Ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis

The ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis were examined by using conventional two-electrode voltage clamp and whole-cell patch clamping methods. Several separable currents were identified. These include: (1) a transient and (2) a steady-state voltage-activated inward current; both are tetrodotoxin (TTX) and saxitoxin (STX) insensitive, partly reduced by decreasing external Ca2+ or Na+ or by addition of 5 mM Co2+, D-600 or verapamil and are totally blocked with 5 mM Cd2+; (3) an early, transient, cation-dependent, outward K+ current (IKCa/Na); (4) a transient, voltage-activated, outward K+ current provisionally identified as IA; (5) a delayed, steady-state, voltage-activated outward K+ current (IK) and (6) a late, transient, outward K+ current which is blocked by Cd2+ and evident only during long voltage pulses. Despite their phylogenic origin, most of these currents are similar to currents identified in many vertebrate smooth and cardiac muscle preparations, and other excitable cells in higher animals.     

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.     

Ionic currents in cardiac myocytes of squid, Alloteuthis subulata

This paper provides the first study of voltage-sensitive membrane currents present in heart myocytes from cephalopods. Whole cell patch clamp recordings have revealed six different ionic currents in myocytes freshly dissociated from squid cardiac tissues (branchial and systemic hearts). Three types of outward potassium currents were identified: first, a transient outward voltage-activated A-current (I_A), blocked by 4-aminopyridine, and inactivated by holding the cells at a potential of −40 mV; second, an outward, voltage-activated, delayed rectifier current with a sustained time course (I_K); and third, an outward, calcium-dependent, potassium current (I_K(Ca)) sensitive to Co²⁺ and apamin, and with the characteristic N-shaped current voltage relationship. Three inward voltage-activated currents were also identified. First, a rapidly activating and inactivating, sodium current (I_Na), blocked by tetrodotoxin, inactivated at holding potentials more positive than −40 mV, and abolished when external sodium was replaced by choline. Second, an L-type calcium current (I_Ca,L) with a sustained time course, suppressed by nifedipine or Co²⁺, and enhanced by substituting Ca²⁺ for Ba²⁺ in the external medium. The third inward current was also carried by calcium ions, but could be distinguished from the L-type current by differences in its voltage dependence. It also had a more transient time course, was activated at more negative potentials, and resembled the previously described low-voltage-activated, T-type calcium current. Accepted: 24 September 1999     

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 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.     

Nitric oxide activates voltage-dependent potassium currents of crustacean skeletal muscle.

Nitric oxide (NO), a radical gas, acts as a multifunctional intra- and intercellular messenger. In the present study we investigated the effects of NO on muscle membrane potassium currents of isolated single muscle fibers from the marine isopods, Idotea baltica, using two-electrode voltage clamp recording techniques. Voltage-activated potassium currents consist of an outward current with fast activation and inactivation kinetics and a delayed, persistent outward current. Both currents were blocked by extracellular 4-aminopyridine and tetraethylammonium; the currents were not blocked by charybdotoxin or apamin. Application of the NO donors S-nitroso-N-acetylpenicillamine (SNAP) or hydroxylamine increased both the early and the delayed outward current in a dose- and time-dependent manner. PTIO, a NO scavenger, suppressed the effect of SNAP. N-Acetyl-dl-penicillamine, a related control compound which does not liberate NO, had no significant effect on outward currents. Methylene blue, a guanylyl cyclase inhibitor, prevented the increase of the outward current while 8-bromo-cGMP increased the current. Our experiments show that potassium currents of Idotea muscle are increased by NO donors. They suggest that NO by stimulating cGMP production mediates the effects on membrane currents involved in regulation of invertebrate muscle excitability.     

Effect of potassium channel blocking agents on the actions of phenylephrine in rabbit taenia caeci

The effects of tetraethylammonium, apamin, 4-aminopyridine and holding potential on the phenylephrine-evoked outward currents in dispersed smooth muscle cells of the rabbit taenia caeci were analyzed using the whole cell patch clamp method. Phenylephrine (10 mumol/l) under the double sucrose gap condition, substantially hyperpolarized the smooth muscle membrane and reduced the input membrane resistance. This concentration of phenylephrine enhanced the frequency and amplitude of spontaneous transient outward currents (s.t.o.c.s) and elicited a low amplitude sustained outward current which were voltage and temperature dependent. In addition, phenylephrine (10 mumol/l) reduced the outward current evoked by voltage steps. Tetratehylammonium (1-5 mmol/l) attenuated the depolarization-evoked outward current, blocked the appearance of s.t.o.c.s, and fully abolished the phenylehrine induced changes in membrane currents. Apamin (0.1-10 mumol/l) only slightly affected the evoked outward current and s.t.o.c.s. However apamin did not change the phenylephrine-induced outward currents. Pretreatment with 4-aminopyridine (0.5-2 mmol/l) did not reduce the phenylephrine-induced sustained outward current and s.t.o.c.s but prevented the phenylephrine induced reduction of the depolarization-evoked outward current. These results are in favour of assumption that the phenylephrine induced hyperpolarization and reduction in the input membrane resistance are consequences of an enhanced potassium current via tetraethylammonium-sensitive, apamin and 4-aminopyridine resistant potassium channels.     

Serotonin decreases a background current and increases calcium and calcium-activated current in pedal neurons ofHermissenda

The effects of serotonin (5-HT) on membrane potential, membrane resistance, and select ionic currents were examined in large pedal neurons (LP1, LP3) of the mollusk Hermissenda. Calcium (Ca) action potentials were evoked in sodium-free artificial seawater containing tetramethylammonium, tetraethylammonium, and 4-aminopyridine (0-Na, 4-AP, TEA ASW). They failed at stimulation rates greater than 0.5/sec and were blocked by cadmium (Cd). Under voltage clamp the calcium current (ICa) responsible for them also failed with repeated stimulation. Thus, ICa inactivation accounts for refractoriness of the Ca action potential. The addition of 10 microM 5-HT to 0-Na, 4-AP, TEA ASW produced a slight depolarization and increased excitability and input resistance. Under voltage clamp the background current decreased. The voltage-dependent inward, late outward, and outward tail currents, sensitive to Cd, increased. ICa inactivation persisted. Under voltage clamp with Ca influx blocked by Cd, the addition of 10 microM 5-HT decreased the remaining current uniformly over membrane potentials of -10 to -100 mV. Thus, 5-HT reduces a background current that is active within the physiological range of the membrane potential, voltage insensitive, independent of Ca influx, noninactivating, and not blocked by 4-AP or TEA.     

Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane currents in a calcium type muscle membrane under voltage clamp.

Four ionic current components were identified in the total membrane current recorded under voltage clamp conditions from the muscle membrane of the crayfish (Astacus fluviatilis). The early inward current component is dependent on the presence of Ca2+ ions, disappears in Ca2+ free solutions and is insensitive to variaton of external Na+ ions and to tetrodotoxin. The outward current consists of at least three components, an early, a late and a slow outward current. The outward currents are sensitive to TEA and their reversal potentials differ. The early potassium current may be separated in a proportion of fibres by a hump from the later potassium current. An insufficient space clamp as a cause of the hump was excluded by comparing the size of the clamped membrane area with the distribution of large membrane clefts in the fibre. The early outward current is critically dependent on the presence of Ca2+ ions and is relatively more sensitive to TEA ions and to conditioning depolarisation than the late outward current.     

Action of quinidine on ionic currents of molluscan pacemaker neurons

The effects of quinidine on the fast, the delayed, and the Ca2+- activated K+ outward currents, as well as on Na+ and Ca2+ inward currents, were studied at the soma membrane from neurons of the marine mollusk Aplysia californica. External quinidine blocks these current components but to different degrees. Its main effect is on the voltage- dependent, delayed K+ current, and it resembles the block produced by quaternary ammonium ions (Armstrong, C. M., 1975, Membranes, Lipid Bilayers and Biological Membranes: Dynamic Properties, 3:325-358). The apparent dissociation constant is 28 microM at V = +20 mV. The blocking action is voltage and time dependent and increases during maintained depolarization. The data are consistent with the block occurring approximately 70-80% through the membrane electric field. Internal injection of quinidine has an effect similar to that obtained after external application, but its time course of action is faster. External quinidine may therefore have to pass into or through the membrane to reach a blocking site. The Ca2+-activated K+ current is blocked by external quinidine at concentrations 20-50-fold higher compared with the delayed outward K+ current. In addition, it prolongs the time course of decay of the Ca2+-activated K+ current. Na+ and Ca2+ inward currents are also blocked by external quinidine, but again at higher concentrations. The effects of quinidine on membrane currents can be seen from its effect on action potentials and the conversion of repetitive "beating" discharge activity to "bursting" pacemaker activity.     

Reduction of calcium currents in identified neurons of Helix pomatia: intracellular injection of D890

The actions of intracellularly applied D890 on membrane currents of the identified neurons B1, B2 and B3 of Helix pomatia were investigated. The TTX-resistant component of the inward current, the inward currents in Na+-free sucrose solution and in Ca2+-free Ba2+ solution were reduced. In Ca2+-free Co2+ solution the inward current was not affected. The late outward currents were strongly reduced. In solutions containing 20 mmol/l NiCl2 the remaining parts of these currents were blocked only to a lesser extent. The early outward current remained unchanged. It is concluded that intracellularly applied D890 mainly exerts its effects on the calcium current.     

Presence of a calcium-activated chloride current in mouse ventricular myocytes

The properties of several components of outward K(+) currents, including the pharmacological and kinetics profiles as well as the respective molecular correlates, have been identified in mouse cardiac myocytes. Surprisingly little is known with regard to the Ca(2+)-activated ionic currents. We studied the Ca(2+)-activated transient outward currents in mouse ventricular myocytes. We have identified a 4-aminopyridine (4-AP)- and tetraethyl ammonium-resistant transient outward current that is Ca(2+) dependent. The current is carried by Cl(-) and is critically dependent on Ca(2+) influx via voltage-gated Ca(2+) channels and the sarcoplasmic reticulum Ca(2+) store. The current can be blocked by the anion transport blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Single channel recordings reveal small conductance channels (approximately 1 pS in 140 mM Cl(-)) that can be blocked by anion transport blockers. Ensemble-averaged current faithfully mirrors the transient kinetics observed at the whole level. Niflumic acid (in the presence of 4-AP) leads to prolongation of the early repolarization. Thus this current may contribute to early repolarization of action potentials in mouse ventricular myocytes.     

The mechanism of rectification of iK1 in canine Purkinje myocytes.

We have characterized the inward rectifying background potassium current, iK1, of canine cardiac Purkinje myocytes in terms of its reversal potential, voltage activation curve, and "steady-state" current-voltage relation. The latter parameter was defined from the difference current between holding currents in the presence and absence of 20 mM cesium. Our data suggest that iK1 rectification does not arise exclusively from voltage-dependent gating or exclusively from voltage-dependent blockade by internal magnesium ions. The voltage activation curve constructed from tail currents fit to a Boltzmann two-state model predicts less outward current than is actually observed. The magnesium-dependent rectification due to channel blockade is too fast to account for the time-dependent gating of iK1 that gives rise to the tail currents. We propose a new model of rectification that assumes that magnesium blockade of the channel occurs simultaneously with voltage-dependent gating. The new model incorporates the kinetic schema elaborated by Matsuda, H. (1988. J. Physiol. 397:237-258) to explain the appearance of subconducting states of the iK1 channel in the presence of blocking ions. That schema suggested that iK1 channels were composed of three parallel pores, each of which could be blocked independently. In our model we considered the consequences of partial blockade of the channel. If the channels are partially blocked at potentials where normally they are mostly gated closed, and if the partially blocked channels cannot close, then blockade will have the paradoxical result of enhancing the current carried by iK1.     

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.     

Endplate channel block by guanidine derivatives

The effects of the n-alkyl derivatives of guanidine on the frog neuromuscular junction were studied using the two-microelectrode voltage clamp and other electrophysiological techniques. Methyl-, ethyl- , and propylguanidine stimulated the nerve-evoked release of transmitter. However, amyl-and octylguanidine had no apparent presynaptic action. All of the derivatives blocked the postsynaptic response to acetylcholine, the potency sequence being octyl-greater than amyl-greater than propyl-, methyl-greater than ethylguanidine. Methyl- and octylguanidine did not protect the receptor from alpha- bungarotoxin block, suggesting that these compounds do not bind to the receptor but probably block the ionic channel. Methyl-, ethyl-, and propylguanidine shortened inward endplate currents but prolonged outward currents. Amylguanidine prolonged both inward and outward endplate currents, and the currents became biphasic at negative membrane potentials. Octylguanidine increased the rate of decay of endplate currents at all potentials. All of the derivatives blocked inward endplate currents more markedly than outward currents, resulting in a highly nonlinear current-voltage relation. Methyl-, ethyl-, and propylguanidine reversed the voltage dependence of endplate current decay, while amyl-and octylguanidine reduced the voltage dependence of endplate current decay. Octylguanidine appears to block the ionic channel in both the open and the closed state. The block of the open channel follows pseudo-first-order kinetics with a forward rate constant of 4-6 X 10(7) M-1 s-1.     

Voltage-dependent conductances in Limulus ventral photoreceptors

The voltage-dependent conductances of Limulus ventral photoreceptors have been investigated using a voltage-clamp technique. Depolarization in the dark induces inward and outward currents. The inward current is reduced by removing Na+ or Ca2+ and is abolished by removing both ions. These results suggest that both Na+ and Ca2+ carry voltage-dependent inward current. Inward current is insensitive to tetrodotoxin but is blocked by external Ni2+. The outward current has a large transient component that is followed by a smaller maintained component. Intracellular tetraethylammonium preferentially reduces the maintained component, and extracellular 4-amino pyridine preferentially reduces the transient component. Neither component is strongly affected by removal of extracellular Ca2+ or by intracellular injection of EGTA. It is concluded that the photoreceptors contain at least three separate voltage-dependent conductances: 1) a conductance giving rise to inward currents; 2) a delayed rectifier giving rise to maintained outward K+ current; and 3) a rapidly inactivating K+ conductance similar to the A current of molluscan neurons.     

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)     

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.     

A calcium-dependent transient outward current in Xenopus laevis oocytes

Membrane currents were investigated in Xenopus laevis oocytes under voltage clamp. Depolarizing pulses, given from a holding potential of about-100 mV, elicited a transient outward current when the membrane potential was made more positive than about-20 mV. As the potential was made increasingly positive the transient outward current first increased and then decreased. The amplitude of the transient current increased when the external Ca2+ concentration was raised; and the current was abolished by Mn2+. It appears that when the membrane is depolarized Ca2+ ions enter the oocyte and trigger an outward current, possibly by opening C1- channels.