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.     

An electrophysiological study of the membrane properties of the immature and mature oocyte of the Batstar,Patiria miniata

Immature oocyte membrane properties of a starfish, Patiria miniata, were investigated by microelectrode techniques. The resting membrane potential in artificial seawater (ASW) was ?78.5 ± 6.7 mV (n = 61, inside negative). This was mainly accounted for by a selective permeability to potassium ions. Potassium ion-selective microelectrodes were used to measure intracellular K⁺ ion activity, which was 350 mM. The sodium to potassium permeability ratio was 0.02 ± 0.01 (n = 4). The current-voltage relation was nonlinear. The I–V curve included both areas of inward and outward rectification. The dependence of inward rectification upon the K⁺ ion electrochemical gradient was demonstrated. The membrane was capable of a regenerative action potential due to permeability changes for Ca²⁺ and Na⁺ ions. The Ca and Na components of the action potential were identified. The Ca component was reversibly suppressed by cobalt and irreversibly blocked by D-600. The Na component was tetrodotoxin (TTX) insensitive. The excitable response of P. miniata oocytes is similar to that described by Miyazaki et al. (1975a) for those of the starfish Asterina pectinifera.Immature oocytes were stimulated to mature with 10^?5M 1-methyladenine (1-MA) during continuous monitoring of the membrane potential. The resting potential in ASW became more inside negative during maturation. This change of the passive membrane property of the oocyte may be accounted for by the increased selectivity to K⁺ ions. The specific membrane resistance near the resting potential increased from 4.2 ± 1.4 to 21 ± 8.7 kΩ·cm² (n = 15) during maturation, while the specific membrane capacitance decreased slightly from 2 ± 0.5 to 1.7 ± 0.6 μF/cm² (n = 5). Maturation had little effect upon the active membrane properties.     

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.     

Maturation of Xenopus oocytes: II. Observations on membrane potential

Large, progesterone-responsive oocytes within their follicles have an average resting potential of about ?25 mV. When manually dissected out of their follicles, most of these oocytes undergo a hyperpolarization over the next 30–60 min to values of about ?60 to ?80 mV. The relatively high negative membrane potentials previously recorded on dissected amphibian oocytes may thus be an artifact in the sense that such measurements do not reflect the electrical characteristics of the oocyte within the follicle. The available evidence indicates that the hyperpolarization reflects the activation of an electrogenic Na⁺,K⁺-transport process. One of the terminal events of oogenesis appears to be a suppression of the generation of the Na⁺,K⁺-transport process when oocytes are ovulated artificially (by dissection) or naturally. Continuous, long-term recordings on dissected oocytes reveal that a rather pronounced depolarization of the membrane potential together with an inflection in the recorded potential around the time of germinal vesicle breakdown takes place in the presence of progesterone. Recordings of oocytes within the follicle reveal similar changes, although reduced in absolute magnitude. In both cases, final membrane potentials of ?10 to ?15 mV are achieved. The electrophysiological changes which accompany the normal maturation process thus do not appear to be as pronounced as previously indicated.     

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.     

Regulation of the amphibian oocyte plasma membrane ion permeability by cytoplasmic factors during the first meiotic division.

The denuded Rana pipiens oocyte depolarizes from 80–90 mV inside negative to 3–5 mV? inside positive during progesterone-induced meiotic maturation, apparently due to decreased K⁺ permeability. Depolarization is dependent upon protein synthesis and coincides with breakdown of the oocyte nucleus, but occurs even in the absence of the nucleus, suggesting cytoplasmic regulation of cation selectivity of the oocyte plasma membrane.     

Electrical response to fertilization in ascidian oocytes

The fertilization potential of the ascidian oocyte has been studied using two intracellular electrodes. Two classes of oocyte were observed; low resting potential (RP) oocytes of ?20 to ?35 mV and high RP oocytes of ?80 to ?90 mV. The two types have comparable membrane resistance, falling in the range of 100–300 MΩ, and both may be fertilized and develop normally, although the fertilization potential (FP) is different in the two cases. High RP oocytes give rise to step-like regenerative potentials which attain positive values, whereas low RP oocytes give rise to slower depolarizations that reach zero level. In both cases the FP was sometimes preceded by a small-step depolarization as normally observed in the sea urchin. Irrespective of the original RP, the membrane resistance always decreased to 1–10% of its initial value during the first few minutes of the FP plateau. In contrast when the membrane was depolarized to a comparable potential by current injection the membrane resistance did not decrease. Polyspermic fertilization was induced by removing the extracellular coats, aging the oocytes and using high densities of sperm. The FP in monospermic and polyspermic oocytes were comparable and we could not correlate additional sperm interactions with additional electrical events. Our results suggest that the plasma membrane in ascidian oocytes lacks intrinsic mechanisms, electrical or otherwise, to prevent polyspermic fertilization.     

Fertilization of the starfish Astropecten aurantiacus

In the starfish Astropecten aurantiacus the acrosome reaction occurs when the spermatozoon contacts the outer surface of the jelly layer. A long thin acrosomal filament is extruded from the anterior region of the spermatozoon and establishes contact with the oocyte surface. This latter interaction initiates the movement of the spermatozoon to the oocyte surface, formation of the fertilization cone and the cortical reaction. The first detectable electrical change across the oocyte plasma membrane during interaction with the spermatozoon is the fertilization potential (FP) which occurs simultaneously with the cortical reaction. The FP is probably the electrical result of the modification of the oocyte plasma membrane during cortical exocytosis. There are no primary step-like depolarizations during fertilization of starfish oocytes, which contrasts with the situation in sea urchin eggs [see 13]. We suggest that the difference in electrical response to fertilization of starfish oocytes and sea urchin eggs may be attributed to the location of the acrosome reaction in these animals and not to their different meiotic states.     

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.     

Membrane potential, action potential and activation potential of eggs of the sea urchin, Lytechinus variegatus

Unfertilized Lytechinus variegatus eggs in sea water in their normal physiological state have membrane potentials that approximate ?70 to ?80 mV. This conclusion is based on microelectrode measurements and on computation from the Na⁺ and K⁺ fluxes. The ?8 to ?15 mV values for the membrane potential previously reported and which are generally measured are the consequence of depolarization by impalement. The activation potential in inseminated eggs with an initial membrane potential more negative than ?60 mV is a compound event involving sperm-induced as well as voltage dependent conductance changes. The sperm-induced mechanism is a two-phase conductance increase which involves both Na⁺ and Ca²⁺ during the first phase, and Na⁺ alone during the second phase. In addition, the sperm-induced depolarization at the beginning of the first phase activates a voltage dependent Ca²⁺-conductance mechanism resulting in generation of an action potential.     

Calcium-activated and voltage-dependent potassium conductances in clonal pituitary cells

Voltage clamp recordings of GH3/B6 pituitary cells reveal the presence of non linear steady state membrane properties at the level of the resting potential (about ?41 mV). Clamping the cells to potentials more depolarized than ?60 mV is associated with a potential dependent increase in membrane conductance and membrane current variance. Tetra-ethylammonium (TEA), Cobalt (Co²⁺) and methoxy-verapamil (D-600) each attenuate these potential-dependent changes. Spectral analysis of membrane current fluctuations shows that power spectral densities calculated for fluctuations occuring over the ? 70 to ? 40 mV range declin? monotonically as a function of frequency, while spectra derived from fluctuations obtained over the ? 20 mV to 0 mV range decline as the square of frequency and are usually well fitted by a single Lorentzian equation. The half-power frequency of these spectra varies from 45 to 65 Hz. If we assume that the activities of two-state (open-closed) ion channels underlie the electrical behaviour of the membrane at the resting potential and at more depolarized levels, then the results suggests the presence of K⁺ ion channels whose activation depends both on potential and Ca²⁺ ions. These K⁺ ion channels have estimated electrical properties (conductance : 15 ps ; duration : 3 msec) similar to those present in other excitable membranes.     

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.     

An electrogenic Na+/K+ pump in the choroid plexus

Intracellular electrical potential and potassium activity was measured by means of microelectrodes in the epithelial cells of choroid plexus from bullfrogs (Rana catesbeiana). Ouabain applied from the ventricular side caused an abrupt depolarisation of 10 mV but only a gradual loss of potassium from the cells. Readministration of potassium to the ventricular solution of plexuses which were previously depleted of potassium, caused a hyperpolarisation of about 4 mV. These two experiments are consistent with the notion of an electrogenic Na⁺/K⁺ pump situated at the ventricular membrane and which pumps potassium into the cell and sodium into the ventricle. The numerical values obtained suggest that 3 sodium ions are pumped for 2 potassium ions. The permeability coefficient for potassium exit from the cell is calculated to be 1.24 · 10^?5 cm^?1 · s^?1 expressed per cm² of flat epithelium.     

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.     

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.     

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.     

An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis

In 27% DeBoer's saline (DBS), which yields maximum fertility rates, Xenopus eggs fertilized in vitro are monospermic, regardless of sperm concentration. One block to polyspermy (the “slow” block), described previously, occurs at the fertilization envelope that is elevated in response to the cortical reaction. This paper describes properties of an earlier, “fast” block at the plasma membrane and evaluates the functional significance of the two blocks at physiological sperm concentrations in natural mating conditions. Unfertilized eggs have a resting membrane potential of ?19 mV in 27% DBS. Fertilization triggers a rapid depolarization to +8 mV (the fertilization potential, FP); the potential remains positive for ca. 15 min. Activation of eggs with the ionophore, A23187, produces a slower but similar depolarization (the activation potential, AP). As in other amphibian eggs, the FP appears to result from a net efflux of Cl^?, since the peak of the FP (or the AP in ionophore-activated eggs) decreases as the concentration of chloride salts in the medium is increased. In 67% DBS no FP or AP is observed; eggs fertilized in 67% DBS become polyspermic and average 2 sperm entry sites per egg. In the 5–37 mM range, I^? and Br^?, but not F^?, are more effective than Cl^? in producing polyspermy. In 20 mM NaI the plasma membrane hyperpolarizes in response to sperm or ionophore; 100% levels of polyspermy and an average of 14 sperm entry sites per egg are observed. NaI does not inhibit or retard elevation of the fertilization envelope; the cortical reaction and fertilization envelope are normal in transmission electron micrographs. In 67% DBS, which also inhibits the fast block, the slow block was estimated to become functional 6–8 min after insemination. Eggs fertilized by natural mating in 20 mM NaI exhibit polyspermy levels of 50–90% and average 5 sperm entry sites per egg. Since eggs become polyspermic when fertilized by natural mating under conditions that inhibit the fast, but not the slow, block to polyspermy, we conclude that the fast block is essential to the prevention of polyspermy at the sperm concentrations normally encountered by the egg.     

Membrane potential of the unfertilized sea urchin egg

The membrane potential, specific resistance, and potassium selectivity of the unfertilized Strongylocentrotus purpuratus egg were determined by two independent methods: tracer flux and microelectrode. The potassium influx was 0.50 ± 0.2 pmole/cm²· sec, which was greater than the sodium, chloride, and calcium influxes by factors of 4, 7, and 75, respectively. By means of the constant-field equations, the flux data were used to calculate membrane potential (?70 mV) and specific resistance (420 kΩ · cm²). The effect of the external potassium concentration on the sodium influx was determined and the results closely fit the result expected if the membrane behaved as a potassium electrode. Microelectrode measurements of the potential and resistance were ?75 ± 3 mV and 380 ± kΩ · cm².     

Intracellular pH Increase Driven by an Na+/H+ Exchanger upon Activation of Surf Clam Oocytes

Intracellular pH (pH_i) measurements were performed in surf clam (Spisula solidissima) oocytes before and after artificial activation or fertilization [evidenced by germinal vesicle breakdown (GVBD)] by the dimethyloxazolidinedione (DMO) and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) methods. Results using both methods showed increases of pH_iof 0.3 pH unit after activation by excess K⁺. Using BCECF, we found an increase of similar magnitude after fertilization or after the addition of serotonin. By contrast, GVBD did not occur when the pH_iwas increased to similar or even higher levels by exposing the oocytes to ammonia. In sodium-free seawater, excess K⁺induced GVBD but the pH_iof K⁺-activated oocytes decreased significantly below the resting level of unactivated oocytes. The pH_iincreases in K⁺-activated oocytes were otherwise proportional to the external Na⁺concentration. The amiloride derivatives dimethylamiloride and hexamethylene amiloride (at 10–50 μM) efficiently inhibited the K⁺-induced increase of pH_ibut did not block GVBD. These two derivatives were able, however, to retard K⁺-induced GVBD, hexamethylene amiloride being the more efficient. This retardation of K⁺-induced GVBD could be abolished by the simultaneous addition of ammonia. Taken altogether, these results show that a pH_iincrease, driven by a typical Na⁺/H⁺exchanger, follows activation of surf clam oocytes but that this pH_iincrease is neither sufficient nor required for GVBD, though it does allow its progression at an optimal rate.