T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

Temperature- and structure-dependent interaction of pyrethroids with the sodium channels in frog node of Ranvier

(1) The interaction of a series of pyrethroid insecticides with the Na+ channels in myelinated nerve fibres of the clawed frog, Xenopus laevis, was investigated using the voltage clamp technique. (2) Out of 11 pyrethroids 9 insecticidally active compounds induce a slowly decaying Na+ tail current on termination of a step depolarization, whereas the Na+ current during depolarization was hardly affected. These tail currents are most readily explained by a selective reduction of the rate of closing of the activation gate in a fraction of the Na+ channels that have opened during depolarization. (3) The rate of decay of the Na+ tail current varies considerably with pyrethroid structure. After alpha-cyano pyrethroids the decay is at least one order of magnitude slower than after non-cyano pyrethroids. The decay always follows a single-exponential time course and is reversibly slowed when the temperature is lowered from 25 to 0 degrees C. Arrhenius plots in this temperature range are linear. (4) These results indicate that the relaxation of the activation gate in pyrethroid-affected Na+ channels is governed by an apparent first order, unimolecular process and that the rate of relaxation is limited by a single energy barrier. Application of transition state theory shows that after alpha-cyano pyrethroids this energy barrier is 9.6 kJ/mol higher than after non-cyano pyrethroids. (5) Differences in rate of decay of the Na+ tail current account for the reported differences in repetitive nerve activity induced by various pyrethroids. In addition, the effect of temperature on the rate of decay explains the increase in repetitive activity with cooling.     

Calcium antagonists nicardipine and verapamil suppress Ca(2+) entry and activate Na(+) entry into platelets

In micromolar concentrations both antagonists suppressed CA2+ entry and simultaneously elevate the agonist-induced plasma membrane depolarization due to Na+ inward current via these channels. Potentiation by nicardipine of the Na+ current induced by the platelet-activating factor, was revealed. Both antagonists caused plasma membrane depolarization suppressed by Na+ and Ca2+ ions. The depolarization disappeared after substitution of NaCl by an isotonic solution of choline chloride. The antagonists nicardipine and verapamil seem to modulate the platelet receptor-operated channels suppressing Ca2+ entry and elevating Na+ current via these channels.     

Properties of a Sodium Channel (Nax) Activated by Strong Depolarization of Xenopus Oocytes

Short (<1 sec) duration depolarization of Xenopus laevis oocytes to voltages greater than +40 mV activates a sodium-selective channel (Na(x)) with sodium permeability five to six times greater than the permeability of other monovalent cations examined, including K+, Rb+, Cs+, TMA+, and Choline+. The permeability to Li+ is about equal to that of Na+. This channel was present in all oocytes examined. The kinetics, voltage dependence and pharmacology of Na(x)distinguish it from TTX-sensitive or epithelial sodium channels. It is also different from the sodium channel of Xenopus oocytes activated by prolonged depolarization, which is more highly selective for Na+, requires prolonged depolarization to be activated, and is blocked by Li+. Intracellular Mg2+ reversibly inhibits Na(x), whereas extracellular Mg2+ does not have an inhibitory effect. Intracellular Mg2+ inhibition of Na(x), is voltage dependent, suggesting that Mg2+ binding occurs within the membrane field. Eosin is also a reversible voltage-dependent intracellular inhibitor of Na(x), suggesting that a P-type ATPase may mediate the current. An additional cytoplasmic factor is involved in maintaining Na(x) since the current runs down in internally perfused oocytes and excised membrane patches. The rundown is reversible by reintroduction of the membrane patch into oocyte cytoplasm. The cytoplasmic factor is not ATP, because ATP has no effect on Na(x) current magnitude in either cut-open or inside-out patch preparations. Extracellular Gd3+ is also an inhibitor of Na(x). Na(x) activation follows a sigmoid time course. Its half-maximal activation potential is +100 mV and the effective valence estimated from the steepness of conductance activation is 1.0. Na(x) deactivates monoexponentially upon return to the holding potential (-40 mV). The deactivation rate is voltage dependent, increasing at more negative membrane potentials.     

Modification of sodium inactivation in myelinated nerve by Anemonia toxin II and iodate. Analysis of current fluctuations and current relaxations

(1) Na+ currents and Na+ current fluctuations were measured in single myelinated nerve fibres of Rana esculenta under voltage-clamp conditions. The process of Na+ inactivation was modified by external treatment with 7 microM Anemonia Toxin II or by internal application of 20 or 40 mM IO3(-). (2) At depolarization of 24 and 32 mV the spectral density of Na+ current fluctuations could be described as the sum of two contributions, Sh(f) and Sm(f), representing the spectrum from fluctuations of the inactivation (h) and activation (m) gates, respectively. At higher depolarizations of 40 and 48 mV the low frequency (h) fluctuations could be better fitted by the sum, Sh1(f)+Sh2(f), of two separate Lorentzian functions. (3) The Na+ current and the variance of Na+ current fluctuations between 150 and 450 ms after depolarization are increased by one order of magnitude after application of Anemonia Toxin II or IO3(-). (4) The kinetics of Na+ current inactivation were described as A1 x exp(-t/tau h1) + A2 x exp(-t/tau h2) + B. The constant, tau h1, of fast Na+ inactivation was the same in normal and modified nerve fibres. The slow inactivation time constant, tau h2, increased with increasing depolarizations in modified fibres but decreased under control conditions. In all cases tau h2 showed a similar voltage dependence as the time constant found by fitting the low frequency fluctuations of Na+ current with one Lorentzian function, Sh(f). (5) It is concluded that Anemonia Toxin II and IO3(-) modify a fraction of Na+ channels in an all-or-none manner. A lower limit of the number of modified Na+ channels is estimated from the Na+ current and the variance Na+ current fluctuations. 7 microM external Anemonia Toxin II modifies more than 17% and 20 or 40 mM internal IO3(-) more than 8% of all Na+ channels. The inactivation gates in modified channels experience an electric field different from that in normal fibres.     

Na+ and K+ transport at basolateral membranes of epithelial cells. III. Voltage independence of basolateral membrane Na+ efflux

Na+ efflux across basolateral membranes of isolated epithelia of frog skin was tested for voltage sensitivity. The intracellular Na+ transport pool was loaded with 24Na from the apical solution and the rate of isotope appearance in the basolateral solution (JNa23) was measured at timed intervals of 30 s. Basolateral membrane voltage was depolarized by either 50 mM K+, 5 mM Ba++, or 80 mM NH+4. Whereas within 30 s ouabain caused inhibition of JNa23, depolarization of Vb by 30-60 mV caused no significant change of JNa23. Thus, both pump-mediated and leak Na+ effluxes were voltage independent. Although the pumps are electrogenic, pump-mediated Na+ efflux is voltage independent, perhaps because of a nonlinear relationship between pump current and transmembrane voltage. Voltage independence of the leak Na+ efflux confirms a previous suggestion (Cox and Helman, 1983. American Journal of Physiology. 245:F312-F321) that basolateral membrane Na+ leak fluxes are electroneutral.     

Extracellular polyvalent cation block of slow Na+ channels in Xenopus laevis oocytes

Sustained depolarization of the Xenopus oocyte membrane elicits a slowly activating Na+ current, thought to be due to the opening of sodium selective channels. These channels are induced to become voltage gated by the depolarization. They show unconventional gating properties and are insensitive to tetrodotoxin (TTX). The present study was undertaken to evaluate the effect of extracellular multivalent cations (Ca2+, Co2+, Cd2+, La3+, Mg2+, Mn2+, Ni2+, Sr2+ and Zn2+) on these TTX-resistant channels, either on membrane potential responses or on current responses. Our data show that all the polyvalent cations used blocked Na+ channels in a concentration-dependent manner. The order of potency of the most efficient cations was Co2+ < Ni2+ < Cd2+ < Zn2+, the respective concentration required to cause a 50% decrease of Na+ current was 0.9+/-0.29; 0.47+/-0.15; 0.36+/-0.09 and 0.06+/-0.02 mmol/l. The comparison of the activation curves from controls and after treatment with the cations indicated a shift towards more positive voltages. These results can be interpreted as due to the screening effect of divalent cations together with an alteration of the Na+ channel gating properties. We also show that divalent cations blocked Na+ channels in an open state without interfering with the induction mechanism of the channels. The possibility that cation block was due to a possible interaction between cations and SH-groups was investigated, but a sulphydryl alkylating reagent failed to abolish Zn2+ block.     

K+ secretion across frog skin. Induction by removal of basolateral Cl-

We examined the development of K+ secretion after removing Cl- from the basolateral surface of isolated skins of Rana temporaria using noise analysis. K+ secretion was defined by the appearance of a Lorentzian component in the power density spectrum (PDS) when Ba2+ was present in the apical bath (0.5 mM). No Lorentzians were observed when tissues were bathed in control, NaCl Ringer solution. Replacement of basolateral Cl- by gluconate, nitrate, or SO4- (0-Clb) yielded Lorentzians with corner frequencies near 25 Hz, and plateau values (So) that were used to estimate the magnitude of K+ secretion through channels in the apical cell membranes of the principal cells. The response was reversible and reproducible. In contrast, removing apical Cl- did not alter the PDS. Reduction of basolateral Cl- to 11.5 mM induced Lorentzians, but with lower values of So. Inhibition of Na+ transport with amiloride or by omitting apical Na+ depressed K+ secretion but did not prevent its appearance in response to 0-Clb. Using microelectrodes, we observed depolarization of the intracellular voltage concomitant with increased resistance of the basolateral membrane after 0-Clb. Basolateral application of Ba2+ to depolarize cells also induced K+ secretion. Because apical conductance and channel density are unchanged after 0-Clb, we conclude that K+ secretion is "induced" simply by an increase of the electrical driving force for K+ exit across this membrane. Repolarization of the apical membrane after 0-Clb eliminated K+ secretion, while further depolarization increased the magnitude of the secretory current. The cell depolarization after 0-Clb is most likely caused directly by a decrease of the basolateral membrane K+ conductance. Ba2(+)-induced Lorentzians also were elicited by basolateral hypertonic solutions but with lower values of So, indicating that cell shrinkage per se could not entirely account for the response to 0-Clb and that the effects of 0-Clb may be partly related to a fall of intracellular Cl-.     

Electrophysiological study of L-lysine transport across Triturus proximal tubule: evidence for Na(+)-independent entry and Na(+)-dependent exit.

Cell membrane depolarization induced by intraluminal injection of lysine was entirely independent of the presence of Na+ in Triturus proximal tubule, confirming our previous observation. The amplitude of the depolarization conformed to Michaelis-Menten kinetics regardless of the presence or absence of Na+ in the perfusion solutions. pH of the intraluminal solution had no effect on the electrical response in its range from 5.5 to 8.5. In a Na(+)-free medium, particularly in a Tris-substituted medium, the depolarization induced by a constant concentration of lysine gradually decreased in its size when injection followed by washout of lysine was repetitively tested. The addition of Na+ to the peritubular side after extinction of the responsiveness resulted in a significant restoration of the voltage response to intraluminal lysine. In addition, influx of Na+ from the peritubular fluid into the cells was significantly greater in lysine-loaded tubules than in nonloaded tubules as indicated by a greater rate of increase in intracellular Na+ activity in the presence of ouabain. The data strongly suggest that lysine enters the cells via an electrogenic uniport mechanism and leaves the cells via Na+:amino acid exchange transport mechanism.     

Conditions for a backward-running Na+/K+ pump in Xenopus oocytes.

Current generated by the electrogenic Na+/K+ pump protein was determined in oocytes of Xenopus laevis as strophantidine-sensitive current measured under voltage clamp. Under conditions of reduced intracellular [Na+] and [ATP], both to values below 1 mM, and in extracellularly K(+)-free medium, the Na+/K+ pump seems to operate in a reversed mode pumping Na+ into the cell and K+ out of the cell. This is demonstrated by strophantidine-induced hyperpolarization of the membrane and inward-directed current mediated by the pump protein. In addition, strophantidine-sensitive uptake of 22Na+ can be demonstrated under these conditions. The pump current decreases with membrane depolarization as expected for a pump cycle that involves inward movement of positive charges during Na+ translocation.     

Effects of metabolic inhibition on ion transport by dog bronchial epithelium

Mammalian bronchial epithelium absorbs Na+ under basal conditions, but Cl- secretion can be induced. We studied the effects of several modes of metabolic inhibition on the bioelectric properties and solute permeability of dog bronchial epithelium mounted in Ussing chambers. Net Na+ absorption and short-circuit current were inhibited by approximately 75% by hypoxia or by 10(-3) M NaCN. The reduced net Na+ absorption was characterized by a decrease in absorptive flux and an increase in backflux. The latter change was proportional to an increase in permeability to [14C]mannitol, implying that solute flow through a paracellular shunt was increased. In contrast, the reduction of conductance expected from exposure to amiloride (0.94 +/- 0.15 ms/cm2 or 12%) was abolished by NaCN pretreatment. Metabolic inhibition also decreased epithelial conductance and unidirectional Cl- fluxes by approximately 25%. NaCN rapidly and reversibly inhibited the hyperpolarization of potential difference (PD) induced by low luminal bath [Cl-]. This effect was mimicked by the Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid. Because the transepithelial Cl- diffusion PD reflects, in part, the depolarization of the Cl- -conductive apical cell membrane, metabolic inhibition appears to affect this path. We conclude that metabolic inhibition not only decreased net ion transport by dog bronchial epithelium but also inhibited cellular Na+- and Cl- -conductive pathways and increased paracellular permeability.     

A pH-sensitive and voltage-dependent proton conductance in the plasma membrane of macrophages

Phagocytes generate large amounts of metabolic acid during activation. Therefore, the presence of a conductive pathway capable of H+ extrusion has been suggested (Henderson, L. M., J. B. Chappell, and O. T. G. Jones. 1987. Biochemical Journal. 246:325-329). In this report, electrophysiological and fluorimetric methods were used to probe the existence of a H+ conductance in murine peritoneal macrophages. In suspended cells, recovery of the cytosolic pH (pHi) from an acid-load in Na+ and HCO3(-)-free medium was detectable in depolarizing but not in hyperpolarizing media. The rate of alkalinization was potentiated by the rheogenic ionophore valinomycin. These findings are consistent with the existence of a conductive H+ (equivalent) pathway. This notion was confirmed by patch-clamping and fluorescence ratio measurements of single adherent cells. When voltage was clamped in the whole-cell configuration, depolarizing pulses induced a sizable outward current which was accompanied by cytosolic alkalinization. Several lines of evidence indicate that H+ (equivalents) carry this current: (a) the conductance was unaffected by substitution of the major ionic constituents of the intra-and/or extracellular media, (b) the reversal potential of the tail currents approached the H+ equilibrium potential; and (c) the voltage-induced currents and pHi changes were both Zn2+ sensitive and had similar time course and potential dependence. The peak whole-cell current displayed marked outward rectification and was exquisitely H+ selective. At constant voltage, the H+ permeability was increased by lowering pHi but was inhibited by extracellular acidification. Together with the voltage dependence of the conductance, these features ensure that H+ extrusion can occur during activation, while potentially deleterious acid uptake is precluded. The properties of the conductance appear ideally suited for pHi regulation during phagocyte activation, because these cells undergo a sustained depolarization and an incipient acidification when stimulated. Comparison of the magnitude of the current with the amount of metabolic acid generated during macrophage activation indicates that the conductance is sufficiently large to contribute to the H+ extrusion required for maintenance of pHi.     

Cesium blockade of delayed outward currents and electrically induced pacemaker activity in mammalian ventricular myocardium

The effects of Cs+, 5-25 mM, were studied in cat and guinea pig papillary muscles using voltage clamp and current clamp techniques. In solutions containing normal K+, the major effects of Cs+ were depolarization of the resting potential and reduction of the delayed outward current (ixl) between -80 and -20 mV. Both inward and outward portions of the isochronal current voltage relation (l-s clamps) were reduced by extracellular Cs+. This resulted in a substantial reduction of inward rectification and, by subtraction from the normal I-V relationship, the definition of a Cs+-sensitive component of current. Under current clamp conditions, 5-10 mM Cs+ produced a dose-dependent slowing of repetitive firing induced by depolarization. At higher concentrations (25 mM) the resting potential was depolarized and repetitive activity could not be induced by further depolarization. However, release of hyperpolarizing pulses was followed by prolonged bursts of repetitive action potentials, suggesting partial reversal of blockade or participation of another pacemaker process. The experimental results and a numerical simulation show that under readily attainable conditions, reduction in an outward pacemaker current may slow pacemaker activity.     

Induction of Na+ channel voltage sensitivity in Xenopus oocytes depends on Ca2+ mobilization

An unusual inward current which is slowly elicited in the Xenopus oocyte membrane during sustained depolarization is reportedly carried by Na+. It is thought that Na+ selective channels are in some way induced to become voltage-sensitive by the depolarization. Earlier studies report that the induction process involves a phospholipase C and a protein kinase C as well as calcium ions. The present work investigated the origins of this calcium in the oocyte. We show that injection of the powerful Ca2+ chelator (BAPTA) in the oocyte, before induction of the Na+ channels, prevented the appearance of the Na+ current, confirming an important role for [Ca2+]i. However, in oocytes perfused with Ca2+ -free medium, induction of the channels could still be obtained, indicating that induction did not depend upon the entry of external Ca2+. Downmodulation of Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive stores with caffeine and with a low molecular weight heparin resulted in decreased or no Na+ currents. The results are discussed in terms of the contributions from other endogenous calcium-dependent conductances which can influence the Na+ current amplitudes and time courses. The results presented support the idea that intracellular Ca2+ increase principally due to Ca2+ released from InsP3-sensitive stores is needed by the enzyme systems to produce the depolarization-induced activation of the Na+ conductance in the Xenopus oocyte.     

G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

Our previous experiments in membranes prepared from rat heart and brain led us to suggest that the binding of agonists to the muscarinic receptors and to the Na+ channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [3H]acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [3H]batrachotoxin to Na+ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22Na+ uptake in the presence and absence of tetrodotoxin, which blocks Na+ channels. Our findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na+ channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components--receptor, G-protein, and Na+ channel--is such that at resting potential the muscarinic receptor induces opening of Na+ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.     

Multiple acute effects on the membrane potential of PC12 cells produced by nerve growth factor (NGF)

We studied whether nerve growth factor (NGF) can affect the membrane potential and conductance of PC12 cells. We demonstrate that NGF depolarizes the membrane of PC12 cells within a minute and by using transfected NIH 3T3-Trk and -p75 cells we show that both the high affinity NGF receptor p140(trk) and the low affinity NGF receptor or p75(NGF) may be involved in the depolarization. Tyrosine kinase inhibitor, K252a, partially inhibited the depolarization, but two agents affecting intracellular calcium movements, Xestospongin C (XeC) and thapsigargin, did not. The early depolarization was eliminated in Na+ free solutions and under this condition, a 'prolonged' (> 2 min) hyperpolarization was observed in PC12 cells in response to NGF. This hyperpolarization was also induced in PC12 cells by epidermal growth factor (EGF). Voltage clamp experiments showed that NGF produced a late (> 2 min) increase in membrane conductance. The Ca2+-dependent BK-type channel blocker, iberiotoxin, and the general Ca2+-dependent K+ channel blocker, TEA, attenuated or eliminated the hyperpolarization produced by NGF in sodium free media. Under pretreatment with the non-selective cation channel blockers La3+ and Gd3+, NGF hyperpolarized the membrane of PC12 cells. These results suggest that three different currents are implicated in rapid NGF-induced membrane voltage changes, namely an acutely activated Na+ current, Ca2+-dependent potassium currents and non-selective cation currents.     

Calcium-dependent sodium currents inParamecium: Mutational manipulations and effects of hyper- and depolarization

Summary The membrane ofParamecium generates a Ca-dependent Na current upon depolarization. There is, however, also a Na current upon hyperpolarization in this membrane. The second Na current was analyzed under voltage clamp and found to have properties identical to those of the first. Both currents could be carried by Na and Li ions and not by K, Cs or choline ion. They were eliminated by either EGTA injection into the cell or Ca removal from the bath. Both currents were eliminated by a single-gene mutation,fast-2, that had no effect on Ca currents. These findings strongly suggest that these two currents are through the same Ca-dependent Na conductance. A hyperpolarization-induced Ca current was also identified, which served to activate the second Na current. These observations support a model that theParamecium membrane has two Ca channels with different voltage dependencies and only one Na channel, which is elicited by a rise of the itternal free Ca²⁺ concentration. The function of the Ca-dependent Na conductance is discussed.     

Voltage-dependent block of cardiac inward-rectifying potassium current by monovalent cations

The inward-rectifying K+ current (IK1) in cat ventricular myocytes, like inward-rectifying K+ currents in many other preparations, exhibited a negative slope conductance region at hyperpolarized membrane potentials that was time-dependent. This was evident as an inactivation of inward current elicited by hyperpolarizing voltage-clamp pulses resulting in a negative slope region of the steady-state current-voltage relationship at potentials negative to -140 mV. Removing extracellular Na+ prevented the development of the negative slope in this voltage region, suggesting that Na+ can block IK1 channels in a time- and voltage-dependent manner. The time and voltage dependence of Cs+-induced block of IK1 was also examined. Cs+ blocked inward current in a manner similar to that of Na+, but the former was much more potent. The fraction of current blocked by Cs+ in the presence of Na+ was reduced in a time- and voltage-dependent manner, which suggested that these blocking ions compete for a common or at least similar site of action. In the absence of Na+, inactivation of IK1 could also be induced by both Cs+ and Li+. However, Li+ was less potent than Na+ in this respect. Calculation of the voltage sensitivity of current block by each of these ions suggests that the mechanism of block by each is similar.     

The role of transmembrane cationic gradients in immune complex stimulation of human polymorphonuclear leukocytes

The role of monovalent cationic gradients in human polymorphonuclear leukocyte (PMNL) stimulation was investigated by monitoring immune complex-stimulated transmembrane depolarization and superoxide production, events which accompany--and have been used as indicators of --PMNL activation. Abolishing only the Na+ gradient by substitution of choline for extracellular Na+ did not affect the resting membrane potential but reduced the rate of stimulus-induced transmembrane depolarization to 50% of control. In contrast, collapsing both Na+ and K+ gradients by suspension in K+ buffer (high K-PRK) depolarized the cells and reduced the stimulus-induced rate of depolarization to 11% of control. Pretreatment of cells suspended in Na+ buffers with 5-(N,N-dimethyl)amiloride hydrochloride (DMA) or with valinomycin reduced by one-half the rate of immune complex induced membrane depolarization. Conversely, in the absence of either or of both Na+ or K+ gradients, or in the presence of valinomycin, immune complex elicited an enhanced rate of superoxide production. However, PMNL prepared via NH4Cl (NH4Cl-PMNL) instead of H2O (H2O-PMNL) lysis of residual red blood cells exhibited an absolute requirement for an intact Na+ gradient in cell stimulation. The present results thus demonstrate that: 1) both Na+ and K+ gradients participate equally in the membrane depolarization elicited by immune complex; 2) neither a Na+ or a K+ gradient is required for immune complex activation, or for activity of the respiratory burst; and 3) an artifactual requirement for an intact Na+ gradient occurs in neutrophils prepared by the NH4Cl lysis technique.     

A slowly inactivating potassium current in native oocytes of Xenopus laevis

Membrane currents were recorded in voltage-clamped oocytes of Xenopus laevis in response to voltage steps. We describe results obtained in oocytes obtained from one donor frog, which showed an unusually large outward current upon depolarization. Measurements of reversal potentials of tail currents in solutions of different K+ concentration indicated that this current is carried largely by K+ ions. It was strongly reduced by extracellular application of tetraethylammonium, though not by Ba2+ or 4-aminopyridine. Removal of surrounding follicular cells did not reduce the K+ current, indicating that it arises across the oocyte membrane proper. Activation of the K+ conductance was first detected with depolarization to about -12 mV, increased with a limiting voltage sensitivity of 3 mV for an e-fold change in current, and was half-maximally activated at about +10 mV. The current rose following a single exponential timecourse after depolarization, with a time constant that shortened from about 400 ms at -10 mV to about 15 ms at +80 mV. During prolonged depolarization the current inactivated with a time constant of about 4 s, which did not alter greatly with potential. The K+ current was independent of Ca2+, as it was not altered by addition of 10 mM Mn2+ to the bathing medium, or by intracellular injection of EGTA. Noise analysis of K+ current fluctuations indicated that the current is carried by channels with a unitary conductance of about 20 ps and a mean open lifetime of about 300 ms (at room temperature and potential of +10 to +20 mV).