External Mg2+ is required for hyperpolarization to occur in ovulated oocytes of the prawn Palaemon serratus

Immediately after dissection, the ovulated oocyte of the prawn Palaemon serratus had a resting potential E_m of −42 ± 2 mV and a membrane resistance R_m of 15 ± 5 MΩ; the membrane was more permeable to Cl⁻ than to K⁺. The oocyte spontaneously hyperpolarized and E_m gradually reached −70 mV 20–30 min after removal of the oocyte from the female, due to increased membrane permeability to K⁺. However, the hyperpolarization occured only if Mg²⁺ was present in the seawater; external Ca²⁺ was not required. Long-term incubation without external Mg²⁺ depolarized the membrane and increased membrane resistance. After preincubation in Mg²⁺-free ASW, oocytes transferred to standard artificial seawater (ASW) transiently hyperpolarized and then repolarized, before gradually hyperpolarizing to a sustained value of −62 ± mV. The respective roles of external Mg²⁺ and fertilization in eliciting the electrical response of the prawn egg at natural spawning are discussed.     

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.     

Characterization of cell ion exchange in the sea anemoneCondylactis gigantea

Summary Maintenance of intracellular ion contents and their relations to transmembrane potential were studied in tentacles ofCondylactis gigantea. Tentacles leached at 2°C in 10 mMK⁺ and 2 mM K⁺ artificial seawaters (10K ASW and 2K ASW) with and without 2 MM ouabain, and in 0K ASW, lost cell K⁺ and gained Na⁺. Rewarming to 25°C in 10K ASW resulted in a marked accumulation of K⁺ and extrusion of Na⁺ in tentacles leached in 10K ASW and in 0K ASW. Initial rate of Na⁺ extrusion was twice the initial rate of K⁺ accumulation, suggesting a pump coupling ratio of 2. In tentacles leached and rewarmed in 2K ASW, no net reaccumulation of K⁺ and little net extrusion of Na⁺ was observed; i.e., the pump just kept pace with the leaks. Ouabain inhibited K⁺ reaccumulation and Na⁺ extrusion. This effect was less marked in 10K ASW than in 2K ASW confirming, in anemone tentacles, the well documented ouabain-K⁺ antagonism observed in other systems. In no case did K _i ⁺ /K ₀ ⁺ equal Cl ₀ ^– /Cl _i ^– ; therefore, the distribution of these ions did not fit a Donnan distribution. Transmembrane potential difference was –22±3 mV in 10K ASW at 25°C. It fitted a modified Nernst equation which includes the pump coupling ratio and a Na⁺ to K⁺ permeability ratio of 0.31.A moderately high permeability of the cell membrane to Na⁺, and a ouabain and K⁺ sensitive ion pump, exchanging 2 Na⁺ for 1 K⁺, appear to be responsible for the observed ionic distribution and transmembrane potential in anemone cells.     

Electrophysiology of phagocytic membranes: intracellular K+ activity and K+ equilibrium potential in macrophage polykaryons

The role of K⁺ as current carrier during the slow membrane hyperpolarizations (SH) elicited by iontophoretic Ca²⁺ injections into macrophage polykaryons is studied. The intracellular K⁺ activity (a_K) and the K⁺ equilibrium potential (E_K) are measured using ion-sensitive microelectrodes. The mean value of a_K is 84 ± 5 mM in a culture medium containing 5.3 mM K⁺, but increases to 100 ± 8 mM when the extracellular K⁺ concentration is raised to 30.3 mM. Under the same conditions the values of E_K obtained from the Nernst equation are −81 ± 2 mV and −40 ± 2 mV, respectively. The reversal potentials (E_R) of the SH are calculated from changes observed in transmembrane potential and input resistance, according to an equivalent model based only on passive ionic fluxes. The mean E_R values obtained are −74 ± 8 mV in the presence of low K⁺ concentration and −37 ± 3 mV for the high K⁺ medium. These values are significantly smaller than the estimated E_K for the corresponding situations. Evidence for the existence of an electrogenic (Na⁺ + K⁺)-ATPase activity is also presented. The evidence indicates that an increase in the membrane potassium permeability can account for about 90% of the total permeability change occurring during the SH.     

Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

Basolateral K+ Conductance Establishes Driving Force for Cation Absorption by Outer Sulcus Epithelial Cells

Outer sulcus epithelial cells were recently found to actively reabsorb cations from the cochlear luminal fluid, endolymph, via nonselective cation channels in the apical membrane. Here we determined the transport properties of the basolateral membrane with the whole-cell patch clamp technique; the apical membrane contributed insignificantly to the recordings. Outer sulcus epithelial cells exhibited both outward and inward currents and had a resting membrane potential of −90.4 ± 0.7 mV (n= 78), close to the Nernst potential for K⁺ (−95 mV). The reversal potential depolarized by 54 mV for a tenfold increase in extracellular K⁺ concentration with a K⁺/Na⁺ permeability ratio of 36. The most frequently observed K⁺ current was voltage independent over a broad range of membrane potentials. The current was reduced by extracellular barium (10⁻⁵ to 10⁻³ m), amiloride (0.5 mm), quinine (1 mm), lidocaine (5 mm) and ouabain (1 mm). On the other hand, TEA (20 mm), charybdotoxin (100 nm), apamin (100 nm), glibenclamide (10 μm), 4-aminopyridine (1 mm) and gadolinium (1 mm) had no significant effect. These data suggest that the large K⁺ conductance, in concert with the Na⁺,K⁺-ATPase, of the basolateral membrane of outer sulcus cells provides the driving force for cation entry across the apical membrane, thereby energizing vectorial cation absorption by this epithelium and contributing to the homeostasis of endolymph.     

Decrease in the electrogenic Contribution of Na,K-ATPase and the resting membrane potential as a possible mechanism of Ca<Superscript>2+</Superscript> accumulation in rat soleus muscle in a short-term gravity unloading

The resting membrane potential and electrogenic contribution of α1- and α2-isoforms of Na⁺/K⁺-ATPase in the rat soleus muscle at early stages of gravity unloading were analyzed. The role of L-type calcium channels in accumulation of calcium ions in the myoplasm under these conditions was estimated. After 3-day antiorthostatic suspension, the resting membrane potential of the muscle fibers decreased from ?71.0 ± 0.5 to ?66.8 ± 0.7 mV, the muscle excitability reduced, and a trend of muscle fatigue acceleration appeared. The electrogenic contribution of ouabain-sensitive α2-isoform of Na⁺/K⁺-ATPase, determined as the depolarization caused by 1μM ouabain, decreased after suspension from 6.2 ± 0.6 to 0.5 ± 0.8 mV. The contribution of ouabain-resistant α1-isoform of Na⁺/K⁺-ATPase, determined as an additional depolarization after addition of 500 μM ouabain, decreased from 4.6 ± 0.6 to 2.6 ± 0.6 mV. The intensity of Fluo-4AM fluorescence in individual muscle fibers increased after suspension more than fourfold, which suggests an elevated calcium concentration in the myoplasm. A local delivery of nifedipine, a blocker of the L-type calcium channels, to the muscle removed this effect. The existence of a selective mechanism suppressing the electrogenic contribution of Na⁺/K⁺-ATPase α2-isoform, which is the main cause of the muscle fiber membrane depolarization after 3-day suspension, is postulated. The depolarization can activate part of potential-sensitive L-type Ca²⁺ channels, causing the accumulation of calcium ions in the muscle fiber myoplasm.     

Intracellular Na+, K+ and Cl− activities in Ehrlich ascites tumor cells

Ehrlich ascites tumor cell membrane potential (V_m) and intracellular Na⁺, K⁺ and Cl⁻ activities were measured under steady-state conditions in normal saline medium (Na⁺ = 154, K⁺ = 6, Cl⁻ = 150 mequiv./l). Membrane potential was estimated to be −23.3 ± 0.8 mV using glass microelectrodes. Intracellular ion activities were estimated with similar glass electrodes rendered ion-selective by incorporation of ion-specific ionophores. Measurements of V_m and ion-activity differences were made in the same populations of cells. Under these conditions the intracellular Na⁺, K⁺ and Cl⁻ activities are 4.6 ± 0.5; 68.3 ± 8.0; and 43.6 ± 2.1 mequiv./l, respectively. The apparent activity coefficients for Na⁺ and K⁺ are 0.18 ± 0.02 and 0.41 ± 0.05 respectively. These are significantly lower than the activity coefficients expected for the ions in physiological salt solutions (0.71 and 0.73, respectively). The activity coefficient for intracellular Cl⁻ (0.67 ± 0.03), however, is close to that of the medium (0.73), and the transmembrane electrochemical potential difference for Cl⁻ is not different from zero. The results establish that the energy available from the Na⁺ electrochemical gradient is much greater than previously estimated from chemical measurements.     

Electrical properties of the Drosophila egg membrane

Electrical properties of the egg membrane of Drosophila melanogaster were examined using intracellular microelectrodes. Unfertilized eggs and fertilized eggs for the period up to the syncytial blastoderm stage were used. Among Na, K, and Cl, K was most permeant to the membrane. The K permeability, however, did not completely determine the membrane potential. The resting potential in standard solution was ?63.5 ± 8.0 mV (mean ± SD) in unfertilized eggs collected within 2–3 days after virgin flies started to lay eggs. The resting potential in fertilized eggs was ?27.0 ± 8.4 mV within 20 min after egg deposition, while it was ?55.1 ± 6.5 mV at the syncytial blastoderm stage. These changes at different developmental stages were associated with changes in the K-dependence of the membrane. The larger amplitude of the resting potential was suggested to be due to increased K permeability but not to decreased nonspecific leakage. The current-voltage relation was linear throughout the stages examined. Action potentials, such as those in eggs of other animals, were not observed. High Ca media significantly increased the amplitude of the resting potential associated with increase in the membrane resistance. A remarkable nonlineality in the I–V relation appeared in high Ca media, which caused continuously increasing hyperpolarization during sustained inward current. Eggs of temperature-sensitive mutants, shi^ts1 and para^ts1 showed properties similar to those in wild-type eggs with transient temperature changes.     

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.     

Ion transport across the skin of a larval salamander

The transport characteristics of the skin of neotenic Ambystoma tigrinum were investigated using ion substitution and circuit analysis. When bathed with sodium Ringer solution on both sides, a transepithelial potential of up to 50 mV (inside positive) and a short-circuit current (I_sc) of up to 10 μA/cm² were observed. When amiloride was added or Na⁺ was replaced by tetramethylammonium in the apical solution, I_sc was decreased from 3.7 ± 0.4 to 1.5 ± 0.2 μA/cm² (n = 10). When K⁺ replaced Na⁺, there was a smaller change in I_sc from 5.8 ± 0.6 to 3.7 ± 0.5 μA/cm² (n = 10). Although barium had no effect when added to 100 K Ringer on normal skin, it inhibited I_sc on skins taken from K⁺-loaded animals. Nystatin caused substantial increases in I_sc with either Na⁺ or K⁺ as the dominant cation in the apical solution. Current voltage analysis using amiloride was used to estimate the resistances and electromotive forces (EMF) associated with ion transport. The EMF for ion transport was partially dependent on K⁺ in the basolateral solution and it was similar to that observed in other epithelia. The resistance of the transport pathway was high, consistent with the low I_sc. These results suggest that there is an amiloride-sensitive Na⁺ channel in parallel with a small K⁺ conductance in the apical membrane of this preparation.     

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.     

Quantitation of corneal endothelial potentials using a carbocyanine dye

The carbocyanine dye, diS-C₃-(5) was used to quantitate the plasma membrane potential of the bullfrog corneal endothelium. It was shown that valinomycin hyperpolarized the endothelial cell and that in the presence of the ionophore the membrane potential largely reflected the K⁺ equilibrium potential. Using calibration curves constructed by changing medium K⁺ concentration in the presence of valinomycin, and nigericin and ouabain to abolish ion gradients and electrogenic pump activity, the cell membrane potential was calculated to be 28.6 ± 4.2 mV. The major source of this potential was a K⁺ diffusion potential, and the membrane Na⁺ conductance reduced the cell potential to less than the apparent K⁺ equilibrium potential of 51.5 ± 5.1 mV. About 20% of the cell potential could be ascribed to the rheogenic (Na⁺+K⁺)-ATPase.     

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.     

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.     

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.