An Analysis of the Leakages of Sodium Ions into and Potassium Ions out of Striated Muscle Cells

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na]_i, and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na]_i and a component that increased linearly with increasing [Na]_i. Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na]_o, and a steady-state balance of the sodium ion fluxes occurred at [Na]_o = 40 mM. When solutions were K-free and contained 10^-4 M ouabain, net sodium influx varied linearly with [Na]_o and a steady state for the intracellular sodium was observed at [Na]_o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na]_i but dependent on [Na]_o. Replacing external NaCl with MgCl₂ brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na]_o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

Potassium and the Recovery of Arterial Smooth Muscle after Cold Storage

The influence of K on the performance of vascular smooth muscle was studied by observing the mechanical performance of the muscle under conditions in which the magnitudes of [K_i] and of the [K_i]:[K_o] ratio varied in opposite directions. During prolonged storage at 4°C the artery strips lost K and their ability to respond to stimuli. Subsequently they were transferred to recovery solutions of various [K_o] at 38°C. The initial rate of K_i reaccumulation and steady state [K_i] were greater in solutions of higher [K_o]. Conversely for any time during recovery, the greater [K_o], the smaller the [K_i]:[K_o] ratio. When the strip was placed in the warm recovery solution it first contracted and then relaxed. The initial contraction was not relatable to [K_o] of the recovery solution but the subsequent relaxation was greater in rate and magnitude as [K_o] was greater. As the muscles recovered further they went into tonic contracture. As the [K_o] in the recovery solutions was greater these contractures occurred after shorter recovery times, and attained greater amplitude at a faster rate. Solution-switching experiments indicated a dependence of responses to electrical shocks on both the [K_i]:[K_o] ratio and [K_i]. Conclusions drawn were: (a) increased [K_i] increases contractility, (b) increased [K_i] increases the rate of relaxation, (c) excitability is decreased by too high or low a [K_i]: [K_o] ratio, and (d) the extent of tonic shortening depends on the [K_i]:[K_o] ratio.     

Effect of metabolic depletion on the furosemide-sensitive Na and K fluxes in human red cells

Summary We report in this paper the effect of metabolic depletion on several modes of furosemide-sensitive (FS) Na and K transport in human red blood cells. The reduction of ATP content below 100 mol/liter cells produced a marked decrease in the maximal activation (V _max) of the outward. FS transport of Na and K into choline medium in the presence of ouabain (0.1 mM) and 1 mM MgCl₂. TheK _0.5 for internal Na to activate the FS Na efflux was not altered by metabolic depletion. However, metabolic depletion markedly decreased the K _i for external K (K _o ) to inhibit the FS Na efflux into choline medium (from 25 to 11 mM). Repletion of ATP content by incubation of cells in a substraterich medium recovered control levels ofV _max of the FS Na and K fluxes and of K _i for external K to inhibit FS Na efflux. TheV _max of FS Na and K influxes was also markedly decreased when the ATP content dropped below 100 mol/liter cells. This was mainly due to a decrease in the inward-coupled transport of K and Na (Na _o -stimulated K influx and the K _o -stimulated Na influx). The FS K _i /K _o exchange pathway of the Na–K cotransport, estimated from the FS K influx from choline-20 mM K _o medium into cells containing 22 mmol Na/liter cells, was also reduced by starvation. Starvation did not inhibit the FS Na _i /Na _o exchange pathway, estimated as FS Na influx from a medium containing 130 mM NaCl into cells containing 22 mmol Na/liter cells. The unidirectional FS²²Na efflux and influx were also measured in control and starved cells containing 22 mmol Na/liter cells, incubated in a Na medium (130 mM) at varying external K (0 to 20 mM). In substrate-fed cells, incubated in the absence of external K, FS Na efflux was larger than Na influx. This FS net Na extrusion (400 to 500 mol/liter cells·hr) decreased when external K was increased, approaching zero around 15 mM K _o . In starved cells the net Na extrusion was markedly decreased and it approached zero at lower K _o than in substrate-fed cells. Our results indicate that the FS Na and K fluxes, and their major component, the gradient driven Na–K–Cl cotransport system, are dependent on the metabolic integrity of the cells.     

Sodium movements in the human red blood cell

Measurements were made of the sodium outflux rate constant, ^o k _Na, and sodium influx rate constant, ⁱ k _Na, at varying concentrations of extracellular (Na_o) and intracellular (Na_c) sodium. ^o k _Na increases with increasing [Na_o] in the presence of extracellular potassium (K_o) and in solutions containing ouabain. In K-free solutions which do not contain ouabain, ^o k _Na falls as [Na_o] rises from 0 to 6 mM; above 6 mM, ^o k _Na increases with increasing [Na_o]. Part of the Na outflux which occurs in solutions free of Na and K disappears when the cells are starved or when the measurements are made in solutions containing ouabain. As [Na_o] increases from 0 to 6 mM, ⁱ k _Na decreases, suggesting that sites involved in the sodium influx are becoming saturated. As [Na_c] increases, ^o k _Na at first increases and then decreases; this relation between ^o k _Na and [Na_c] is found when the measurements are made in high Na, high K solutions; high Na, K-free solutions; and in (Na + K)-free solutions. The relation may be the consequence of the requirement that more than one Na ion must react with the transport mechanism at the inner surface of the membrane before transport occurs. Further evidence has been obtained that the ouabain-inhibited Na outflux and Na influx in K-free solutions represent an exchange of Na_c for Na_o via the Na-K pump mechanism.     

Influence of external barium and potassium on potassium efflux in depolarized frog sartorius muscles

Summary Efflux of⁴²K⁺ was measured in frog sartorius muscles equilibrated in depolarizing solutions with external K⁺ concentrations ([K⁺] _o ) between 75 and 300mm and NaCl concentrations of 60, 120, or 240mm. For several combinations of KCl and NaCl, steady-state internal potentials (V _i) were the same for different [K⁺] _o . For the range ofV _i examined, K⁺ efflux occurs principally through the K⁺ inward rectifier channels. When external K⁺ is removedV _i remains constant for 2 to 3 hr because of the high membrane conductance to Cl^–, but K⁺ efflux drops by about one order of magnitude.External Ba²⁺ in the presence or absence of external K⁺ produces an inhibition of K⁺ efflux described by a relation of the formu=(u₁/(1+C)[Ba²⁺] _o ))+u ₂, whereu is the uninhibited fraction of K⁺ efflux;u ₁, u₂ andC are constants; andu ₁+u₂=1.C depends both on [K⁺] _o andV _i. When [K⁺] _o 75mm, increasing [K⁺] _o at constantV _i reduces Ba²⁺ sensitivity. For constantV _i–30 mV, Ba²⁺ sensitivity is less when [K⁺] _o =0 than when [K⁺] _o 75mm. When [K⁺] _o =0, Ba²⁺ sensitivity decreases asV _i is made more positive. The dependence of the Ba²⁺ sensitivity onV _i at constant [K⁺] _o is greater when [K⁺] _o =0 than when [K⁺] _o 75mm.Both the activation of K⁺ efflux by external K⁺ and the Ba²⁺ inhibition of K⁺ efflux can be explained on the basis of two membrane control sites associated with each channel. When both sites are occupied by K⁺, the channels are in a high flux state. When one or both sites are empty, the channels are in a low, nonzero flux state. When Ba²⁺ occupies either site, K⁺ efflux is further reduced. The reduction of Ba²⁺-sensitivity by increasing [K⁺] _o at high [K⁺] _o is attributable to the displacement of Ba²⁺ from the control sites by K⁺. The increased Ba²⁺ sensitivity produced by going from [K⁺] _o =0 to [K⁺] _o >-75mm whenV _i–30 mV is attributable to states in which Ba²⁺ occupies one site and K⁺ the other when [K⁺] _o 0. The smallerV _i dependence of the Ba²⁺ sensitivity when [K⁺] _o 75mm compared to [K⁺] _o =0 is attributable to the necessity that Ba²⁺ displace K⁺ at the control sites when [K⁺] _o is high but not when [K⁺] _o =0.     

Interaction of External K, Na, and Cardioactive Steroids with the Na-K Pump of the Human Red Blood Cell

The interaction of extracellular Na (Na_o), K (K_o), and strophanthidin with the Na-K pump of the human red blood cell has been investigated. Inhibition by submaximal concentrations of strophanthidin rapidly reaches a level which does not increase further over a relatively long period of time. Under these circumstances, it is possible to apply a steady-state kinetic analysis to the interaction of Na_o, K_o, and strophanthidin with the pump. In Na-free solutions, strophanthidin increases the apparent K_1/2 of the pump for K_o, but does not change the form of the relation between the reciprocal of the active K influx (ⁱM_K^P–1) and the reciprocal of [K_o] ([K_o]^–1); the relation both in the presence and absence of strophanthidin is adequately described by a straight line. In solutions containing Na, strophanthidin changes the form of the curve describing the relation between ⁱM_K^P–1 vs. [K_o]^–1; the curve becomes more parabolic in solutions containing strophanthidin. The rate of ouabain binding to K-free cells has also been measured; in the absence of K, the rate of binding is unaffected by Na_o. The data are considered in terms of a simple kinetic model. The findings can be explained if it is supposed that at low external K the form of the pump combined with one Na_o is more likely to combine with strophanthidin than is the uncombined form of the pump. The uncombined form of the pump is more likely to combine with K even at very low K_o than with strophanthidin.     

The phosphorylation of intramitochondrial AMP: A suggestion for the compartmentation of endogenous Pi pool

1. The mitochondrial level of AMP gradually diminishes during incubation of mitochondria with glutamate but does not with succinate. This decline of AMP, associated with stoichiometric increase in ADP and/or ATP, is accelerated by the addition of electron acceptors or 2,4-dinitrophenol, while arsenite, arsenate and rotenone are inhibitory. These results are in agreement with the view that AMP is phosphorylated to ADP in the inner space of rat liver mitochondria via succinyl-CoA synthetase (succinate: CoA ligase (GDP), EC 6.2.1.4) and GTP:AMP phosphotransferase dependent on the oxidation of 2-oxoglutarate, which is promoted by the transfer of electron from NADH to the respiratory chain.2. Studies of the periodical changes of chemical quantities of adenine nucleotides as well as of their labelling with ³²P_i reveals the following characteristics concerning mitochondrial phosphorylation. (i) In contrast to the mass action ratio of ATP to ADP, the ratio of ADP to AMP is not affected by the intramitochondrial concentration of P_i. (ii) ³²P_i, externally added, is incorporated into ADP much more slowly than into γ-phosphate of ATP. (iii) Conversely, ATP loses its radioactivity from γ-phosphate position more rapidly than [³²P]ADP when ³²P-labelled mitochondria are incubated with non-radioactive P_i.3. In order to elucidate the above characteristic properties of phosphorylation, a hypothetical scheme is proposed which postulates the two separate compartments in the intramitochondrial pool of P_i; one readily communicates with external P_i and is utilized for the phosphorylation of ADP in oxidative phosphorylation, while the other less readily communicates with external P_i and serves as the precursor of ADP via succinyl-CoA synthetase and GTP:AMP phosphotransferase.     

Steady-state kinetics of ADP-arsenate and ATP-synthesis in Rhodospirillum rubrum chromatophores

(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) V_m for ADP-arsenate synthesis was about 2-times lower than V_m for ATP-synthesis. With saturating [ADP], K(As_i) was about 20% higher than K(P_i). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of $\text{1}\text{v}$ vs. $\text{1}\text{[}\text{substrate}\text{]}$ were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio. High concentrations of the fixed substrate cause a similar increase in $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (As_i) stimulate ATP synthesis supported by low concentrations of P_i and ADP about 2-fold. (5) At high ADP concentrations, the apparent K_i of As_i for inhibition of ATP-synthesis was 2–3-times higher than the apparent K_m of As_i for arsenylation; the apparent K_i of P_i for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent K_m of P_i for ATP synthesis. (6) The results are discussed in terms of a model in which P_i and As_i compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion.     

Fractionation of Sodium Efflux in Frog Sartorius Muscles by Strophanthidin and Removal of External Sodium

The influence of strophanthidin, ouabain, and the removal of external sodium on the sodium efflux from frog sartorius muscle was measured. In freshly dissected muscles strophanthidin and ouabain in maximally effective concentrations reduced the efflux of sodium by about 50%. Of the sodium efflux which is strophanthidin-insensitive about 75% is inhibited after complete replacement of external sodium by lithium. In the absence of strophanthidin replacement of external sodium by lithium, calcium, or magnesium produces an initial rise in the sodium efflux, followed by a fall in the efflux as the exposure of the muscles to sodium-free media is continued. When the muscles are exposed for prolonged periods in sodium-free media, the fraction of internal sodium lost per minute is higher when returned to normal Ringer fluid than it was initially. The activation of sodium efflux by external sodium after long periods in sodium-free solutions is partly strophanthidin-sensitive and partly strophanthidin-insensitive. The internal sodium concentration is an important factor in these effects. The effects of temperature on the sodium efflux were also measured. Above 7°C the Q₁₀ of both the strophanthidin-sensitive and strophanthidin-insensitive sodium efflux is about 2.0. Below 7°C the strophanthidin-insensitive sodium efflux has a Q₁₀ of about 7.4.     

The kinetics of sodium extrusion in striated muscle as functions of the external sodium and potassium ion concentrations

After a 20 min initial washout, the rate of loss of radioactively labeled sodium ions from sodium-enriched muscle cells is sensitive to the external sodium and potassium ion concentrations. In the absence of external potassium ions, the presence of external sodium ions increases the sodium efflux. In the presence of external potassium ions, the presence of external sodium ions decreases the sodium efflux. In the absence of external potassium ions about one-third of the Na⁺ efflux that depends upon the external sodium ion concentration can be abolished by 10^-5 M glycoside. The glycoside-insensitive but external sodium-dependent Na⁺ efflux is uninfluenced by external potassium ions. In the absence of both external sodium and potassium ions the sodium efflux is relatively insensitive to the presence of 10^-5 M glycoside. The maximal external sodium-dependent sodium efflux in the absence of external potassium ions is about 20% of the magnitude of the maximal potassium-dependent sodium efflux. The magnitude of the glycoside-sensitive sodium efflux in K-free Ringer solution is less than 10% of that observed when sodium efflux is maximally activated by potassium ions. The inhibition of the potassium-activated sodium efflux by external sodium ions is of the competitive type. Reducing the external sodium ion concentration displaces the plots of sodium extrusion rate vs. [K]_o to the left and upwards.     

Adenine nucleotide efflux in mitochondria induced by inorganic pyrophosphate

The efflux of mitochondrial adenine nucleotide which is induced by addition of PP_i to suspensions of rat liver mitochondria has been investigated. This efflux of adenine nucleotide is greatly stimulated by the uncoupler FCCP at 1 μM, V_max being 6.7 nmol/min per mg protein as compared to 2.0 nmol/min per mg protein in its absence. The depletion process is inhibited by carboxyatractyloside. The K_m for PP_i of 1.25 mM is essentially unchanged when uncoupler is added. Quantitation of the individual adenine nucleotide species (ATP, ADP and AMP) and their relationship to the rate of efflux suggests that ADP is the predominant species being exchanged for PP_i.     

Ligand-Dependent Effects on the Conformational Equilibrium of the Na,K-ATPase As Monitored by Voltage Clamp Fluorometry

Voltage clamp fluorometry was used to monitor conformational changes associated with electrogenic partial reactions of the Na⁺,K⁺-ATPase after changes in the concentration of internal sodium (Na⁺_i) or external potassium (K⁺_o). To probe the effects of the Na⁺_i concentration on the Na⁺ branch of the Na⁺,K⁺-ATPase, oocytes were depleted of Na⁺_i and then loaded with external sodium (Na⁺_o) using the amiloride-sensitive epithelial sodium channel. The K⁺ branch of the Na⁺,K⁺-ATPase was studied by exposing the oocytes to different K⁺_o concentrations in the presence and absence of Na⁺_o to obtain additional information on the apparent affinity for K⁺_o. Our results demonstrate that lowering the concentration of Na⁺_i or increasing the amount of K⁺_o in the external solution shifts the equilibrium toward E₁/E₁P. Furthermore, the K⁺_o-induced relocation toward E₁ occurs at a much lower K⁺_o concentration when Na⁺_o is absent, indicating a higher apparent affinity. Finally, voltage-dependent steps associated with the K⁺ branch or the Na⁺ branch of the Na⁺,K⁺-ATPase are affected by the K⁺_o concentration or the Na⁺_i concentration, respectively.     

Effect of internal and external K+ on Na+−Ca2+ exchange in dialyzed squid axons under voltage clamp conditions

The effect of external and internal K⁺ on Na_o⁺-dependent Ca²⁺ efflux was studied in dialyzed squid axons under constant membrane potential. With axons clamped at their resting potentials, external K⁺ (up to 70 mM) has no effect on Na⁺?Ca²⁺ exchange. Removal of K_i⁺ causes a marked inhibition in the Na_o⁺-dependent Ca²⁺ efflux component. Internal K⁺ activates the Na⁺?Ca²⁺ exchange with low affinity $\text{(K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 90 mM)}$. Activation by K_i⁺ is similar in the presence or in the absence of Na_i⁺, thus ruling out a displacement of Na_i⁺ from its inhibitory site. Axons dialyzed with ATP also show a dependency of Ca²⁺ efflux on K_i⁺. The present results demonstrate that K_i⁺ is an important cofactor (partially required) for the proper functioning of the forward Na⁺?Ca²⁺ exchange.     

A 31P-NMR study of the cross-membrane pH gradient induced by ATP hydrolysis in mitochondria

³¹P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P_i. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P_i peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P_i and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P_i increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.     

Synthesis and possible character of a high-energy intermediate in bacterial photophosphorylation

1. In photophosphorylation with chromatophores from Rhodospirillum rubrum, evidence is presented for the synthesis of activated precursors of ATP in the energy-conversion system coupled to photosynthetic electron transport. 2. A significant amount of ATP is synthesized when a reaction mixture containing chromatophores and ADP is illuminated and then incubated with P_i in the dark. ATP is not synthesized to an appreciable extent, either when a reaction mixture containing chromatophores and P_i is illuminated and then incubated with ADP in the dark, or when one containing chromatophores alone is illuminated and then incubated with ADP and P_i in the dark. The amount of ATP thus synthesized is influenced markedly by concentrations of ADP. 3. The chromatophores illuminated with ADP, if allowed to stand in the dark at 30°, gradually lose the ability to form ATP with P_i in the dark. No loss of the ability occurs when the chromatophores illuminated with ADP are allowed to stand in the dark at 13° or in a frozen state. 4. Mg²⁺ is absolutely required for chromatophores to form ATP in the dark after illumination in the presence of ADP, and for the chromatophores to achieve ATP formation with P_i in the dark. 5. Antimycin A, 2-heptyl-4-hydroxyquinoline N-oxide and o-phenanthroline strongly inhibit the light-dependent acquisition of the ability to form ATP with P_i in the dark, but not the consequent ATP formation with P_i in the dark. Arsenate, 2,4-dinitrophenol, quinacrine hydrochloride, quinine hydrochloride and pyrophosphate inhibit the former or the latter, or both. Oligomycin inhibits the former somewhat more than the latter. 6. From these findings it is suggested that a high-energy intermediate is formed in photosynthetic ATP formation, and that its formation is dependent on ADP but not P_i.     

Ion transport and regulation ofTetrahymena pyriformis in isotonic and hypotonic media

Summary The ion and volume regulatory mechanisms ofTetrahymena pyriformis were studied in normal or hypotonic nutrient media and in isotonic inorganic media with different Na/K ratios, in conjunction with the effects of a general metabolic inhibitor (low temperature) and a specific inhibitor (iodoacetate). For K two mechanisms of active influx were found: The first is sensitive to IAc and seems to be the basic mechanism for the maintenance of the K_i/K_o gradient. The second is sensitive to cooling and related to the function of the contractile vacuole; it is also responsible for the high intracellular levels of K. The passive K efflux seems to be a basic factor for volume regulation, together with the contractile vacuole. It increases in hypotonic media and this seems to be related to structural changes of the membranes occurring in hypotonic media. For Na two mechanisms of active transport were also found: One for active Na efflux with highK _m, which is associated with the contractile vacuole and another, for active Na influx with lowK _m, which is inhibited by high levels of intracellular K.The electrochemical potentials of Na and K and the membrane potential (Cl Nernst potential) were also studied in isotonic inorganic media. The membrane is negatively polarized, except if Na_o<5 mM when it becomes positive. In normal conditions, Na is transported outwards actively and leaks passively, while K is transported inwards actively and leaks 56 times more rapidly than Na ions.A model for the overall transport and regulation of ions inTetrahymena is proposed.Abbreviations IAc iodoacetate - PCV packed cell volume - Na _i,K _i,Cl intracellular concentrations ofNa ⁺,K ⁺,Cl ^–, respectively - Na _o,K _o, Cl_o extracellular concentrations of Na⁺, K⁺, Cl^–, respectively - DR distribution ratio - HyN hypotonic nutrient medium - IsN isotonic nutrient medium - HyS IsS hypotonic, and isotonic salt medium, respectively     

Separate Gating Mechanisms Mediate the Regulation of K2P Potassium Channel TASK-2 by Intra- and Extracellular pH

TASK-2 (KCNK5 or K_2P5.1) is a background K⁺ channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pH_o) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pH_i) is independent from that under the command of pH_o. It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pH_o had normal pH_i-dependent gating. Increasing extracellular K⁺ concentration acid shifts pH_o activity curve of TASK-2 yet did not affect pH_i gating of TASK-2. pH_o modulation of TASK-2 is voltage-dependent, whereas pH_i gating was not altered by membrane potential. These results suggest that pH_o, which controls a selectivity filter external gate, and pH_i act at different gating processes to open and close TASK-2 channels. We speculate that pH_i regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys²⁴⁵) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pH_i. We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pH_i-dependence curve of TASK-2 as expected from its lower pK_a. We conclude that intracellular pH, together with pH_o, is a critical determinant of TASK-2 activity and therefore of its physiological function.     

D2O and the sodium pump in squid nerve membrane

Summary In 10 K artificial seawater (ASW). D₂O replacement reduced the Na efflux of squid axons by about one third. In 0 K ASW, D₂O replacement had little effect. D₂O reduced the K⁺ sensitivity of the efllux but increased the affinity for K⁺. A 4° decrease in temperature mimicked the effects of D₂O. When axons were injected with arginine, to decrease the ATP/ADP ratio, they lost K⁺ sensitivity in normal ASW, as expected. Their efflux into 0 K ASW became D₂O sensitive. The results are discussed in terms of conformational changes in the Na pump molecular complex.     

The Influence of H+ on the Membrane Potential and Ion Fluxes of Nitella

The resting membrane potential of the Nitella cell is relatively insensitive to [K]_o, but behaves like a hydrogen electrode. K⁺ and Cl^- effluxes from the cell were measured continuously, while the membrane potential was changed either by means of a negative feedback circuit or by external pH changes. The experiments indicate that P_K and P_Cl are independent of pH but are a function of membrane potential. Slope ion conductances, G_K, G_Cl, and G_Na were calculated from efflux measurements, and their sum was found to be negligible compared to membrane conductance. The possibility that a boundary potential change might be responsible for the membrane potential change was considered but was ruled out by the fact that the peak of the action potential remained at a constant level regardless of pH changes in the external solution. The conductance for H⁺ was estimated by measuring the membrane current change during an external pH change while the membrane potential was clamped at K⁺ equilibrium potential. In the range of external pH 5 to 6, H⁺ chord conductance was substantially equal to the membrane conductance. However, the [H]_i measured by various methods was not such as would be predicted from the [H]_o and the membrane potential using the Nernst equation. In artificial pond water containing DNP, the resting membrane potential decreased; this suggested that some energy-consuming mechanism maintains the membrane potential at the resting level. It is probable that there is a H⁺ extrusion mechanism in the Nitella cell, because the potential difference between the resting potential and the H⁺ equilibrium potential is always maintained notwithstanding a continuous H⁺ inward current which should result from the potential difference.     

Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles

Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pH_i, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, ‘bulk pH values’, pH_o, were measured by a combination electrode. While pH_i = pH_o for neutral, pH_i < pH_o for anionic and pH_i > pH_o for cationic liposomes prepared in 5.0 · 10^?3 M phosphate buffers. pK_a values for the ionization of pyranine were 7.22 ± 0.04 and 6.00 ± 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-d-α-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calculated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established.