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.     

Studies on Electrogenesis under High K+ Concentrations in Internodal Cells of Chara corallina

+ concentration ([K⁺]_o) on the membrane potential (E_m) of Chara corallina was studied. E_m more negative than -100 mV was maintained even at 100 mM [K⁺]_o. Addition of Ca²⁺ to the external medium further increased this tendency. However, E_m responded sensitively to the increase in [K⁺]_o, when the electrogenic proton pump of the plasma membrane was inhibited by treating cells with dicyclohexylcarbodiimide, an inhibitor of proton pump. Analysis using equivalent circuit model of the plasma membrane suggested that the electrogenic proton pump was activated by the increase in [K⁺]_o. In the presence of 100 mM K⁺, action potentials were generated by electric stimuli. The ionic mechanism of generation of action potentials in the presence of K⁺ at high concentration was discussed. Received 3 October 2000/ Accepted in revised form 6 January 2001     

Voltage dependent potassium fluxes and the significance of action potentials in Acetabularia

Membrane potential, V_m, and K⁺(⁸⁶Rb⁺) fluxes have been measured simultaneously on individual cells of Acetabularia mediterranea. During resting state (resting potential approx. ?170 mV) the K⁺ influx amounts to 0.24–0.6 pmol · cm^?2 · s^?1 and the K⁺ efflux to 0.2–1.5 pmol · cm^?2 s^?1. According to the K⁺ concentrations inside and outside the cell (40 : 1) the voltage dependent K⁺ flux (zero at V_m = E_K = ?90 mV) is stimulated approx. 40-fold for V_m more positive than E_K.It is calculated that during one action potential (temporary depolarization to V_m more positive than E_K) a cell looses the same amount of K⁺, which leaks in during 10–20 min in the resting state (V_m = ?170 mV). Since action potentials occur spontaneously in Acetabularia, they are therefore suggested to have a significant function for the K⁺ balance of this alga.     

Dependence of membrane potential on Ca2+ transport in cultured cytotrophoblasts of human immature placentas

The electrical membrane properties of cultured human cytotrophoblast were examined by means of a standard electrophysiological technique. The mean values of the membrane potential (E_m) and the membrane resistance in a physiological medium were around ?49 mV and 12 MΩ, respectively. The membrane potential was dependent, to a large extent, on the external Ca²⁺ concentration ([Ca²⁺]₀). Deprivation of external Ca²⁺ reduced membrane potential to about ?20 mV, and an increase in [Ca²⁺]₀ caused a hyperpolarization in a saturable manner. The Ca²⁺-dependency of membrane potential was affected remarkably by [K⁺]₀, but not by [Na⁺]₀ or [Cl^?]₀. The intracellular Ca²⁺ injection hyperpolarized the membrane in a Ca²⁺-free medium. A Ca²⁺ channel blocker, verapamil, completely abolished the Ca²⁺-dependent E_m. The Ca²⁺-dependent E_m was also suppressed by cooling or by the application of metabolic inhibitors. It is suggested that the Ca²⁺-dependent E_m in cultured human cytotrophoblast is caused by a Ca²⁺ influx which, in turn, increases the K⁺ conductance of the cell membrane, presumably due to stimulation of Ca²⁺-activated K⁺ channel.     

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.     

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.     

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.     

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.     

The electrical changes accompanying fertilization and cortical vesicle secretion in the medaka egg

The electrical properties of the egg of the medaka, Oryzias latipes, were studied before, during, and after fertilization. The resting potential of the unfertilized egg averaged ?39 ± 9 mV in Yamamoto's Ringers (Y. Ringers), but 20% of the values were between ?50 and ?60 mV. Fertilization triggers a small depolarization of 4 ± 3 mV in 10% Y. Ringers with an average duration of 20 ± 10 sec. The amplitude of this depolarization is independent of [Na⁺]_o, [Ca²⁺]_o, and [Cl^?]_o, so it appears to be due to a nonspecific leak triggered by sperm-egg fusion. The depolarization is followed by a longer hyperpolarizing phase with an average amplitude of 31 ± 12 mV. Recovery from this hyperpolarization has a fast phase lasting 155 ± 18 sec, followed by a slower phase which reaches a steady average membrane potential of ?19 ± 1 mV by 9 min after fertilization. The membrane resistance falls 10-fold during the first 2 min after fertilization, from 40 (1520 kΩ-cm²) to 3 MΩ. This is largely due to an increase in the K⁺ conductance. At the peak of the hyperpolarization, the membrane potential exhibits a 28 mV/decade [K⁺]_o dependence and a 6 mV/decade [Na⁺]_o dependence. The membrane resistance slowly recovers over the next 8 min to a value about 30% larger than before fertilization. The relation of current vs voltage was linear before, during, and after fertilization and indicated a reversal potential of ?98 ± 20 mV for the hyperpolarization peak. The egg's capacitance averaged 0.04 ± 0.01 μF (0.9 μF/cm²) before fertilization and approximately doubles within 90 sec after fertilization. It then decreases over a 9-min period, reaching a value 25% smaller than before fertilization.     

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.     

Transmembrane potential-mediated coupling between H+ pump operation and K+ fluxes in Elodea densa leaves hyperpolarized by fusicoccin, light or acid load

In isolated Elodea densa leaves, the relationships between H⁺ extrusion (-ΔH⁺), K⁺ fluxes and membrane potential (E_m) were investigated for two different conditions of activation of the ATP-dependent H⁺ pump. The ‘basal condition’ (darkness, no pump activator present) was characterized by low values of-ΔH⁺ and K⁺ uptake (ΔK⁺), wide variability of the ?ΔH⁺/ΔK⁺ ratio, relatively low membrane polarization and E_m values more positive than EK for external K⁺ concentrations (|K⁺]_o of up to 2mol m^?3. A net K⁺ uptake was seen already at [K⁺]_o below 1 mol m^?3, suggesting that K⁺ influx in this condition was a thermodynamically uphill process involving an active mechanism. When the H⁺ pump was stimulated by fusicoccin (FC), by cytosol acidification, or by light (the ‘high polarization condition’), K⁺ influx largely dominated K⁺ and C^? efflux, and the ?ΔH⁺/ΔK⁺ ratio approached unity. In the range 50 mmol m^?3?5 mol m^?3 [K⁺]₀, E_m was consistently more negative than E_K. The curve of K⁺ influx at [K⁺]₀ ranging from 50 to 5000mmol m^?3 fitted a monophasic, hyperbolic curve, with an apparent half saturation value = 0–2 mol m^?3. Increasing |K⁺]₀ progressively depolarized E_m, counteracting the strong hyperpolarizing effect of FC. The effects of K⁺ in depolarizing E_m were well correlated with the effects on both K⁺ influx and ?ΔH⁺, suggesting a cause-effect chain: K⁺₀ influx → depolarization → activation of H⁺ extrusion. Cs⁺ competitively inhibited K⁺ influx much more strongly in the ‘high polarization’ than in the ‘basal’ condition (50% inhibition at [Cs⁺]/[K⁺]₀ ratios of 1:14 and 1:2, respectively) thus confirming the involvement of different K⁺ uptake systems in the two conditions. These results suggest that in E. densa leaves two distinct modes of interactions rule the relationships between H⁺ pump, membrane polarization and K⁺ transport. At low membrane polarization, corresponding to a low state of activation of the PM H⁺-ATP^ase and to E_m values more positive than E_K, K⁺ influx would mainly     

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 membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification

The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K⁺ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (E_m) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their E_m, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized E_m in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in E_m was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K⁺ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K⁺ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in E_m and membrane conductance, the expected effect of acidification on K⁺ fluxes was calculated. It was concluded that apoplastic acidification will increase the K⁺-efflux in the depolarized state and reduce the K⁺-influx in the hyperpolarized state. Received: 28 April 1997 / Accepted: 10 November 1997     

The independence of membrane potential and potassium activation of the sodium pump in muscle

The membrane potential (E_m) of sartorius muscle fibers was made insensitive to [K⁺] by equilibration in a 95 mM K⁺, 120 mM Na⁺ Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with E_m sensitive to [K⁺]. In addition, in the presence of 10 mM K⁺, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10^?4 M ouabain. This suggests that the membrane potential does not play a major role in the potassium activation of the sodium pump in muscles.     

Transport Systems of Ventricaria ventricosa: I/V Analysis of Both Membranes in Series as a Function of [K+] o

The current-voltage (I/V) profiles of Ventricaria (formerly Valonia) membranes were measured at a range of external potassium concentrations, [K⁺] _o , from 0.1 to 100 mm. The conductance-voltage (G/V) characteristics were computed to facilitate better resolution of the profile change with time after exposure to different [K⁺] _o . The resistance-voltage (R/V) characteristics were computed to attempt resolution of plasmalemma and tonoplast. Four basic electrophysiological stages emerged: (1) Uniform low resistance between −60 and +60 mV after the cell impalement. (2) High resistance between +50 and +150 for [K⁺] _o from 0.1 to 1.0 mm and hypotonic media. (3) High resistance between −150 and −20 mV for [K⁺] _o of 10 mm (close to natural seawater) and hypertonic media. (4) High resistance between −150 and +170 mV at [K⁺] _o of 100 mm. The changes between these states were slow, requiring minutes to hours and sometimes exhibiting spontaneous oscillations of the membrane p.d. (potential difference). Our analysis of the I/V data supports a previous hypothesis, that Ventricaria tonoplast is the more resistive membrane containing a pump, which transports K⁺ into the vacuole to regulate turgor. We associate state (1) with the plasmalemma conductance being dominant and the K⁺ pump at the tonoplast short-circuited probably by a K⁺ channel, state (2) with the K⁺ pump ``off' or short-circuited at p.d.s more negative than +50 mV, state (3) with the K<sup>+</sup> pump ``on,' and state (4) with the pump dominant, but affected by high K⁺. A model for the Ventricaria membrane system is proposed. Received: 5 November 1998/Revised: 11 May 1999     

K+-Sensitive Gating of the K+ Outward Rectifier in Vicia Guard Cells

The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K⁺ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K⁺ current was monitored under voltage clamp in 0.1–30 mm K⁺ and in equivalent concentrations of Rb⁺, Cs⁺ and Na⁺. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K⁺ activated current through outward-rectifying K⁺ channels (I _{K, out}) at the plasma membrane in a voltage-dependent fashion. Increasing [K⁺] _o shifted the voltage-sensitivity of I _{K, out} in parallel with the equilibrium potential for K⁺ across the membrane. A similar effect of [K⁺] _o was evident in the kinetics of I _{K, out} activation and deactivation, as well as the steady-state conductance- (g _K ⁻) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K⁺, −80 mV in 1.0 mm K⁺, and −20 mV in 10 mm K⁺. Similar data were obtained with Rb⁺ and Cs⁺, but not with Na⁺, consistent with the relative efficacy of cation binding under equilibrium conditions (K⁺≥ Rb⁺ > Cs⁺ > > Na⁺). Changing Ca²⁺ or Mg²⁺ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of g _K or on I _{K, out} activation kinetics, although 10 mm [Ca²⁺] _o accelerated current deactivation at voltages negative of −75 mV. At any one voltage, increasing [K⁺] _o suppressed g _K completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K⁺ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively. These, and additional data indicate that extracellular K⁺ acts as a ligand and alters the voltage-dependence of I _{K, out} gating; the results implicate K⁺-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K⁺ ions outside affects the distribution between closed states of the channel. Received: 27 November 1996/Revised: 4 March 1997     

Simultaneous Measurements of Cytoplasmic K Concentration and the Plasma Membrane Electrical Parameters in Single Membrane Samples of Chara corallina

The electrophysiological properties of cytoplasm-rich fragments (single membrane samples) prepared from internodal cells of Chara corallina were explored in conjunction with K⁺-sensitive microelectrode and current-voltage (I-V) measurements. This system eliminated the problem of the inaccessible cytoplasmic layer, while preserving many of the electrical characteristics of the intact cells. In 0.1 millimolar external K concentration (K_o⁺), the resting conductance (membrane conductance G_m, 0.85 ± 0.25 Siemens per square meter (±standard error)) of the single membrane samples, was dominated by the proton pump, as suggested by the response of the near-linear I-V characteristic to changes in external pH. Initial cytoplasmic K⁺ activities (a_K+), judged most reliable, gave values of 117 ± 67 millimolar; stable a_K+ values were 77 ± 31 millimolar. Equilibrium potentials for K⁺ (Nernst equilibrium potential) (E_K) calculated, using either of these data sets, were near the mean membrane potential (V_m). On a cell-to-cell basis, however, E_K was generally negative of the V_m, despite an electrogenic contribution from the Chara proton pump. When K_o⁺ was increased to 1.0 millimolar or above, G_m rose (by 8- to 10-fold in 10 millimolar K_o⁺), the steady state I-V characteristics showed a region of negative slope conductance, and V_m followed E_K. These results confirm previous studies which implicated a K_o⁺-induced and voltage-dependent permeability to K⁺ at the Chara plasma membrane. They provide an explanation for transitions between apparent K_o⁺-insensitive and K_o⁺-sensitive (`K⁺ electrode') behavior displayed by the membrane potential, as recorded in many algae and higher plant cells.     

Potential difference responses due to K+, Na+ and Cl− changes in Bullfrog antrum with and without HCO3−

The effect of changing [K⁺], [Na⁺] and [Cl^?] in nutrient solution was studied in bullfrog antrum with and without HCO₃^? in nutrient. In 25 mM HCO₃^? (95% O₂/5% CO₂) and in zero HCO₃^? (100% O₂), nutrient pH was maintained at 7.3. Changing from 4 to 40 mM K⁺ or from 81 to 8.1 mM Cl^? gave a decrease 10 min later in transmucosal PD (nutrient became more negative) — a normal response. These responses were less in zero than in 25 mM HCO₃^?. A decrease from 102 to 8 mM Na⁺ decreased PD (anomalous response of electrogenic NaCl symport). This effect was attenuated or eliminated in zero HCO₃^?. In contrast, change from 4 to 40 mM K⁺ gave initial anomalous PD response and change from 102 to 8 mM Na⁺, initial normal PD response with either zero or 25 mM HCO₃^?. Both responses were associated with (Na⁺ + K⁺)-ATPase pump and were greater in zero than in 25 mM HCO₃^?. Initial PD increases in zero HCO₃^? are explained as due to increase in the resistance of passive conductance and/or NaCl symport pathways. Thus, removal of HCO₃^? modifies conductance pathways of nutrient membrane.     

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

Synechococcus R-2 is a unicellular blue-green alga. The cells will grow on Rb⁺ as a substitute for K⁺ but at a slower rate (t₂～ 15 h versus 12 h). Potassium is not, strictly speaking, an essential element for Synechococcus. Rubidium duxes (using ⁸⁶Rb⁺) are much slower than those of potassium, about 1 nmol m^?2 s^?1 in the light (0.35 mol m^?3 Rb⁺). ⁸⁶Rb⁺ fluxes in the dark are about 0.1 nmol m^?2 s^?1. These fluxes are very slow compared to those of Na⁺ and other ions. Isotopic influx of Rb⁺ can supply sufficient Rb⁺ to keep up with the demands for growth, but the net dux needed to keep up with growth in the light is a large proportion of the total observed dux. Kinetic studies of Rb⁺ uptake versus [Rb⁺] show two uptake phases consistent with a high-affinity and a low-affinity system. Both systems appear to be light-activated. Transport of Rb⁺ appears to be passive at pH_o 10 in the light and dark. There is no case for active transport of Rb⁺ at pH_o 7.5 in the light, but a marginal case for active uptake in the dark (about 3 kJ mol^?1). There is only a small effect of Na⁺ upon Rb⁺ transport. ⁸⁶Rb⁺ should not be used in place of ⁴²K⁺ in K⁺ nutrition studies as the details of Rb⁺ transport are different to those of K⁺ transport.     

Large-conductance Calcium-activated Potassium Channels of Cultured Rat Melanotrophs

A large conductance, Ca²⁺-activated K⁺ channel of the BK type was examined in cultured pituitary melanotrophs obtained from adult male rats. In cell-attached recordings the slope conductance for the BK channel was ≈190 pS and the probability (P _o ) of finding the channel in the open state at the resting membrane potential was low (<<0.1). Channels in inside-out patches and in symmetrical 150 mm K⁺ had a conductance of ≈260 pS. The lower conductance in the cell-attached recordings is provisionally attributed to an intracellular K⁺ concentration of ≈113 mm. The permeability sequence, relative to K⁺, was K⁺ > Rb⁺ (0.87) > NH⁺ ₄ (0.17) > Cs⁺≥ Na⁺ (≤0.02). The slope conductance for Rb⁺ was much less than for K⁺. Neither Na⁺ nor Cs⁺ carried measurable currents and 150 mm internal Cs⁺ caused a flickery block of the channel. Internal tetraethylammonium ions (TEA⁺) produced a fast block for which the dissociation constant at 0 mV (K _D (0 mV)) was 50 mm. The K _D (0 mV) for external TEA⁺ was much lower, 0.25 mm, and the blocking reaction was slower as evidenced by flickery open channel currents. With both internal and external TEA⁺ the blocking reaction was bimolecular and weakly voltage dependent. External charybdotoxin (40 nm) caused a large and reversible decrease of P _o . The P _o was increased by depolarization and/or by increasing the concentration of internal Ca²⁺. In 0.1 μm Ca²⁺ the half-maximal P _o occurred at ≈100 mV; increasing Ca²⁺ to 1 μm shifted the voltage for the half-maximal P _o to −75 mV. The Ca²⁺ dependence of the gating was approximated by a fourth power relationship suggesting the presence of four Ca²⁺ binding sites on the BK channel. Received: 23 October/Revised: 15 December 1995