The effect of pentylenetetrazol on the metacerebral neuron of Helix pomatia

The effects of Pentylenetetrazol (PTZ) on the metacerebral giant cell (MCC) of the snail, Helix pomatia were studied. Actions on membrane resistance, time constant, resting and action potentials, outward and inward ionic currents were examined. Superfusion with PTZ in concentrations of 25 to 50 mmol/l, induced a gradually evolving convulsive state, which could be studied by intracellular recording from the MCCs. In the pre-convulsive state an acceleration of the spontaneous activity developed and was followed by paroxysmal depolarization shifts (PDSs), in the convulsive phase. PTZ prolonged the membrane time constant by about 10 percent, but this could not be traced back to alterations in membrane resistance or capacity. The resting membrane potential was not significantly altered; the action potentials were prolonged by slowing down of both the rising and decaying phases. The outward potassium currents were repressed by PTZ in a voltage dependent manner. The decrease of the IA current became more pronounced at increasingly positive command pulses, while IK was relieved from depression especially at longer pulse durations. Inward currents were isolated with the aid of suppression of outward currents by 50 mmol/l TEA. Under these conditions sodium currents, measured in calcium deficient Ringer solution were moderately depressed, while the calcium currents, examined during sodium-free superfusion, were mildly enhanced by PTZ. It is concluded that PTZ effects on ionic conductances, on membrane parameters, on the resting potential and ionic currents explain only modifications of spike potentials occurring in the convulsive state and do not account for the PDS, the central phenomenon of the convulsive electrographic activity, at least in this thoroughly examined type of neuron.     

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.     

Thyroid hormone regulates mRNA expression and currents of ion channels in rat atrium

Atrial fibrillation is one of the common arrhythmias associated with hyperthyroidism. This study examined the effects of thyroid hormone (T3) on mRNA expression and currents of major ionic channels determining the action potential duration (APD) in the rat atrium using the RNase protection assay and the whole-cell patch-clamp technique, respectively. T3 increased the Kv1.5 mRNA expression and decreased the L-type calcium channel mRNA expression, while the Kv4.2 mRNA expression did not change. APD was shorter in hyperthyroid than in euthyroid myocytes. The ultrarapid delayed rectifier potassium currents were remarkably increased in hyperthyroid than in euthyroid myocytes, whereas the transient outward potassium currents were unchanged. L-type calcium currents were decreased in hyperthyroid than in euthyroid myocytes. T3 shifted the current-voltage relationship for calcium currents negatively. In conclusion, T3 increased the outward currents and decreased the inward currents. The resultant changes of ionic currents shortened APD, providing a substrate for atrial fibrillation.     

Transmembrane ion currents in pulmonary artery smooth muscle

Transmembrane ionic currents were investigated in the rabbit pulmonary artery smooth muscle under voltage clamp conditions with the use of the double sucrose gap method. With depolarizing pulses, there developed a fast inactivated outward current that was followed by a steady-state outward current. Tetraethylammonium (TEA) partly suppressed the outward current, and the fast inward current that preceded the fast outward one could be seen in these conditions. Appearance of the fast inward current in TEA-containing solution suggests the overlapping of the fast inward and outward currents. It appears that the resultant transmembrane current has an outward direction since in normal conditions the permeability of the fast potassium channels exceeds that of calcium channels. Conditioning hyperpolarization increased and depolarization decreased the fast outward current indicating that at the resting membrane potential a part of the potassium channels is inactivated and this inactivation is removed by hyperpolarization.     

Ionic mechanisms underlying excitation-to-frequency transduction: studies by voltage clamp methods

The transduction of synaptic activity to impulse generation is controlled by the active and passive properties of neurons. The voltage dependent conductances of cat motoneurons, as we understand them, are presented and related to repetitive firing behavior. Both outward potassium and inward calcium currents are activated in the subthreshold region. Accomodation of the initial segment allows tonic activation of these currents during repetitive firing and the response properties of the neuron depend upon the balance of inward and outward currents. The effects of putative neurotransmitters and changes in ionic concentration upon the active ionic currents and upon the response properties of neurons are also discussed.     

Extrasynaptic action of vasopressin,oxytocin, and vasotocin neuropeptides on electrically operated ionic channels at the mollusk neuronal membrane

Techniques of intracellular dialysis and neuronal perfusion in the visceral ganglion ofLymnaea stagnalis used during voltage-clamping at the neuronal membrane helped to ascertain that a concentration of 1×10^–16–1×10^–6 M neuroactive peptides (vasopressin, oxytocin, and vasotocin) alter the amplitude of electrically-operated transmembrane ionic currents considerably without affecting the kinetics of current activation and inactivation and surface potential at the membrane. The experimental conditions applying made it possible to record incoming sodium and calcium currents separated from each other as well as outward delayed and transient potassium currents. It was found that electrically-operated cerebral currents could either increase or decline in amplitude under the effects of peptides applied at different concentrations to the membrane of the same unit. Receptors of the peptides investigated in this study are thought to be located within the structure of electrically-operated channels at the neuronal membrane.A. I. Gertsen Teaching Institute, Leningrad. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 526–533, July–August, 1990.     

Cholesterol influences voltage-gated calcium channels and BK-type potassium channels in auditory hair cells

The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitability are unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassium current by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (~30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.     

Intracellular and voltage clamp studies of capsaicin induced effects on a sensory neuron model

The acute effects of capsaicin (CAP) were studied on membrane properties, the action potential (AP) and the membrane ionic currents in the giant serotoninergic neuron of the cerebral ganglion (MCC) in the snail of Helix pomatia L. CAP (30-300 microM) depolarized the MCC, decreased the amplitude, the rate of rise and the rate of fall of the action potential. CAP prolonged the AP-duration, increased the membrane slope resistance, decreased the hyperpolarizing afterpotential and the posttetanic hyperpolarization both in normal and Na-free media. All the effects were reversible and could be evoked repeatedly. CAP attenuated the outward membrane currents with decreasing potency in the sequence of the transient potassium (IA) voltage-dependent potassium (IK), Ca-dependent potassium (IC) and leakage currents (IL). CAP decreased or increased the peak amplitude of the Ca-current (ICa), depending on the extracellular Ca concentration. CAP increased the inactivation of the ICa, decreased the Ca-conductance (GCa) in normal and high Ca solutions and shifted the Ca-equilibrium potential (VCa) to more positive voltage in 30 mM Ca-solution. CAP decreased the electrically activated Na-current and blocked the acetylcholine (ACh) activated increase in Na-K conductances. It is concluded that CAP profoundly affects the electrically and some transmitter-activated cationic conductances. Further studies are needed to clarify the significance of these changes with respect to the mechanism of the selective neurotoxic effects of CAP.     

Blocking of Ca-dependent outward current in molluscan neuron somatic membrane by raised intracellular pH

The action of a raised intracellular pH (pH_i) on transmembrane ionic currents was investigated on isolated unidentified neurons ofHelix pomatia under intracellular dialysis and membrane voltage clamping conditions. With a rise in pH_i from 7.3 to 9.0 and in the simultaneous presence of an inward calcium current, the outward potassium current was considerably reduced and the current-voltage characteristic curve was shifted toward more positive membrane potential values. The inward calcium current was practically unchanged in this case. If, however, the calcium current was inhibited by the action of cadmium ions, no decrease in the outward current was observed, only a shift of the I_K(V) curve toward more positive values of membrane potential. It is suggested that an increase in pH_i selectively blocks the Ca-dependent component of the outward potassium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 4, pp. 426–430, July–August, 1982.     

Ion channels in rabbit cultured fibroblasts

Large outward currents are recorded with the whole-cell patch-clamp technique on depolarization of rabbit cultured fibroblasts. Our findings suggest that these outward currents consist of two voltage-dependent components, one of which also depends on cytoplasmic calcium concentration. Total replacement of external Cl- by the large anion ascorbate does not affect the amplitude of the currents, indicating that both components must be carried by K+. Consistent with these findings with whole-cell currents, in single channel recordings from fibroblasts we found that most patches contain high-conductance potassium-selective channels whose activation depends on both membrane potential and the calcium concentration at the cytoplasmic surface of the membrane. In a smaller number of patches, a second population of high-conductance calcium-independent potassium channels is observed having different voltage-dependence. The calcium- and voltage-dependence suggest that these two channels correspond with the two components of outward current seen in the whole-cell recordings. The single channel conductance of both channels in symmetrical KCl (150 mM) is 260-270 pS. Both channels are highly selective for K+ over both Na+ and Cl-. The conductance of the channels when outward current is carried by Rb+ is considerably smaller than when it is carried by K+. Some evidence is adduced to support the hypothesis that these potassium channel populations may be involved in the control of cell proliferation.     

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.     

Effect of sotalol on transmembrane ionic currents responsible for repolarization in cardiac ventricular myocytes from rabbit and guinea pig

The effects of sotalol, a β-adrenoceptor blocker and class III antiarrhythmic agent, on transmembrane ionic currents were examined in single rabbit and guinea pig ventricular myocytes using whole-cell voltage-clamp techniques. In neither of these species did 60 μM sotalol appreciably effect the inward rectifier, the transient outward or the inward calcium currents. In addition, sotalol did not elicit a slowly inactivating component of the sodium current as did 1 μg/ml veratrine. In guinea pig ventricular myocytes, sotalol also significantly depressed the outward delayed rectifier current. An outward delayed rectifier current was not observed in rabbit ventricular myocytes examined at room temperature; and, under these conditions sotalol did not lengthen action potential duration. Sotalol induced lengthening of cardiac action potential duration can, therefore, be explained by depression the outward delayed rectifier current.     

Outward currents in olfactory receptor neurons activated by odorants and by elevation of cyclic AMP

We studied the outward currents elicited by an odorous compound, isoamyl acetate, in isolated olfactory receptor neurons of the grass frog under whole-cell perforated-patch voltage-clamp recording. Odorant-induced outward currents were relatively rare, occurring in about 16% of the responding cells. Responses had smaller amplitudes and shorter time courses when compared to the more commonly found odorant-induced inward currents. There was a high correlation between odorant-induced outward current and responses evoked by either 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphate, a membrane-permeant cyclic adenosine monophosphate analog, or 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. The outward current responses to all three substances increased in amplitude when the membrane potential was more negative than -60 mV and decreased in amplitude when the membrane potential was more positive. Responses were still present when the potential was held at -100 mV, indicating that the responses are not the result of a potassium conductance. Removal of external calcium from the perfusion medium abolished the outward currents. Our results indicate that the odorant-induced outward current is a calcium-dependent event that may be mediated by cyclic adenosine monophosphate.     

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.     

Some electrical properties of the membrane of the barnacle muscle fibers under internal perfusion

Summary Intracellular perfusion technique has been applied to the muscle fibers of the barnacle species,Balanus nubilus. In these fibers, generation and the form of the calcium spike was governed by the frequency of stimulation and intra- and extracellular calcium concentrations. Voltage-clamp experiments showed that the magnitude of the potassium outward current was controlled by the intracellular calcium concentration whose increase, nearly 10³-fold, raised the resting membrane conductance and the outward potassium current. On the other hand, application of 10mm zinc ions inside the muscle fiber had no effect on either the resting potential or the outward potassium current but suppressed the early inward calcium current. Similarly, the inward calcium current was decreased by low concentration of sodium ions in the extracellular fluid only when its ionic strength was made low by substituting sucrose for the sodium salt. Measurement of outward current with the muscle fiber in calcium-free ASW solution and intracellularly perfused with several cationic solutions established the selectivity sequence TEA相似文献   

Effects of Taurine–Magnesium Coordination Compound on Ionic Channels in Rat Ventricular Myocytes of Arrhythmia Induced by Ouabain

Taurine-magnesium coordination compound (TMCC) has anti-arrhythmic effects. The aim of the present study was to explore the targets of the anti-arrhythmic effect of TMCC and the electrophysiological effects of TMCC on ouabain-induced arrhythmias in rat ventricular myocytes. Sodium current (I(Na)), L-type calcium current (I(ca, L)), and transient outward potassium current (I(to)) were measured and analyzed using whole-cell patch-clamp recording technique in normal rat cardiac myocytes and rat ventricular myocytes of arrhythmia induced by ouabain. In isolated ventricular myocytes, I(Na) and I(to) were blocked by TMCC (100, 200, 400 μM) in a concentration-dependent manner, and the effects of TMCC (400 μM) were equal to that of amiodarone. However, I (ca, L) was moderately increased by TMCC (400 μM) while significantly decreased by amiodarone. Ouabain (5 μM) significantly decreased sodium, L-type calcium, and transient outward potassium currents. TMCC (100 μM) relieved abnormal sodium currents induced by ouabain through facilitation of steady-state inactivation. TMCC (200 and 400 μM) relieved abnormal L-type calcium currents induced by ouabain through facilitation of steady-state activation and retardation of steady-state inactivation. TMCC failed to further inhibit abnormal transient outward potassium currents induced by ouabain. However, amiodarone inhibited the decreasing sodium, L-type calcium, and transient outward potassium currents further. These data suggest that I(Na), I(ca, L), and I(to) may be the targets of the antiarrhythmic effect of TMCC, which can antagonize ouabain-induced changes of ionic currents in rat ventricular myocytes.     

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.     

Effects of Internal Divalent Cations on Voltage-Clamped Squid Axons

We have studied the effects of internally applied divalent cations on the ionic currents of voltage-clamped squid giant axons. Internal concentrations of calcium up to 10 mM have little, if any, effect on the time-course, voltage dependence, or magnitude of the ionic currents. This is inconsistent with the notion that an increase in the internal calcium concentration produced by an inward calcium movement with the action potential triggers sodium inactivation or potassium activation. Low internal zinc concentrations (~1 mM) selectively and reversibly slow the kinetics of the potassium current and reduce peak sodium current by about 40% with little effect on the voltage dependence of the ionic currents. Higher concentrations (~10 mM) produce a considerable (ca. 90%) nonspecific reversible reduction of the ionic currents. Large hyperpolarizing conditioning pulses reduce the zinc effect. Internal zinc also reversibly depolarizes the axon by 20–30 mV. The effects of internal cobalt, cadmium, and nickel are qualitatively similar to those of zinc: only calcium among the cations tested is without effect.     

Some electrical properties of the membrane of the barnacle muscle fibers under internal perfusion.

Intracellular perfusion technique has been applied to the muscle fibers of the barnacle species, Balanus nubilus. In these fibers, generation and the form of the calcium spike was governed by the frequency of stimulation and intra- and extracellular calcium concentrations. Voltage-clamp experiments showed that the magnitude of the potassium outward current was controlled by the intracellular calcium concentration whose increase, nearly 10(3)-fold, raised the resting membrane conductance and the outward potassium current. On the other hand, application of 10 mM zinc ions inside the muscle fiber had no effect on either the resting potential or the outward potassium current but suppressed the early inward calcium current. Similarly, the inward calcium current was decreased by low concentration of sodium ions in the extracellular fluid only when its ionic strength was made low by substituting sucrose for the sodium salt. Measurement of outward current with the muscle fiber in calcium-free ASW solution and intracellularly perfused with several cationic solutions established the selectivity sequence TEA less than Cs less than Li less than Tris less than Rb less than Na less than K for the potassium channel.     

Transmitter control of voltage-dependent currents

At one time neurotransmitters were thought of as chemical agents that simply depolarized or hyperpolarized a postsynaptic cell. Now it is known that transmitters can do much more. Biochemical processes, most notably the consequences of activation of adenylate cyclase, are subject to neurotransmitter control. Transmitters can alter a cell's sensitivity to another neurotransmitter; this is exemplified by the action of aspartate in enhancing responses to glutamate. Another action of transmitters is the subject of this review: control of voltage-dependent neuronal currents.Voltage-dependent processes are necessary for the normal function of neuronal systems. Potassium, sodium, and calcium currents that turn on and off with changes in membrane potential are responsible for action potentials and slow-wave (or burst firing) activity. Transmitter control of these ionic currents allows direct synaptic regulation of basic electrophysiological events.Discussion of the voltage-dependent actions of transmitters in neuronal systems will be divided into four areas: (a) broadening and narrowing of action potentials, (b) modulation of burst firing activity, (c) blockade of a voltage-dependent potassium conductance, and (d) induction of a voltage-dependent calcium current. The membrane currents underlying voltage-dependent events will be reviewed only as necessary to understanding transmitter effects. The reader is referred to a recent review for further details on some of these currents (1).