent affinity of the exchanger for Na⁺ was 19mM at an intracellular pH of 7.1 and 60mM at an intracellular pH of 6.6. Inhibition of Na⁺/H⁺ exchange activity by EIPA was competitive with respect to extracellular [Na⁺] and theK _i was 3.4 M. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pH _i and processes associated with their polarized expression and regulation.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

The membrane potential in whole cells of Methanobacterium thermoautotrophicum

of whole cells of Methanobacterium thermoautotrophicum was estimated under varying conditions using an electrode sensitive to the lipophilic cation tetraphenylphosphonium chloride (TPP⁺). Since was found to be extremely sensitive to air, a special reaction vessel was developed to maintain strict anaerobiosis. The cells took up TPP⁺ under energization by H₂ and CO₂ thus allowing to calculate the from the distribution of TPP⁺ inside and outside the cells. The unspecific uptake of deenergized cells was around 10% of the total uptake of energized cells. TPP⁺ itself slightly diminished the , but had no effect on the formation of methane. Typical values of were in the range of-150 to-200 mV. showed a quantitative dependence on both the electron donor H₂ and the electron acceptor CO₂. NaCl stimulated the extent of the , whereas KCl slightly diminished it. Valinomycin resulted in a linear decline of , whereas the methane production rate was only slightly affected. In contrast, monensin reduced both methanogenesis and .Abbreviations pmf proton motive force - membrane potential - TPP⁺ tetraphenylphosphonium (chloride salt) - TPMP⁺ triphenylmethylphosphonium (chloride salt, if not otherwise indicated) - d.w. dry weight - t _d doubling time - PVC polyvinylchloride     

Active transport in phototrophic bacteria

Phototrophic bacteria utilize light-driven, cyclic electron flow to pump protons out of their cytoplasm, creating an electrochemical proton gradient, _H+, outside acid and positive. These bacteria exchange external protons for internal cations (Na⁺, K⁺ and Ca⁺²), allowing the cells to maintain a nearly constant internal pH while maintaining the electrical component of _H+. Na⁺/H⁺ exchange also establishes an electrochemical Na⁺ gradient. Phototrophic bacteria are able to utilize these electrochemical gradients as energy sources for the uptake of a wide variety of metabolites (e.g., sugars, organic acids and amino acids) via metabolite/cation symports.     

Monovalent Cations Contribute to T-type Calcium Channel (Ca<Subscript>V</Subscript>3.1 and Ca<Subscript>V</Subscript>3.2) Selectivity

Low voltage-activated (LVA) Ca²⁺ channels regulate chemical signaling by their ability to select for Ca²⁺. Whereas Ca²⁺ is the main permeating species through Ca²⁺ channels, Ca²⁺ permeation may be modified by abundant intra- and extracellular monovalent cations. Therefore, we explored monovalent cation regulation of LVA Ca²⁺ permeation in the cloned T-type Ca²⁺ channels 1G (Ca_V3.1) and 1H (Ca_V3.2). In physiological [Ca²⁺], the reversal potential in symmetrical Li⁺ was 19 mV in 1G and 18 mV in 1H, in symmetrical Cs⁺ the reversal potential was 36 mV in 1G and 37 mV in 1H, and in the bi-ionic condition with Li⁺ in the bath and Cs⁺ in the pipette, the reversal potential was 46 mV in both 1G and 1H. When Cs⁺ was used in the pipette, replacement of external Cs⁺ with Li⁺ (or Na⁺) shifted the reversal potential positive by 5–6 mV and increased the net inward current in 1G. Taken together the data indicate that in physiological [Ca²⁺], external Li⁺ (or Na⁺) permeates more readily than external Cs⁺, resulting in a positive shift of the reversal potential. We conclude that external monovalent cations dictate T-type Ca²⁺ channel selectivity by permeating through the channel. Similar to Li⁺, we previously reported that external [H⁺] can regulate T-type Ca²⁺ channel selectivity. 1Hs selectivity was more sensitive to external pH changes compared to 1G. When Cs⁺ was used in the pipette and Li⁺ was used in the bath external acidification from pH_o 7.4 to 6.0 caused a negative shift of the reversal by 8 mV in 1H. Replacement of internal Cs⁺ with Li⁺ reduced the pH-induced shift of the reversal potential to 2 mV. We conclude that, similar to other external monovalent cations, H⁺ can modify T-type Ca²⁺ channel selectivity. However, in contrast to external monovalent ions that readily permeate, H⁺ regulate T-type Ca²⁺ channel selectivity by increasing the relative permeability of the internal monovalent cation. Present address: B.P. Delisle, Department of Medicine, The University of Wisconsin, Madison, WI 53706, USA