Ionic currents in two strains of rat anterior pituitary tumor cells

The ionic conductance mechanisms underlying action potential behavior in GH3 and GH4/C1 rat pituitary tumor cell lines were identified and characterized using a patch electrode voltage-clamp technique. Voltage-dependent sodium, calcium, and potassium currents and calcium-activated potassium currents were present in the GH3 cells. GH4/C1 cells possess much less sodium current, less voltage-dependent potassium current, and comparable amounts of calcium current. Voltage-dependent inward sodium current activated and inactivated rapidly and was blocked by tetrodotoxin. A slower-activating voltage-dependent inward calcium current was blocked by cobalt, manganese, nickel, zinc, or cadmium. Barium was substituted for calcium as the inward current carrier. Calcium tail currents decay with two exponential components. The rate constant for the slower component is voltage dependent, while the faster rate constant is independent of voltage. An analysis of tail current envelopes under conditions of controlled ionic gradients suggests that much of the apparent decline of calcium currents arises from an opposing outward current of low cationic selectivity. Voltage-dependent outward potassium current activated rapidly and inactivated slowly. A second outward current, the calcium-activated potassium current, activated slowly and did not appear to reach steady state with 185-ms voltage pulses. This slowly activating outward current is sensitive to external cobalt and cadmium and to the internal concentration of calcium. Tetraethylammonium and 4-aminopyridine block the majority of these outward currents. Our studies reveal a variety of macroscopic ionic currents that could play a role in the initiation and short-term maintenance of hormone secretion, but suggest that sodium channels probably do not make a major contribution.     

巨孔匙(虫戚)(Megathura)未受精卵细胞膜离子流的特征

用双微电极电压钳技术在巨孔匙(虫戚)(Megathura)未受精卵细胞膜上记录到多种离子流。主要有一种内向的两价离子流和几种钾离子流:包括钡离子激活的钾离子流,迅速激活又迅速失活的钾离子流(类似于I_A)和异常整流钾离子流。不同细胞的离子流大小不同。在一些卵可能会缺少其中某一种离子流。此外,还观察到浴槽溶液中氯和钠离子浓度改变对膜电位及膜电导的影响。     

Ionic currents in avian granulosa cells

Transmembrane ionic currents have been recorded in single granulosa cells from the laying hen using the whole-cell patch-clamp technique. Under voltage-clamp conditions, depolarizing voltage steps evoked currents composed of a fast inactivating inward component and a delayed outward component. The former was activated at voltages more positive than -50 mV and was fully inactivated within 500 ms. It was blocked by D600 (methoxyverapamil) and by cobalt, suggesting that it is a calcium current. The latter displayed inward rectification and did not inactivate during long duration pulses. It was blocked by tetraethylammonium indicating that it is a potassium current. This is the first evidence of the existence of potassium and calcium transmembrane currents in granulosa cells.     

Slow components of potassium tail currents in rat skeletal muscle

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.     

Effects of conditioning polarization on the membrane ionic currents in rat myometrium

Summary Membrane ionic currents were measured in pregnant rat uterine smooth muscle under voltage clamp conditions by utilizing the double sucrose gap method, and the effects of conditioning pre-pulses on these currents were investigated. With depolarizing pulses, the early inward current was followed by a late outward current. Cobalt (1mm) abolished the inward current and did not affect the late outward currentper se, but produced changes in the current pattern, suggesting that the inward current overlaps with the initial part of the late outward current. After correction for this overlap, the inward current reached its maximum at about +10 mV and its reversal potential was estimated to be +62 mV. Tetraethylammonium (TEA) suppressed the outward currents and increased the apparent inward current. The increase in the inward current by TEA thus could be due to a suppression of the outward current. The reversal potential for the outward current was estimated to be –87 mV. Conditioning depolarization and hyperpolarization both produced a decrease in the inward current. Complete depolarization block occurred at a membrane potential of –20 mV. Conditioning hyperpolarization experiments in the presence of cobalt and/or TEA revealed that the decrease in the inward current caused by conditioning hyperpolarization was a result of an increase in the outward current overlapping with the inward current. It appears that a part of the potassium channel population is inactivated at the resting membrane potential and that this inactivation is removed by hyperpolarization.     

Two Fast Transient Current Components during Voltage Clamp on Snail Neurons

Voltage clamp currents from medium sized ganglion cells of Helix pomatia have a fast transient outward current component in addition to the usually observed inward and outward currents. This component is inactivated at normal resting potential. The current, which is carried by K+ ions, may surpass leakage currents by a factor of 100 after inactivation has been removed by hyperpolarizing conditioning pulses. Its kinetics are similar to those of the inward current, except that it has a longer time constant of inactivation. It has a threshold close to resting potential. This additional component is also present in giant cells, where however, it is less prominent. Pacemaker activity is controlled by this current. It was found that inward currents have a slow inactivating process in addition to a fast, Hodgkin-Huxley type inactivation. The time constants of the slow process are similar to those of slow outward current inactivation.     

Comparison of effects of selected local anesthetics on sodium and potassium channels in mammalian neuron

Effects of procaine, trimecaine, and a new carbanilate local anesthetic, carbizocaine, on early sodium inward current and fast and slow components of potassium outward current in the membrane of the rat dorsal root ganglion neuron were studied using the internal dialysis and potential clamp techniques. All the currents studied were depressed in the presence of the drugs tested. However, for inhibition of the inward current concentrations lower by approximately one to more than two orders were sufficient compared to those required for similar inhibition of the outward currents. Carbizocaine was the most effective, procaine the least effective drug. Almost identical ratios of the negative logarithms of mean effective concentrations for blocking the inward and the outward current respectively, were found for each of the drugs tested. None of the drugs could be characterized as a specific blocker of sodium or potassium channels. It is concluded that the mechanisms of action of these three local anesthetics in all the three types of ion channels studied in the neuronal membrane are very similar regardless of both the type of the chemical bond in the intermediary chain of the molecules (ester, anilide, carbanilate) and the structure of the aromatic moiety, or the absolute potency of the drug.     

Investigation of the TEA-resistant outward current in the somatic membrane of perfused nerve cells

Outward currents remaining after addition of 20–50 mM of tetraethylammonium (TEA) ions to the extracellular or intracellular solution, were investigated in perfused isolatedHelix neurons. After this addition, the inactivated inward current carried by potassium ions, the potential-dependent and kinetic characteristics of which differ from those of potassium outward currents suppressed by TEA, is preserved in the membrane. A component dependent on the inward calcium current was found in this TEA-resistant outward current; it was abolished by replacement of the extra-cellular calcium ions by magnesium ions, by blocking of the calcium channels by extracellular cadmium ions, and by their destruction by intracellular fluoride ions. Increasing the intracellular concentration of free calcium ions by perfusing the cell with solutions containing calcium-EGTA buffer potentiated the TEA-resistant component of the outward current, whereas removal of these ions with EGTA weakened it. It is concluded that a system of outward current channels whose activation depends on the presence of calcium ions near the inner surface of the membrane is present in the somatic membrane. It is suggested that to keep these channels capable of being activated, calcium ions must bind with the structures forming their internal opening.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 460–468, September–October, 1979.     

Blockade of sodium and potassium channels in the node of Ranvier by ajmaline and N-propyl ajmaline

The inhibition of sodium and potassium currents in frog myelinated fibres by ajmaline (AM) and its quaternary derivative, N-propyl ajmaline (NPA), depends on voltage-clamp pulses and the state of channel gating mechanisms. The permanently charged NPA and protonated AM interact only (or mainly) with open channels, while unprotonated AM affects preferently inactivated Na channels. Inhibition of Na currents by NPA and AM does not depend on the current direction and Na ion concentration in external or internal media. In contrast only the outward potassium currents can be blocked by NPA and AM; the inward potassium currents in high K+ ions external media are resistant to the blocking action of these drugs. The voltage dependence of ionic current inhibition by charged drugs suggests the location of their binding sites in the inner mouths of Na and K channels. Judging by the kinetics of current restoration after cessation of pulsing, the drug-binding site complex is much more stable in Na than in potassium channels. Batrachotoxin and aconitine, unlike veratridine and sea anemone toxin, decrease greatly the affinity of Na channel binding sites to NPA and AM. The effects of NPA and AM are compared with those of local anesthetics and other amine blocking drugs.     

Whole-cell clamp of dissociated photoreceptors from the eye of Lima scabra

Voltage-dependent membrane currents were investigated in enzymatically dissociated photoreceptors of Lima scabra using the whole-cell clamp technique. Depolarizing steps to voltages more positive than -10 mV elicit a transient inward current followed by a delayed, sustained outward current. The outward current is insensitive to replacement of a large fraction of extracellular Cl- with the impermeant anion glucuronate. Superfusion with tetraethylammonium and 4-aminopyridine reversibly abolishes the outward current, and internal perfusion with cesium also suppresses it, indicating that it is mediated by potassium channels. Isolation of the inward current reveals a fast activation kinetics, the peak amplitude occurring as early as 4-5 ms after stimulus onset, and a relatively rapid, though incomplete inactivation. Within the range of voltages examined, spanning up to +90 mV, reversal was not observed. The inward current is not sensitive to tetrodotoxin at concentrations up to 10 microM, and survives replacement of extracellular Na with tetramethylammonium. On the other hand, it is completely eliminated by calcium removal from the perfusing solution, and it is partially blocked by submillimolar concentrations of cadmium, suggesting that it is entirely due to voltage-dependent calcium channels. Analysis of the kinetics and voltage dependence of the isolated calcium current indicates the presence of two components, possibly reflecting the existence of separate populations of channels. Barium and strontium can pass through these channels, though less easily than calcium. Both the activation and the inactivation become significantly more sluggish when these ions serve as the charge carrier. A large fraction of the outward current is activated by preceding calcium influx. Suppression of this calcium-dependent potassium current shows a small residual component resembling the delayed rectifier. In addition, a transient outward current sensitive to 4-aminopyridine (Ia) could also be identified. The relevance of such conductance mechanisms in the generation of the light response in Lima photoreceptors is discussed.     

Membrane properties of isolated mudpuppy taste cells

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.     

Voltage gated ionic channels in rat cultured astrocytes, reactive astrocytes and an astrocyte-oligodendrocyte progenitor cell

Astrocytes (both type 1 and type 2), cultured from the central nervous system of newborn or 7 day old rats show voltage gated sodium and potassium channels that are activated when the membrane is depolarized to greater than -40 mV. The sodium channels in these cells have an h-infinity curve similar to that of nodal membranes but the activation (peak current-voltage) curves are shifted along the voltage axis by about +30 mV. These sodium currents are blocked only by high concentrations of tetrodotoxin. The voltage activated potassium currents in both types of astrocyte show at least two components; an inactivating component that is suppressed at holding potentials of greater than -40 mV and a persistent, non-inactivating current. Several types of single channel currents were observed in outside-out membrane patches from type 2 astrocytes. One type of potassium channel showed inactivation on depolarization and may contribute to the whole-cell inactivating current. In contrast, oligodendrocytes showed no obvious voltage gated membrane channels. The properties of the type 2 astrocyte-oligodendrocyte progenitor cell were investigated in two ways: 1) by examination of cells just beginning to differentiate along the "electrically silent" oligodendrocyte pathway or 2) by recording from progenitor cells cultured for 24 hours in the presence of cycloheximide to block the appearance of new membrane channels. In both cases, voltage gated inward (sodium) and outward (potassium) currents were noted. The outward current response showed both an inactivating and a non-inactivating component. Similar voltage activated inward and outward membrane currents were noted in reactive astrocytes freshly isolated (3-6 hours) from lesioned areas of adult rat brains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioids activate both an inward rectifier and a novel voltage-gated potassium conductance in the hippocampal formation

Opioid receptors were found to activate two different types of membrane potassium conductance in acutely dissociated neurons from the CA1/subiculum regions of the adult rat hippocampal formation. Opioid-responsive neurons were distinguished based on their morphology and electrophysiological responses. In one population of neurons having a multipolar, nonpyramidal cell shape, mu-selective opioid agonists increased an inward rectifying potassium current. Opioid activation of the inward rectifying conductance resulted in small outward potassium currents at resting membrane potentials and increased inward currents at hyperpolarized potentials. In a second population of nonpyramidal neurons, mu opioid agonists increased a novel voltage-gated potassium current. This current was blocked by internal CsCl2, unaffected by external BaCl2 or CdCl2, irreversibly activated by intracellular GTP-gamma-S, and inactivated by sustained depolarization. In contrast to the inward rectifying conductance, the voltage-gated conductance was not activated at resting membrane potentials or hyperpolarized potentials. The opioid-activated, voltage-gated conductance represents a new class of G protein-regulated potassium current in the brain.     

Calcium currents and calcium-activated potassium currents at the frog motor nerve endings

Intracellular recordings were made of evoked electrical response of the nerve endings during experiments on the frog cutaneous pectoral muscle. A delayed inward current was discovered when superfusing the neuromuscular preparation with a calcium-free solution containing tubocurarine in the response evoked at the nerve endings, using CaCl₂-filled electrodes. This was replaced by the opposite (outward) type of current when 4-aminopyridine was added to the external solution. The outward current was dependent on the calcium concentration at the electrode, decreased after local increase on potassium concentration at the electrode, and disappeared under the effects of cobalt. Local iontophoretic application of tetraethylammonium led to the disappearance of the outward current and the appearance of a powerful and protracted inward current. Similar readings of inward and outward currents were obtained when recording electrical signals using electrodes filled with SrCl₂, BaCl₂, and MgCl₂. It was deduced that the late inward current is carried through voltage-dependent calcium channels and outward delayed current through calcium-activated potassium channels at the nerve terminal. The part played by these currents in transmitter secretion from the motor nerve ending is discussed, together with the relationship between them.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR. V. I. Ul'yanov-Lenin State University, Kazan'. Translated from Neirofiziologiya, Vol. 19, No. 4, 1987, pp. 467–473, July–August, 1987.     

Voltage-dependent membrane ionic currents in lamprey striated muscle

Membrane ionic currents in striated muscle bundles of lamprey suction apparatus were recorded using a double sucrose gap technique. Transmembrane currents in a single muscle fiber and a fiber bundle in the frog were compared so as to check the validity of current measurement in multicell preparations. It was found that fast inward sodium currents arise in the lamprey muscle membrane in response to depolarization together with a delayed outward potassium current, with steady-state characteristics resembling those of membrane currents in frog muscle. The only difference consisted of a flatter curve for steady-state inactivation of potassium current, probably indicative of greater density of potassium channels. Both the changes in reversal potential and the speed of potassium current deactivation occurring during protracted stimuli point to the presence of two fractions in this current. No functioning voltage-dependent calcium channels are found in the lamprey muscle membrane.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 629–636, September–October, 1986.     

Separation of potassium and calcium channels in the nerve cell soma membranes

Investigation of isolated neurons ofHelix pomatia during intracellular dialysis revealed differences in the sensitivity of the channels for the outward potassium and inward calcium currents to changes in pH of the external medium. As a result of this difference, considerable separation of the regions of activation of the currents was obtained along the potential axis in solutions with low pH and the characteristics of the inward and outward currents could be studied during their minimal application. Channels for the outward current were shown to have some permeability for tris ions (P_Tris:P_K=0.05), which is the reason why it is impossible to block this current completely by replacing the intracellular potassium by Tris. Channels for the inward calcium current are characterized by slow inactivation, with a first-order kinetics; their momentary voltage-current characteristic curve reveals significant Goldman's rectification. The selectivity of the calcium channels for other bivalent cations is: Ba:Sr:Ca:Mg=2.8:2.6:1.0:0.2.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 6, pp. 645–653, November–December, 1978.     

Potentiated L-type Ca2+ channels rectify

Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type alpha1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-alpha1C) or repeat III (E3K-alpha1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-alpha1C and E3K-alpha1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (-)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-alpha1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-alpha1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels.     

Effects of capsaicin on molluscan neurons: a voltage clamp study

The effects of capsaicin (CAP) on membrane ionic currents of identified and non-identified neurons were investigated by use of the single electrode clamp (SEC). CAP (300 microM, 22 degrees C, pH 7.4) caused a 25-50% reduction of the inward current and a 50-80% reduction of the outward current in normal or Na-free (Tris) solution. The Na current (INa) was moderately decreased (about 10%) in LPa2 neuron, but a 50% reduction of the peak Ca current (ICa) was observed. The action of CAP on ICa varied from cell to cell but an enhanced inactivation of the fast calcium current was found in all neurons studied. CAP (150 microM, 10 min) highly attenuated the long-lasting component of the inward current in LPa2 recorded in Na-free (TEA) Ba solutions. CAP attenuated the fast outward current (IA) and voltage-dependent outward current (IK) in 100 and 300 microM concentrations for the half blocking dose (ID50) in LPa2 neuron, respectively. CAP decreased the slow outward tail currents but hardly influenced the leakage current (IL). We suggest that the acute action of CAP coupled with a series of events in the neuronal membrane can modify the conductance via electrically excitable calcium, potassium and sodium channels differentially.     

A Fast Transient Outward Monovalent Current in Rat Saphenous Myocytes Passing Through Ca2+ Channels

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of -100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 +/- 0.8 ms and 23.9 +/- 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at -55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1-5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.     

Inward and outward currents in isolated dendrites of crustacea coxal receptors

Segments from the nonspiking peripheral dendrites of a crustacean coxal receptor (T fiber) were studied using the voltage clamp technique. The peripheral endings of the T fiber are sensitive to stretch applied to a specialized receptor muscle by rotation of the coxa. The intraganglionary portion of the T fiber is presynaptic to the motor neurons innervating the coxal muscle. Depolarizing commands activated three separate fast channels: (i) a transient inward sodium current, INa, which is blocked by tetrodotoxin (TTX); (ii) a transient outward current, Io1 , having the same voltage-dependent characteristics as INa; and (iii) a second, longer-lasting, outward current, Io2 . Both INa and Io1 were inactivated when segments were clamped at voltages more positive than -50 mV, whereas Io2 could be activated at voltages more positive than -50 mV. Io1 and Io2 were blocked by 4-aminopyridine (4-AP) and by tetraethylammonium (TEA), although Io2 shows a greater sensitivity to TEA than Io1 . It is suggested that Io1 may be a factor in determining the nonspiking behavior of the dendrites and that Io2 may limit the stretch-induced depolarization in the dendrite to a value more negative than that at which the maximum rate of transmitter release occurs. In addition to the three fast currents, the presence of a slow inward and slow outward current could also be demonstrated. The effects of the slow currents were longer in segments cut from the proximal part of the dendrites.