Amine transport at the plasmalemma of Riccia fluitans

Green thallus cells of the aquatic liverwort, Riccia fluitans, are rapidly depolarized in the presence of 1–20 μM NH₄Cl and 5–100 μM CH₃NH₃Cl, respectively. Simultaneously, the membrane conductance is increased from 0.41 to 1.2 S · m^?2. Uptake of [¹⁴C]methylamine is stimulated by increasing [K⁺]_o and inhibited by increasing [Na⁺]_o or [H⁺]_o, is highly voltage sensitive, and saturates at low amine concentrations.Double-reciprocal plots of (a) maximal membrane depolarization and (b) methylamine uptake vs. external amine concentration give apparent K_m values of 2 ± 1 μM ammonia and 25–50 μM methylamine; K_m values for changes in conductance and membrane current are greater and voltage dependent. Whereas the amine transport into the cell is strongly inhibited by CN^?, the amine efflux is stimulated.The current-voltage characteristics of the ammonia transport are represented by a sigmoid curve with an equilibrium potential of ?60 mV, and this is understood as a typical carrier curve with a saturation current of about 70 mA · m^?2. It is further concluded that the evidently carrier-mediated transport is competitive for the two amines tested, and that ammonia and methylamine are transported in the protonated form as NH₄⁺ and CH₃NH₃⁺ into the cytoplasm.     

Cesium,Na+-K+ pump and pacemaker potential in cardiac purkinje fibers

The mechanisms of the hyperpolarizing and depolarizing actions of cesium were studied in cardiac Purkinje fibers perfused in vitro by means of a microelectrode technique under conditions that modify either the Na⁺-K⁺ pump activity or I_f. Cs⁺ (2 mM) inconsistently increased and then decreased the maximum diastolic potential (MDP); and markedly decreased diastolic depolarization (DD). Increase and decrease in MDP persisted in fibers driven at fast rate (no diastolic interval and no activation of I_f). In quiescent fibers, Cs⁺ caused a transient hyperpolarization during which elicited action potentials were followed by a markedly decreased undershoot and a much reduced DD. In fibers depolarized at the plateau in zero [K⁺]_o (no I_f), Cs⁺ induced a persistent hyperpolarization. In 2 mM [K⁺]_o, Cs⁺ reduced the undershoot and suppressed spontaneous activity by hyperpolarizing and thus preventing the attainment of the threshold. In 7 mM [K⁺]_o, DD and undershoot were smaller and Cs⁺ reduced them. In 7 and 10 mM [K⁺]_o, Cs⁺ caused a small inconsistent hyperpolarization and a net depolarization in quiescent fibers; and decreased MDP in driven fibers. In the presence of strophanthidin, Cs⁺ hyperpolarized less. Increasing [Cs⁺]_o to 4, 8 and 16 mM gradually hyperpolarized less, depolarized more and abolished the undershoot. We conclude that in Purkinje fibers Cs⁺ hyperpolarizes the membrane by stimulating the activity of the electrogenic Na⁺-K⁺ pump (and not by suppressing I_f); and blocks the pacemaker potential by blocking the undershoot, consistent with a Cs⁺ block of a potassium pacemaker current.     

The second component of the fertilization potential in sea urchin (Pseudocentrotus depressus) eggs involves both Na+ and K+ permeability

The fertilization potential of the Pseudocentrotus depressus egg involved three transiently depolarizing components which had a different time course and a peak value. Three peaks were at less than 10 sec, 43 ± 4 sec (mean ± SD), and 182 ± 22 sec after the onset of the fertilization potential. Their peak values (mean ± SD) were 37 ± 4, 17 ± 3, and −31 ± 5 mV in standard artificial sea water. The effect of external ions on the membrane potential at the peak of the second component was measured with a conventional voltage-recording microelectrode. The peak value changed 51 mV with a 10-fold change in external Na⁺ concentration. However, it was about 65 mV more negative than the equilibrium potential of Na⁺, assuming that the internal Na⁺ concentration was 13.5 mM. H⁺, Ca²⁺, Mg²⁺, and Cl⁻ did not contribute to the peak value. The peak value was sensitive to the external K⁺ concentration. These data fitted a theoretical line obtained from the Goldman-Hodgkin-Katz equation, using a ratio of P_Na:P_K:P_Cl = 1.1:1.0:0. This means that the permeability to both Na⁺ and K⁺ is responsible for the second component of the fertilization potential. The fertilization potential was also measured in the artificial sea water containing Li⁺ or Cs⁺. The egg at the second component of the fertilization potential was almost equally permeable to Li⁺ as well as Na⁺ or K⁺ and somewhat permeable to Cs⁺. By contrast, the resting membrane potential before fertilization depended to a large extent upon K⁺ permeability.     

Activation and Inactivation of Oxytocin and Vasopressin Release from Isolated Nerve Endings (Neurosecretosomes) of the Rat Neurohypophysis

Neurosecretory terminals (neurosecretosomes, NSS) were isolated from rat neurohypophyses. High [K⁺]_oor veratridine stimulated secretion of vasopressin and oxytocin by up to ～ 100-fold. Stimulated secretion was dependent on calcium and temperature, and could be elicited from NSS maintained in culture for 4 days. After overnight culture of the NSS, secretion was still inhibited by calcium channel blockers (cobalt, dihydropyridines, ω-conotoxin, D 600) and K opiates (dynorphin and U50488). Ionomycin evoked dose and calcium-dependent hormone release, with a Hill coefficient for calcium of 1.74. High [K⁺]_o enhanced the 5 μMionomycin-induced secretion, apparently through calcium entry rather than depolarization, as the increase in secretion was abolished by 100 μM D 600. During prolonged depolarization the hormone secretion peaked within 2 min, then declined to near basal levels. Depolarization for 25 min without calcium neither activated secretion nor prevented subsequent secretion on readdition of calcium, suggesting that the decline in secretion was not due to membrane depolarization. Indeed, the rates of decline in secretion were similar for different levels of depolarization (0.070 ± 0.003 and 0.081 ± 0.003 min^?1 for 25 and 45 mM [K⁺]_o, respectively). Four minutes after the onset of continuous depolarization (45 mM[K⁺]_o) in the presence of calcium, the declining secretion was still dependent on voltage-activated calcium influx through channels sensitive to D 600 and nitrendipine. The results presented here suggest that the decline in secretion during prolonged depolarizing stimuli may be due to exhaustion, inactivation, or desensitization of a calcium-triggered event.     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.     

Resting potential of the mouse neuroblastoma cells: I. The presence of K+ channels activated at high K+ concentration but closed at low K+ concentration including the physiological concentration

The effects of changes in extracellular K⁺ concentration ([K⁺]_o) on the resting membrane potential, the input resistance and ⁸⁶Rb efflux (as a marker of K⁺ efflux) were examined with use of the cultured mouse neuroblastoma cells (N-18 clone). The results obtained are as follows. (1) The membrane potential was depolarized, with an increase in [K⁺]_o at concentrations above 10–20 mM at a rate of 55–58 mV per 10-fold change in [K⁺]_o, but practically unchanged with varying [K⁺]_o below this concentration. (2) Above the critical [K⁺]_o of 10–20 mM, the input membrane resistance decreased sharply by a factor of $\text{1}\text{4}\text{?}\text{1}\text{5}$ with an increase in [K⁺]_o. A similar decrease in the resistance occurred even under the conditions that the membrane potential was held at control level (about ?55 mV) by a steady-state current passage. (3) Elimination of Na⁺ and Cl^? from the external solution brought about practically no change in the membrane potential. (4) A fractional escape rate of ⁸⁶Rb from N-18 cells remained constant at relatively low level (0.125%/min on average) in the low [K⁺]_o range, but increased sharply with increasing [K⁺]_o above 15 mM (e.g., approx. 3.4- and 4.5-fold at 30 and 100 mM [K⁺]_o, respectively). (5) The high K⁺-induced ⁸⁶Rb efflux was not practically inhibited by 1 mM tetraethylammonium or 0.1 mM 4-aminopyridine, indicating that the K⁺ channels activated by an elevation of [K⁺]_o are not the delayed (voltage-dependent) K⁺ channels. The present results favoured the conclusion that N-18 cells carry K⁺ channels which open at high [K⁺]_o but are closed at low [K⁺]_o including the physiological range for the mouse neuroblastoma cells (around 5.4 mM). This conclusion leads to the notion that in the mouse neuroblastoma N-18 cells the K⁺ permeability does not mainly contribute to determining the resting membrane potential under physiological conditions.     

A study of the current-voltage relationships of electrogenetic active and passive membrane elements in riccia fluitans

Potassium- and proton-dependent membrane potential, conductance, and current-voltage characteristics (I–V curves) have been measured on rhizoid cells of the liverwort Riccia fluitans. The potential difference (E_m) measured with microelectrodes across plasmalemma and tonoplast is depolarized to the potassium-sensitive diffusion potential (E_D) in the presence of 1 mM NaCN, 1 mM NaN₃, or at temperatures below 6°C. Whereas the temperature change from 25°C to 5°C decreases the membrane conductance (g_m) from 0.71 to 0.43 S ? m^?2, 1 mM NaCN increases g_m by about 25%. The membrane displays potassium-controlled rectification which gradually disappears at temperatures below 5°C. The potassium pathway can be described by an equivalent circuit of a diode and an ohmic resistor in parallel. In the potential interval of E_D ± 100 mV the measured I-V curves roughly fit the theoretical curves obtained from a modified diode equation. ⁸⁶Rb⁺(K⁺)-influx is voltage sensitive: In the presence of 1 mM NaCN, ⁸⁶Rb⁺-influx follows a hyperbolic function corresponding to a low conductance at low [K⁺]_o and high conductance at high [K⁺]_o. On the contrary ⁸⁶Rb⁺-influx is linear with [K⁺]_o when pump activity is normal. It is believed that there are two K⁺-transport pathways in the Riccia membrane, one of which is assigned to the low conductance (0.2 S · m^?2), the other to a temperature-dependent facilitated diffusion system with a higher conductance (7.7 S · m^?2). The electrogenic pump essentially acts as a current source and consumes about 39% of the cellular ATP-turnover. In the presence of 30 μM CCCP the saturation current of 0.1 A · m^?2 is doubled to about 0.2 A · m^?2, and the electromotive force of ?360 mV switches to ?250 mV. It is suggested that this may be due to a change in stoichiometry from one to two transported charges per ATP hydrolyzed.     

Effect of various ionic compositions upon the membrane potentials during activation of sea urchin eggs

The membrane potentials of sea urchin (Hemicentrotus pulcherrimus) eggs before and after fertilization and their changes during the membrane elevation induced by intracellular electrical stimulation were recorded in solutions of various ionic compositions. Upon fertilization, the membrane potential (?10 mV) depolarized and reversed polarity by a few mV, then gradually returned to a new steady level ranging between ?50 and ?60 mV. The activation potential is closely associated with a transient increase in the membrane permeability. The potential of the unfertilized egg is hyperpolarized by monovalent anions (Br^?, Cl^? and NO₃^?) and depolarized slightly by K⁺. In contrast, the membrane of the fertilized egg is markedly depolarized by K⁺. Suppression of depolarization associated with an increase of the membrane permeability was recorded in Na-free medium (Tris-HCl). The selective increase in permeability to monovalent anions is thought to alternate with the selective increase in permeability to K⁺through the mediation of a transient increase of Na⁺-permeability at the time of fertilization. No causal relationship between the membrane elevation and the depolarization was established because the breakdown of the cortical granules occurs without depolarization or an increase in membrane permeability.     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Fertilization in crabs: IV. The fertilization potential consists of a sustained egg membrane hyperpolarization

The electrophysiological properties of immature and mature oocytes of two crabs were analyzed. Growing immature oocytes of Carcinus maenas and fully grown immature oocytes of Maia squinado had essentially K⁺ dependent resting potentials, Em, of ?61 ? 1 mV, n=19, and ?67.3 ± 0.5 mV, n=68, respectively. Fully grown immature oocytes of Carcinus maenas showed an Em of ?40 ± 1.5 mV, n=19, that was k⁺ and Cl^? dependent. In mature oocytes of both species, the plasma membrane became exclusively permeable to cl^? and the Em attained–41 ± 1 mV, n=49 and ?34 ± 1.5 mV, n=27 for Carcinus maenas and Maia squinado, respectively. After in vitro insemination, a dramatic increase in egg membrane permeability to K⁺ was observed. This instantaneously caused a sustained hyperpolarization constituting, for these crabs, the fertilization potential. We observed that concurrently with this electrical response to fertilization, sperm reinitiated the oocyte meiotic maturation previously arrested at the first metaphase. The triggering mechanism of the fertilization potential as well as the possible occurrence of a physiological polyspermy are discussed.     

Kinetics of chloride transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

The kinetics of the light-driven Cl^? uptake pump of Synechococcus R-2 (PCC 7942) were investigated. The kinetics of Cl^? uptake were measured in BG-11 medium (pH_o, 7·5; [K⁺]_o, 0·35 mol m^?3; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 mol m^?3) or modified media based on the above. Net³⁶Cl^? fluxes (?Cl^?_o,i) followed Michaelis-Menten kinetics and were stimulated by Na⁺ [18 mol m^?3 Na⁺ BG-11 ?Cl^?_max= 3·29±0·60 (49) nmol m^?2 s^?1 versus Na⁺-free BG-11 ?Cl^?_max= 1·02±0·13 (54) nmol m^?2 s^?1] but the Km was not significantly different in the presence or absence of Na⁺ at pH_o 10; the Km was lower, but not affected by the presence or absence of Na⁺ [Km = 22·3±3·54 (20) mmol m^?3]. Na⁺ is a non-competitive activator of net ?Cl^?_o,i. High [K⁺]_o (18 mol m^?3) did not stimulate net ?Cl^?_o,i or change the Km in Na⁺-free medium. High [K⁺]_o (18 mol m^?3) added to Na⁺ BG-11 medium decreased net ?Cl^?_o,i [18 mol m^?3K⁺ BG-11; ?Cl^?_max= 2·50±0·32 (20) nmol m^?2 s^?1 versus BG-11 medium; ?Cl^?_max= 3·35±0·56 (20) nmol m^?2 s^?1] but did not affect the Km 55·8±8·100 (40) mmol m^?3]. Na⁺-stimulation of net ?Cl^?_o,i followed Michaelis-Menten kinetics up to 2–5 mol m^?3 [Na⁺]_o but higher concentrations were inhibitory. The Km for Na⁺-stimulation of net ?Cl^?_o,i [K_1/2(Na⁺)] was different at 47 mmol m^?3 [Cl^?]_o (K_1/2[Na⁺] = 123±27 (37) mmol m^?3]. Li⁺ was only about one-third as effective as Na⁺ in stimulating Cl^? uptake but the activation constant was similar [K_1/2(Li⁺) = 88±46 (16) mmol m^?3]. Br^? was a competitive inhibitor of Cl^? uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na⁺. The overall Ki was 297±23 (45) mmol m^?3. The discrimination ratio of Cl^? over Br^? (δCl^?/δBr^?) was 6·38±0·92 (df = 147). Synechococcus has a single Na⁺-stimulated Cl^? pump because the Km of the Cl^? transporter and its discrimination between Cl^? and Br^? are not significantly different in the presence and absence of Na⁺. The Cl^? pump is probably driven by ATP.     

Contributions of K+, Na+, and Cl− to the membrane potential of intact hamster vascular endothelial cells

The transmembrane potential (V_m) of vascular endothelial cells (EC) is an important property that may be involved in intra- and intercellular signal transduction for various vascular functions. In this study, V_m of intact aortic and vena caval EC from hamsters were measured using conventional microelectrodes. Vascular strips with the luminal surface upwards were suffused in a tissue chamber with krebs solution in physiological conditions. The resting V_m of aortic and vena caval EC was found to be ?40± 1 mV (n = 55) and ?43± 1 mV (n = 15), respectively. The V_m recordings were confirmed to have originated from EC by scanning and transmission electron microscopy combined with the comparison of electrical recordings between normal and endothelium-denuded aortic strips. The input resistance varied from 10–240 MΩ, which implied the presence of electrical coupling between vascular EC. Elevating the K⁺ level in the suffusate from 4.7 mM to 50 and 100 mM depolarized aortic EC by 19% and 29% and vena caval EC by 18% and 29%, respectively. These low percentages indicated a relatively small contribution of [K⁺] to the resting V_m of vascular EC. A positive correlation (r> 0.69) between the resting V_m and the magnitude of depolarization by the high [K⁺]₀ may be related to the involvement of voltage-dependent K⁺ channels. The hyperpolarization caused by lowering both [Na⁺]₀ and [CI^?]₀ suggested the disengagement of some electrogenic transport systems in the membrane, such as a Na⁺ -K⁺ -CI^? cotransporter. The transference number (t_ion), as an index of membrane conductance for specific ions, was calculated for K⁺ (15-20%), Na⁺ (16%), and Cl^? (9-15%), demonstrating that both Na⁺ and Cl^? as well as K⁺ contribute to the overall resting V_m. Our study documented some basic electrophysiology of the vascular EC when both structural and functional properties of the cell were maintained, thus furthering the understanding of the essential role of endothelial cells in mediating vascular functions. © 1993 Wiley-Liss, Inc.     

The Ionic Permeability Changes during Acetylcholine-Induced Responses of Aplysia Ganglion Cells

ACh-induced depolarization (D response) in D cells markedly decreases as the external Na⁺ is reduced. However, when Na⁺ is completely replaced with Mg⁺⁺, the D response remains unchanged. When Na⁺ is replaced with Tris(hydroxymethyl)aminomethane, the D response completely disappears, except for a slight decrease in membrane resistance. ACh-induced hyperpolarization (H response) in H cells is markedly depressed as the external Cl^- is reduced. Frequently, the reversal of the H response; i.e., depolarization, is observed during perfusion with Cl^--free media. In cells which show both D and H responses superimposed, it was possible to separate these responses from each other by perfusing the cells with either Na⁺-free or Cl^--free Ringer's solution. High [K⁺]₀ often caused a marked hyperpolarization in either D or H cells. This is due to the primary effect of high [K⁺]₀ on the presynaptic inhibitory fibers. The removal of this inhibitory afferent interference by applying Nembutal readily disclosed the predicted K⁺ depolarization. In perfusates containing normal [Na⁺]₀, the effects of Ca⁺⁺ and Mg⁺⁺ on the activities of postsynaptic membrane were minimal, supporting the current theory that the effects of these ions on the synaptic transmission are mainly presynaptic. The possible mechanism of the hyperpolarization produced by simultaneous perfusion with both high [K⁺]₀ and ACh in certain H cells is explained quantitatively under the assumption that ACh induces exclusively an increase in Cl^- permeability of the H membrane.     

Effect of Alkali Metal Cations on Slow Inactivation of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. The kinetics of decaying outward Na⁺ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na⁺]_o from 10 to 150 mM increases the rate of recovery from slow inactivation at −140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na⁺]_o is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na⁺]_o. However raising [Na⁺]_o decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na⁺]_o, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate.     

Fractional contribution of major ions to the membrane potential of Drosophila melanogaster oocytes

In ovarian follicles of Drosophila melanogaster, ion substitution experiments revealed that K⁺ is the greatest contributor (68%) in setting oocyte steady‐state potential (E_m), while Mg²⁺ and a metabolic component account for the rest. Because of the intense use made of Drosophila ovarian follicles in many lines of research, it is important to know how changes in the surrounding medium, particularly in major diffusible ions, may affect the physiology of the cells. The contributions made to the Drosophila oocyte membrane potential (E_m) by [Na⁺]_o, [K⁺]_o, [Mg²⁺]_o, [Ca²⁺]_o, [Cl^?]_o, and pH (protons) were determined by substitutions made to the composition of the incubation medium. Only K⁺ and Mg²⁺ were found to participate in setting the level of E_m. In follicles subjected to changes in external pH from the normal 7.3 to either pH 6 or pH 8, E_m changed rapidly by about 6 mV, but within 8 min had returned to the original E_m. Approximately half of all follicles exposed to reduced [Cl^?]_o showed no change in E_m, and these all had input resistances of 330 kΩ or greater. The remaining follicles had smaller input resistances, and these first depolarized by about 5 mV. Over several minutes, their input resistances increased and they repolarized to a value more electronegative than their value prior to reduction in [Cl^?]_o. Together, K⁺ and Mg²⁺ accounted for up to 87% of measured steady‐state potential. Treatment with sodium azide, ammonium vanadate, or chilling revealed a metabolically driven component that could account for the remaining 13%. © 2009 Wiley Periodicals, Inc.     

Early changes in membrane potential of Saccharomyces cerevisiae induced by varying extracellular K+, Na+ or H+ concentrations

Recently we introduced a fluorescent probe technique that makes possible to convert changes of equilibrium fluorescence spectra of 3,3’-dipropylthiadicarbocyanine, diS-C₃(3), measured in yeast cell suspensions under defined conditions into underlying membrane potential differences, scaled in millivolts (Plasek et al. in J Bioenerg Biomembr 44: 559–569, 2012). The results presented in this paper disclose measurements of real early changes of plasma membrane potential induced by the increase of extracellular K⁺, Na⁺ and H⁺ concentration in S. cerevisiae with and without added glucose as energy source. Whereas the wild type and the ?tok1 mutant cells exhibited similar depolarization curves, mutant cells lacking the two Trk1,2 potassium transporters revealed a significantly decreased membrane depolarization by K⁺, particularly at lower extracellular potassium concentration [K⁺]_out. In the absence of external energy source plasma membrane depolarization by K⁺ was almost linear. In the presence of glucose the depolarization curves exhibited an exponential character with increasing [K⁺]_out. The plasma membrane depolarization by Na⁺ was independent from the presence of Trk1,2 transporters. Contrary to K⁺, Na⁺ depolarized the plasma membrane stronger in the presence of glucose than in its absence. The pH induced depolarization exhibited a fairly linear relationship between the membrane potential and the pH_o of cell suspensions, both in the wild type and the Δtrk1,2 mutant strains, when cells were energized by glucose. In the absence of glucose the depolarization curves showed a biphasic character with enhanced depolarization at lower pH_o values.     

Protection from Cell Death by mcl-1 Is Mediated by Membrane Hyperpolarization Induced By K+ Channel Activation

Mcl-1, a member of the Bcl-2 family, has been identified as an inhibitor of apoptosis induced by anticancer agents and radiation in myeloblastic leukemia cells. The molecular mechanism underlying this phenomenon, however, is not yet understood. In the present study, we report that hyperpolarization of the membrane potential is required for prevention of mcl-1 mediated cell death in murine myeloblastic FDC-P1 cells. In cells transfected with mcl-1, the membrane potential, measured by the whole-cell patch clamp, was hyperpolarized more than −30 mV compared with control cells. The membrane potential was repolarized by increased extracellular K⁺ concentration (56 mV per 10-fold change in K⁺ concentration). Using the cell-attached patch-clamp technique, K⁺ channel activity was 1.7 times higher in mcl-1 transfected cells (NP _o = 22.7 ± 3.3%) than control cells (NP _o = 13.2 ± 1.9%). Viabilities of control and mcl-1 transfected cells after treatment with the cytotoxin etoposide (20 μg/ml), were 37.9 ± 3.9% and 78.2 ± 2.0%, respectively. Suppression of K⁺ channel activity by 4-aminopyridine (4-AP) before etoposide treatment significantly reduced the viability of mcl-1 transfected cells to 49.0 ± 4.6%. These results indicate that as part of the prevention of cell death, mcl-1 causes a hyperpolarization of membrane potential through activation of K⁺ channel activity. Received: 30 March 1999/Revised: 20 July 1999     

Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K⁺]_o was increased from 1.2 to 10 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. As [K⁺]_o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K⁺]_o was increased from 60 to 122 mM, an increase in [K⁺]_i and [Cl^–]_i and a decrease in [Na⁺]_i were observed.Inneurons, as [K⁺]_o was increased from 1.2 to 2.8 mM, [Na⁺]_i and [Cl^–]_i were decreased, whereas [K⁺]_i was increased. As [K⁺]_o was increased from 2.8 to 30 mM, [K⁺]_i, [Na⁺]_i and [Cl^–]_i showed no significant change. When [K⁺]_o was increased from 30 to 122 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. Inastrocytes, pH_i increased when [K⁺]_o was increased. Inneurons, there was a biphasic change in pH_i. In lower [K⁺]_o (1.2–2.8 mM) pH_i decreased as [K⁺]_o increased, whereas in higher [K⁺]_o (2.8–122 mM) pH_i was directly related to [K⁺]_o. In bothastrocytes andneurons, changes in [K⁺]_o did not affect the extracellular water content, whereas the intracellular water content increased as the [K⁺]_o increased. Transmembrane potential (E_m) as measured with Tl-204 was inversely related to [K⁺]_o between 1.2 and 90 mM, a ten-fold increase in [K⁺]_o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The E_m values measured with Tl-204 were close to the potassium equilibrium potential (E_k) except those in neurons at lower [K⁺]_o. However, they were not equal to the chloride equilibrium potential (E_Cl) at [K⁺]_o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K⁺]_o produced different changes in [K⁺]_i, [Na⁺]_i, [Cl^–]_i, and pH_i in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K⁺]_o, in such a way to maintain a more suitable environment for neurons.     

Factors controlling the K+ conductance inChara

Summary Previous current/voltage (I/V) investigations of theChara K⁺ state have been extended by increasing the voltage range (up to +200 mV) through blocking the action potential with La³⁺. A region of negative slope was found in theI/V characteristics at positive PD's, similar to that already observed at PD's more negative than the resting level. These decreases in membrane currents at PD's more negative than –150 mV and at PD's close to 0 or positive are thought to arise from the K⁺ channel closure. Both the negative slope regions could be reversibly abolished by 0.1mm K⁺, 20mm Na⁺, more than 10mm Ca²⁺ or 5mm tetraethylammonium (TEA). The K⁺ channels are therefore blocked by TEA, closed by low [K⁺] _o or high [Ca²⁺] _o and are highly selective to K⁺ over Na⁺. With the K⁺ channels closed, the remainingI/V profile was approximately linear over the interval of 400 mV (suggesting a leakage current), but large rectifying currents were observed at PD's more positive than +50 mV. These currents showed a substantial decrease in high [Ca²⁺] _o , sometimes displayed a slight shift to more positive PD's with increasing [K⁺] _o and were unaffected by TEA or changes in [Na⁺] _o . The slope of the linear part of theI/V profile was steeper in low [K⁺] _o than in TEA or high [Na⁺] _o (indicating participation of K⁺, but not Na⁺, in the leak current). Diethylstilbestrol (DES) was employed to inhibit the proton pump, but it was found that the leakage current and later the K⁺ channels were also strongly affected.