Outward Tail Current Research Articles

Sources of Ca2+ for different Ca(2+)-activated K+ conductances in neurones of the rat superior cervical ganglion.

1. The role of various Ca(2+)-activated K+ conductances were investigated using intracellular recording and single-electrode voltage clamp in neurones of superior cervical ganglia isolated in vitro from young adult rats. 2. Following replacement of Ca2+ with Co2+ (2 mM) or the addition of Cd2+ (100 microM), action potential amplitude and half-width either increased or decreased (in different cells), but both the after-hyperpolarization (AHP) and the outward tail current following a suprathreshold voltage step were markedly attenuated (by about 75%). 3. Addition of charybdotoxin (60 nM) or nifedipine (10 microM) increased action potential half-width (by about 25%) but had no significant effect on the AHP or tail current. 4. Addition of apamin (100 nM) or omega-conotoxin GVIA (100 nM) reduced the AHP and tail current (by about 60%) but did not significantly affect the action potential. A prolonged apamin-resistant component of the AHP present in 50% of neurones was blocked by ryanodine (20 microM). 5. Omega-Conotoxin MVIIC (150 nM) and omega agatoxin IVA (200 nM) had no significant effects on the action potential half-width or the AHP. 6. None of the Ca2+ channel blockers affected the prolonged ryanodine-sensitive component of the AHP and tail current. 7. We conclude that, in rat sympathetic neurones, Ca2+ entry via L-type channels selectively activates large conductance Ca(2+)-activated K+ channels (BK type) contributing to action potential repolarization, whereas Ca2+ entry via N-type channels selectively activates small conductance Ca(2+)-activated K+ channels (SK type) contributing to the AHP. Ca2+ entry via R-type Ca2+ channels prolongs the AHP by activating Ca2+ release from intracellular stores.

Dantrolene depolarizes hippocampal neurons in slices from rats

In intracellular recordings by current clamp, dantrolene (sodium salt, 20 microM) regularly depolarized CA1 neurons (by approximately 13 mV), even in the presence of tetrodotoxin. Dantrolene almost abolished slow (but not medium) afterhyperpolarizations (AHP) (down by approximately 80%). As in previous experiments, dantrolene strongly depressed the hyperpolarization and resistance drop caused by hypoxia. Under voltage clamp (at approximately -70 mV), dantrolene accordingly elicited an inward current of approximately -100 pA and suppressed a voltage-dependent slow outward current and tail current, as well as the outward current generated by hypoxia. In addition, dantrolene enhanced Q-current (IQ) inward relaxations; but it had no effect on high voltage activated Ca currents and did not prevent their suppression by hypoxia. We conclude that depression of slow AHP current and increase in IQ probably account for the depolarizing action of dantrolene.

A rapidly activating delayed rectifier K+ channel in rabbit sinoatrial node cells

The single-channel current of the delayed rectifier K+ current (IK) was recorded in rabbit sinoatrial node cells. In the cell-attached patch, depolarization from -70 mV to potentials more positive than -50 mV activated the IK channel while repolarization deactivated it. The single-channel conductance was 7.8 pS for the outward current and 10.8 pS for the inward current (n = 6). The steady-state open probability (NPo) was maximum at around -30 mV and markedly decreased at more positive potentials. On repolarization from positive potentials, the channel was initially closed and then rapidly opened. The ensemble average showed an initial rise to a peak followed by the deactivation time course. Because the channel events were completely blocked by E-4031, the drug-sensitive component was examined in the whole cell current. The steady-state current-voltage relation of the drug-sensitive current showed a marked negative slope at potentials more positive than -10 mV. Upon repolarization, the drug-sensitive current initially increased (removal of inactivation) to the peak of the outward tail current, which was in agreement with the ensemble average of the single-channel current. We conclude that IK in the sinoatrial node cells is largely composed of the rapidly activating IK (IK,r) channels and that the inward rectification of IK,r, which is more marked than had been assumed in previous studies, is due to the decrease in NPo.

Potassium currents in adult rat intracardiac neurones.

1. Properties of K+ currents were studied in isolated adult rat parasympathetic intracardiac neurones with the use of single-electrode voltage-clamp techniques. 2. A hyperpolarization-activated inward rectifier current was revealed when the membrane was clamped close to the resting level (-60 mV). The slowly developing inward relaxation had a mean amplitude of 450 pA at -150 mV, an activation threshold of -60 to -70 mV and a relaxation time constant of 41 ms at -120 mV. The current was reversibly blocked by Cs+ (1 mM) and became smaller with reduced [K+]o and [Na+]o, indicating that this inward rectifier current probably is a time- and voltage-dependent Na(+)-K+ current. 3. Step depolarizations from the holding potential of -80 mV evoked a transient (< 100 ms at -40 mV) outward K+ current (IA) which was blocked by 4-aminopyridine (4-AP, 1 mM). The time constants for IA inactivation were 20 ms at -50 mV and 16 ms at -20 mV. The steady-state activation and (removal of) inactivation curve showed a small overlap between -70 and -40 mV; the reversal potential of IA was close to EK. 4. Step hyperpolarizations from the depolarized potentials, i.e. -30 mV, revealed a slow inward relaxation associated with the deactivation of a time- and voltage-dependent current. The inward relaxation became faster at more hyperpolarized potentials and reversed at -85 and -53 mV in 4.7 and 15 mM [K+]o. This current was blocked by muscarine (20 microM) and Ba2+ (1 mM) but not affected by Cs+ (1 mM); this current may correspond to the M-current (IM). 5. Depolarization-activated outward K+ currents were evoked by holding the membrane close to the resting potential in the presence of tetrodotoxin (TTX, 3 microM), 4-AP (1 mM) and Ba2+ (1 mM). The amplitude of the outward relaxation and the tail current became smaller as the [K+]o was elevated. The outward tail current was reduced in a Ca(2+)-free solution and the residual current was eliminated by the addition of tetraethylammonium (TEA, 10 mM); the reversal potential was shifted in a direction predicted by the Nernst equation. These findings suggest the presence of delayed rectifier K+ current and Ca(2+)-activated K+ current. 6. Superfusion of TEA, Ba2+ and 4-AP, but not Cs+, induced rhythmic discharges in some of the otherwise quiescent intracardiac neurones.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of chloride currents in repolarizing rabbit atrial myocytes.

Rabbit atrial cells manifest a prominent transient outward K+ current (Ito1), but this current recovers slowly from inactivation and is unlikely to be important at physiological rates (3-5 Hz). Depolarization of rabbit atrial cells also elicits a transient Ca(2+)-dependent outward Cl- current (Ito2). To compare the relative magnitude of these transient outward currents at various rates, we applied whole cell voltage-clamp techniques to isolated rabbit atrial myocytes. Whereas peak Ito1 exceeded Ito2 at slow rates (0.1 Hz), Ito1 was strongly reduced as rate was increased (by 97 +/- 2%, mean +/- SE, at 4 Hz), while Ito2 was slightly reduced (by 28 +/- 4%, 4 Hz). The reversal potential of transient outward tail currents at 0.07 Hz was -49 +/- 9 mV, while at 2.5 Hz the reversal potential became -18 +/- 7 mV (calculated Cl- reversal potential -18 mV). The addition of the Cl- transport blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 150 microM) or the replacement of external Cl- with methanesulfonate inhibited a large part of the transient outward current elicited by depolarization at 4 Hz. DIDS and Cl- replacement increased action potential duration in both single rabbit atrial cells and multicellular rabbit atrial preparations. We conclude that the Ca(2+)-dependent Cl- current is substantially larger than the transient K+ current at physiological rates in the rabbit and is likely to play a more important role in action potential repolarization than the latter current in this tissue in vivo.

Cesium Abolishes the Barium‐Induced Pacemaker Potential and Current in Guinea Pig Ventricular Myocytes

The ability of cesium to block barium-induced diastolic depolarization ("Ba-DD") and pacemaker current was tested in isolated ventricular myocytes. Because Ba-DD is due to decreasing K+ conductance and there is no I(f) at the resting potential, this approach permits verification of whether Cs+ is a specific blocker of i(f) or if it instead also blocks a K+ pacemaker current. Guinea pig isolated ventricular myocytes were studied by a discontinuous, single electrode, voltage clamp method. During hyperpolarizing voltage clamp steps from -80 up to -140 mV in Tyrode's solution, the inward current increased as a function of voltage but did not change as a function of time (no I(f) or K+ depletion). Cesium (4 mM) reduced the current size during the hyperpolarizing steps but did not induce or unmask time-dependent currents. Barium (0.05 to 0.1 mM) induced diastolic depolarization, and, in its presence, depolarizing voltage clamp steps were followed by an outward tail current that reversed at -92.0 +/- 1.3 mV. Outward tail currents were larger at -50 mV than at the resting potential, and inward tail currents decayed more rapidly and to a larger extent during larger hyperpolarizing steps. In the presence of Ba2+, Cs+ (4 mM) had little effect on the steady-state current but markedly reduced or abolished undershoot, Ba-DD, and time-dependent tail currents at potentials both positive and negative to the resting potential. Cs+ had a smaller effect on the steady-state current-voltage (I-V) relation in the presence than in the absence of Ba2+, as part of the IK1 channels were already blocked by Ba2+ and the time-dependent changes induced by Ba2+ were not present. Both Ba2+ and Cs+ had little blocking effect on the steady-state current positive to the negative slope region of the I-V relation. In ventricular myocytes, Cs+ abolishes the Ba(2+)-induced pacemaker current by blocking the time-dependent change in K+ conductance, not by blocking I(f). Because Cs+ can also block a decaying K+ pacemaker current, the abolition of a pacemaker current by Cs+ in other cardiac tissues cannot be taken as unequivocal proof that the blocked current is I(f).

Cation‐dependent gating of the hyperpolarization‐activated cation current in the rabbit sino‐atrial node cells.

1. The gating properties of the hyperpolarization-activated cation current (I(f) or Ih) were investigated in single pacemaker cells dissociated from the rabbit sino-atrial node. 2. The whole-cell I(f) was recorded in the presence of different external cations. The inward I(f) was increased when external Na+ was replaced with K+, and was decreased in Li+ or Rb+ solution. In Tris+ and Cs+ solutions, the inward I(f) was negligible. The outward tail current recorded upon depolarization was largest in Li+ solution and smaller in a sequence of Na+, Tris+ and K+ solutions. In Rb+ and Cs+ solutions, only a small tail current was recorded. 3. The outward tail current had a 'shoulder' in Na+ solution, which was much delayed by replacing Na+ with Li+. In K+ solution, the decay of the tail current was much faster, and no obvious shoulder was recorded. The tail current was slowest in Li(+)-rich and 0 mM K+ solution, and was progressively accelerated by adding K+ over the range from 0 to 3 mM. The tail current at 30 mM [K+]o showed only a small shoulder. A common binding site to modulate the I(f) deactivation was suggested for monovalent cations. 4. The shoulder of the I(f) tail became more evident as I(f) was activated to a larger extent either by prolonging the duration or by increasing the amplitude of the preceding hyperpolarization in both Na+ and Li+ solutions. 5. The I(f) was first activated by hyperpolarizing the membrane to -110 mV, and then deactivated by depolarization. The inward tail current at -50 mV showed a single exponential decay. At more positive potentials, the shoulder of the outward tail currents became more evident and the rate of the final decay was increased. 6. The time course of I(f) activation was well fitted with the sum of two exponential functions. Time constants of both components were not affected by the external cation (Na+, K+ or Li+) replacement. Likewise, the quasi-steady state activation was conserved when external Na+ was replaced with Li+. 7. Two closed and three open states were assumed in a sequential state model of the I(f) channel. The cation effects were well simulated by assuming that the deactivation rate was selectively modulated. The flow of I(f) during the spontaneous action potential was calculated. The activation of I(f) started on repolarization to the maximum diastolic potential and reached a maximum in the middle of the diastolic period. Its peak amplitude was 14% of the net inward current during the diastolic period.

Voltage-dependent potassium channels in activated rat microglia.

1. Voltage-dependent currents of untreated (proliferating) and lipopolysaccharide (LPS)-treated rat microglial cells in culture were recorded using the whole-cell patch-clamp technique. 2. Membrane potentials showed prominent peaks at -35 mV and -70 mV. Membrane potentials of LPS-treated cells alternated between the two values. This may be due to a negative slope region of the I-V relation resulting in two zero current potentials. 3. From a holding potential of -70 mV, hyperpolarizing steps evoked an inwardly rectifying current both in proliferating and in LPS-treated cells, while depolarizing steps below -50 mV evoked an outwardly rectifying current only in LPS-treated microglia. The currents were K+ selective, as indicated by their reversal potential of approximately 0 mV in symmetric K+ concentrations (150 mM both intra- and extracellularly) and the reversal potential of the outward tail currents of approximately -90 mV at a normal extracellular K+ concentration (4.5 mM). 4. The activation of the outward current could be fitted by Hodgkin-Huxley-type n4 kinetics. The time constant of activation depended on voltage. 5. The inactivation of the inward and outward currents could be fitted by a single exponential. The time constant of the inward current inactivation was dependent on voltage, whereas the time constant of the outward current inactivation was virtually independent of voltage, except near the threshold of activation. Recovery of the outward from inactivation was slow and could be fitted by two exponentials. Responses to depolarizing steps were stable at 0.125 Hz, but greatly decreased from the first to the second pulse at 1 Hz. 6. The inactivation of the inward, but not of the outward, current disappeared in a low Na(+)-containing medium (5 mM). The inward current was selectively inhibited by extracellular Cs+ and Ba2+. The outward current was selectively inhibited by Cd2+, 4-aminopyridine and charybdotoxin. Replacement of intracellular K+ by an equimolar concentration of Cs+, and the extracellular application of tetraethylammonium and quinine inhibited both currents. 7. An increase of extracellular Ca2+ from 2 to 20 mM resulted in outwardly rectifying K+ channels activating at more positive potentials. Omission of Ca2+ from the extracellular medium had the opposite effect. When the intracellular free Ca2+ was increased from 0.01 to 1 microM, the outward current amplitudes were depressed. The Ca2+ ionophore A23187 had a similar effect. 8. LPS-treated microglial cells possess inwardly and outwardly rectifying K+ channels. The physiological and pharmacological characteristics of these two channel populations are markedly different.(ABSTRACT TRUNCATED AT 400 WORDS)

Brief increases in intracellular Ca 2+ activate K + current and non-selective cation current in rat nucleus basalis neurons

Neurons were acutely dissociated from the rat nucleus basalis, and membrane currents (whole-cell patch-clamp) and intracellular free Ca 2+ concentrations (Fura-2) were measured simultaneously from large neurons (approximately 25 μm in diameter). A brief depolarization from −60 to 0 mV for 200ms evoked an increase in intracellular free calcium and a slow outward tail current (72 ± 8pA, n = 30). The outward current reversed polarity at −75.5 ± 2.7mV ( n = 14). The tail current declined and the intracellular calcium recovered its resting level exponentially with time-constants of 1.0 ± 0.1s and 2.5 ± 0.2s, respectively ( n = 17). In neurons loaded with Cs-gluconate, a similar depolarizing pulse evoked a similar increase in intracellular free calcium, but this was now followed by an inward tail current ( 118 ± 8pA, n = 44). The inward tail current reversed polarity at −27.8 ± 3.8mV (n = 7), and was suppressed by removal of external sodium ions. Neither outward nor inward tail currents were observed, when the external solution was calcium-free or when the pipette solution contained EGTA (10 mM). These results indicate that a depolarization causes a calcium entry and that this consequently increases both K + conductance and non-selective cation conductance.

Muscarinic modulation of conductances underlying the afterhyperpolarization in neurons of the rat basolateral amygdala

The excitability level of pyramidal neurons in the basolateral amygdala (BLA) is greatly increased following muscarinic receptor activation, an effect associated with an increased rate of action potential firing and reduction of the afterhyperpolarization (AHP). We impaled BLA pyramidal neurons in slices of rat ventral forebrain with a single microelectrode to examine the currents underlying the AHP and spike frequency accomodation and determine their sensitivities to muscarinic modulation. In voltage-clamp, depolarizing steps were followed by biphasic outward tail currents, consisting of rapidly decaying ( I Fast) and slowly decaying ( I Slow) current components. These corresponded temporally with the medium and slow portions of the AHP, respectively. The reversal potential or the I Fast component of the AHP tail current shifted in the depolarizing direction with increases in the extracellular K + concentration. The amplitude of I Fast was reduced during perfusion of 0-Ca 2+ medium or by superfusion of TEA (1–5 mM) or carbachol (10–40 μM). It is suggested that I Fast was produced by the rapidly decaying Ca 2+-activated K + current ( I C) and the muscarinic-sensitive M-current ( I M). The I Slow tail current component reversed at the estimated values for E K in medium containing either normal or elevated K + levels. This component was eliminated by perfusion of 0-Ca 2+ medium or inclusion of cyclic-AMP in the recording electrode. It was not blocked by TEA (5 mM) or apamin (50–500 nM), but was reduced by carbachol in a dose-dependent manner ( IC 50=0.5 μM). Electrical stimulation cholinergic afferent pathways to the BLA produced inhibition of I Slow, an effect which was enhanced by eserine and prevented by atropine. Loss of the I Slow component was always accompanied by similar reductions in accomodation and the slow AHP. It was concluded that this tail current component resulted from the slowly decaying Ca 2+-activated K + current, I AHP. Thus, the muscarinic inhibition of I AHP contributes to the enhanced excitability exhibited by BLA pyramidal neurons following cholinergic stimulation.

Inactivation characteristics of a sustained, Ca(2+)-independent K+ current of rat hippocampal neurones in vitro.

1. Current or voltage clamp recordings from CA3 neurones of the adult rat hippocampal slice were performed to study the inactivation properties of a slow outward K+ current identified as the delayed rectifier (IK). 2. In current clamp experiments, burst firing evoked from resting membrane potential by intracellular current injection was reduced or blocked by conditioning hyperpolarizing pre-pulses of 20-40 mV amplitude. This effect was inhibited by tetraethylammonium (TEA; 20 mM) but was unaffected by Cs+ (3 mM), 4-aminopyridine (4-AP; 2 mM), carbachol (30-50 microM), mast cell degranulating peptide (MCDP; 300 nM), thyrotrophin releasing hormone (TRH; 1 microM) or by a Ca(2+)-free solution containing Mn2+ or Co2+ (2 mM). 3. Single-electrode voltage clamp experiments were carried out on neurones superfused with Ca(2+)-free solution, containing tetrodotoxin (TTX; 1 microM), Mn2+ or Co2+ (2 mM), 4-AP (2 mM), Cs+ (3 mM) and carbachol (30 microM). Step depolarizations from a holding potential of -55 mV activated an outward current which reached a plateau after 200 ms, followed by an outward tail current. Such an outward current had the characteristics of IK. 4. The outward currents were significantly potentiated by conditioning hyperpolarizing pre-pulses suggesting the IK was reduced by a voltage-dependent inactivation process. Removal of inactivation was a function of the amplitude of the conditioning hyperpolarizing pre-pulse. At a holding potential of -55 mV removal of inactivation was time dependent with a time constant of 211 ms. High K+ (12.5 or 21.5 mM) solutions did not affect the inactivation characteristics of IK. 5. Tetraethylammonium (20 mM) or low concentrations of Ba2+ (0.1 mM) readily depressed the outward current without significantly affecting the inactivation process. Dendrotoxin (200 nM) also depressed such a slow current but, in addition, increased the inactivation process of IK. 6. It is suggested that removal of inactivation of IK by hyperpolarization can modulate cell excitability by fully restoring the ability of IK to inhibit burst firing of CA3 hippocampal neurones.

Substance P inhibits activation of calcium-dependent potassium conductances in guinea-pig myenteric neurones.

1. Intracellular recordings were made from myenteric AH neurones of the guinea-pig ileum in vitro. Some experiments were done with a single-electrode voltage clamp to measure membrane currents. 2. Substance P (SP) applied by superfusion (10 nM-300 nM), pressure ejection (100 nM-10 microM, 760 mmHg, for 10-20 ms) or ionophoresis (1 mM, 100 nA, for 0.2 s) caused a membrane depolarization and an inward current, associated with a decrease in potassium conductance. 3. The SP-induced depolarization was abolished within 15 min by superfusion with calcium-free/high-magnesium (10 mM) solution or solutions containing cobalt, manganese or nickel at 1-3 mM. The response persisted even after 40-60 min of superfusion with calcium-free/normal-magnesium (1.2 mM) solution. In all these solutions, synaptic potentials were abolished within 5 min. 4. SP inhibited a slowly developing outward current and an outward tail current during and after a long depolarizing command pulse (2-10 s), and an outward after-current following single or multiple brief depolarizing command pulses (10-50 ms). These outward currents were suppressed in calcium-free/high-magnesium solution. 5. SP depressed both a calcium-dependent slow after-hyperpolarization following the action potential and an outward after-current preceded by a brief depolarizing command. Both the SP-induced depolarization and the SP-induced inward current were augmented when the peptide was pressure-ejected during the recovery phase of the slow after-hyperpolarization and during that of the slow outward after-current, but both of them were inhibited or almost abolished when SP was applied immediately after spike initiation or a brief depolarizing command. 6. The SP-induced response was depressed by barium (1-2 mM). The SP response was not inhibited by tetraethylammonium at low concentrations (5-10 mM), but was depressed at high concentration (20 mM). 7. Superfusion (1-10 nM) or pressure application of a calcium ionophore, A23187, inhibited or even reversed the SP depolarization and the SP-induced inward current. 8. These results indicate that SP inhibits activation of a calcium-dependent potassium conductance which contributes to both the slow after-hyperpolarization and the resting membrane potential. SP may affect the process by which calcium activates this potassium conductance.

Role of membrane potential in Ba2+ induced automatieity in guinea pig cardiac myocytes

The aim was to study in isolated myocardial cells the role of membrane potential in barium induced spontaneous activity and the ionic mechanism of the underlying pacemaker current. The membrane potential and resistance of single myocytes were studied at different voltage levels by means of current and voltage clamp steps in the absence and presence of barium (Ba). The membrane potentials and currents of single guinea pig ventricular myocytes were recorded by means of an intracellular microelectrode through which current could also be passed. In the presence of Ba (0.1-0.2 mM), stepwise depolarisations induced a transient overshoot and initiated action potentials followed by an undershoot, diastolic depolarisation and spontaneous discharge. During progressive depolarisations, membrane resistance (Rm) increased, decreased transiently at the end of the action potential, and reincreased during diastole. Stepwise repolarisations had opposite effects. Hyperpolarisations reversed diastolic depolarisation and could unmask oscillatory potentials (Vos). Voltage clamp steps to +20 mV were followed by outward tail currents during which Rm increased. Larger or longer depolarisations were followed by larger outward tail currents at resting potential level. The outward tail current reversed at potentials negative to EK. In the presence of Ba, applied depolarisation facilitates the induction of spontaneous activity through an interplay between voltage dependent and time dependent Ba block and unblock of gK1, voltage dependent increase in Rm, increased potassium driving force, and negative shift in the slow inward current threshold and sometimes Vos. The pacemaker potential underlying spontaneous activity is due to the slow re-establishment of Ba block of IK1 during diastole.

Slowly inactivating potassium current in cultured bull‐frog primary afferent and sympathetic neurones.

1. Cultured bull-frog dorsal root ganglion cells were voltage clamped in the whole-cell configuration. The cells were superfused with a nominally calcium-free Ringer solution containing tetrodotoxin (3 microM), magnesium (10 mM), cobalt (1 mM), barium (2 mM), 4-aminopyridine (3 mM) and caesium (2 mM). 2. Step depolarizations (10-40 mV, 100-300 ms) from a holding potential close to the rest (typically -70 mV) evoked an outward current (IK) followed by an outward tail current. The peak amplitude of the current was reduced to less than 10% by tetraethylammonium (30 mM). 3. IK developed to its peak in 200 ms at -30 mV. Tail currents reversed at potentials that changed according to the logarithm of the extracellular potassium concentrations. 4. Tail currents declined to the baseline according to an exponential function of time (tau congruent to 40 ms at -60 mV) and its reciprocal time constant increased e-fold with a 13 mV hyperpolarization. 5. The current inactivated during sustained (1-20 s) depolarizing pulses according to a single exponential function (tau congruent to 3 s). 6. The peak amplitude of IK at -30 mV was progressively increased as the holding potential was made more negative than -70 mV reaching the maximum with step depolarizations from -120 mV. Reversed phenomenon was observed as the holding potential was made less negative than -70 mV. 7. The removal of the steady-state inactivation occurred along with a single exponential function and the time constant was decreased from 70 ms at -70 mV to 10 ms at -120 mV. 8. It is suggested that a slowly inactivating potassium current which we called IK in amphibian sensory neurones could be a class of a 'delayed' rectifier potassium current. A potassium current with properties indistinguishable from those which have been described for the sensory IK also occurred in cultured bull-frog sympathetic neurones. 9. Forskolin (1-30 microM) and 1,9-dideoxy forskolin (10 microM) reduced the amplitude of IK by up to 85% but these actions were not mimicked by any of 8-bromo-cyclic AMP (1 mM), dibutyryl cyclic AMP (1 mM) and 3-isobutyl-1-methylxanthine (1 mM). A hydrophilic forskolin analogue, 7-O-hemisuccinyl-7-deacetyl forskolin (10 microM), was about one-tenth as potent as forskolin (10 microM).

Electrophysiologic Properties of UK-66,914, a Novel Class III Antiarrhythmic Agent

A class III antiarrhythmic agent that preferentially increases the effective refractory period without altering conduction velocity holds considerable promise for the treatment of life-threatening cardiac arrhythmias dependent on a reentrant mechanism. In the present study, the cellular electrophysiologic effects of a novel class III antiarrhythmic agent, UK-66,914, were evaluated. UK-66,914 prolonged action potential duration and extended the effective refractory period in isolated canine ventricular muscle and Purkinje fibers in a concentration-dependent manner, beginning at a threshold concentration of 0.1 microM. Analogous effects were found in isolated rabbit atrium beginning at a threshold concentration of 2 microM. At concentrations of UK-66,914 up to 20 microM there was no effect on the maximum rate of phase 0 depolarization (Vmax) or the amplitude of the action potential. In guinea pig papillary muscles. UK-66,914 at concentrations from 0.1 to 20 microM increased the effective refractory period at stimulation frequencies of 1 or 5 Hz, but did not slow conduction velocity. Therefore, UK-66,914 exhibits high selectivity for a class III antiarrhythmic effect in normal tissue. To elucidate the mechanisms responsible for the increase in effective refractory period, voltage clamp procedures were used in guinea pig ventricular myocytes. UK-66,914 reduced the amplitude of outward tail currents following depolarizing clamp steps with little effect either on the background K+ current or calcium currents, indicating that UK-66,914 selectively blocked the time-dependent potassium current. In anesthetized dogs, UK-66,914 (10 micrograms/kg to 1 mg/kg i.v.) prolonged both atrial and ventricular effective refractory periods, but in contrast to the studies performed in vitro, the minimum effective doses required to increase the effective refractory period in atria and ventricle were the same. Therefore, UK-66,914 is a potent selective class III antiarrhythmic agent, which owes its electrophysiologic profile to blockade of the time-dependent potassium current.

Membrane currents in visually identified motoneurones of neonatal rat spinal cord.

1. Ionic currents induced by depolarization of motoneurones were analysed by tight-seal, whole-cell recording in thin slices of neonatal rat lumbar spinal cord. Identification of motoneurones viewed under Nomarski optics was confirmed by retrograde labelling with the fluorescent dye, Evans Blue. 2. Under whole-cell voltage clamp, depolarizing command pulses from a holding potential of about -70 mV evoked a fast inward current followed by an outward current. The former was suppressed either by lowering external Na+ concentration or by application of tetrodotoxin (TTX). The apparent dissociation constant of TTX was about 13 nM. 3. The outward current remaining after TTX application was activated by depolarization above -50 mV, showing marked outward rectification in the current-voltage relation. Outward tail currents reversed in polarity near the K+ equilibrium potential calculated from the external and pipette K+ concentrations. 4. When external Ca2+ was replaced by Mg2+, the outward K+ current was suppressed markedly and reversibly. Subtraction of current recorded in Ca2+-free-Mg2+ solution from that in control solution revealed a Ca2(+)-dependent K+ current, IK(Ca) with both a transient, IC, and a sustained component IAHP; its tail current lasted for several hundred milliseconds. 5. The sustained outward current observed in Ca2(+)-free-Mg2+ solution was largely suppressed by external application of tetraethylammonium chloride (30 mM), suggesting that it was mostly the delayed rectifier current, IK. In Ca2(+)-free-Mg2+ solution containing TEA and TTX, another transient outward current was observed, which was inactivated by depolarizing pre-pulses in a time- and voltage-dependent manner. The steady-state inactivation curve indicated 50% inactivation at about -77 mV. 4-Aminopyridine (4-AP, 4 mM) largely and reversibly suppressed this current, whereas it did not affect IK observed in the absence of TEA. It is suggested that the transient outward current corresponds to the A-current (IA). 6. Action potentials were recorded in current-clamp mode. Replacement of external Ca2+ by Mg2+ markedly diminished the after-hyperpolarization. Concomitantly, the repolarizing phase of action potentials was slightly prolonged. In Ca2(+)-free-Mg2+ solution, application of 4-AP markedly prolonged action potential repolarization. In Ca2(+)-free-Mg2+ solution containing 4-AP, addition of TEA-Cl further prolonged the duration of the action potential. It is concluded that three different potassium currents, IC, IA and IK may all contribute to action potential repolarization in rat spinal motoneurones.

Action potentials and membrane currents of isolated single smooth muscle cells of cat and rabbit colon

Membrane potentials, action potentials and macroscopic currents in enzymatically dispersed, single smooth muscle cells of the circular layer of cat and rabbit colon were investigated. The cells did not exhibit spontaneous depolarizations and repolarizations (slow waves) or spontaneous action potentials. Single action potentials of smooth muscle cells were evoked by depolarizing current pulses of 5 ms to 3 s duration. A repetitive action potential discharge and an increase in the duration of the action potential was observed in cells during long depolarizing current pulses by superfusion with tetraethylammonium (TEA) or 4-aminopyridine (4-AP). Tetrodotoxin (TTX) did not alter the configuration of the action potential. Voltage-clamp experiments revealed two major outward macroscopic currents: a quasi-instantaneous (time-independent) and a time-dependent outward current. Both currents were identified as potassium (K) currents due to their pharmacological sensitivity to K antagonists [TEA, 4-AP and cesium (Cs)] and due to the reversal potential of outward tail currents. Barium selectively blocked the time-independent current. A time-dependent outward K current in colon cells was observed which appeared to be dependent upon entry of calcium ions (Ca2+) through voltage-dependent Ca-channels, since it was blocked by cadmium and low concentrations of nifedipine. The majority of cells did not exhibit transient outward currents. Inward currents were exposed in some of the cells when the K currents were blocked by external TEA and by replacement of K by Cs and TEA in the recording pipette. Inward currents were presumably carried by Ca2+, since they were not altered by TTX, were sensitive to external Ca concentrations and were abolished by the Ca channel antagonist, nifedipine. Carbachol augmented the amplitude of the inward Ca current.

Effects of histamine on dentate granule cells in vitro

Hippocampal slices from rat brain were exposed to histamine and related substances in a perfusion chamber. Granule cells of the dentate gyrus were studied with conventional extra- and intracellular recording and a single electrode voltage clamp. Histamine caused, through activation of H 2-receptors, a small depolarization, an increase in the number of synaptic and action potentials, a block of the long lasting (but not the early) component of spike afterhyperpolarizations and a reduction of the accommodation of action potential firing. These effects were mimicked by forskolin (suggests activation of adenylate cyclase). In voltage clamp, histamine blocked a long lasting calcium-dependent outward tail current without any reduction of inward current. Thus histamine selectively blocks the late calcium-dependent potassium current in dentate granule cells which receive histaminergic input from the posterior hypothalamus. Histamine also reduces the field excitatory postsynaptic potential evoked by perforant path stimulation. These actions allow for a powerful modulation of excitatory signals and an effective regulation of hippocampal excitability.

Activation of N-methyl- d-aspartate (NMDA) receptors augments repolarizing responses in lamprey spinal neurons

Current- and voltage-clamp techniques were used to analyze the mechanisms underlying the repolarization during N-methyl- d-aspartate (NMDA)-induced, tetrodotoxin-resistant pacemaker-like oscillations in lamprey spinal neurons. Long-lasting depolarizing current pulses (15–40 mV, 50–400 ms, tetrodotoxin and tetraethylammonium present) were followed by hyperpolarizing afterpotentials even when NMDA receptors were blocked, but they were markedly enhanced by application of N-methyl- d,l-aspartate (NM(DL)A). The afterpotentials were depressed by replacing Ca 2+ with Ba 2+. During voltage-clamp NM(DL)A enhanced a Ba 2+-sensitive outward tail current following voltage steps of 15–40 mV. The outward current remained after injection of Cl −, as did the NMDA-induced membrane oscillations observed under current-clamp. These results suggest that the repolarization during NMDA-induced oscillations is due to Ca 2+ entry both via NMDA-gated channels and conventional voltage-gated Ca 2+ channels, leading to an activation of Ca 2+-dependent K + channels. The afterhyperpolarization following single action potentials, which is also due to Ca 2+-dependent K + channels, was not significantly altered by NMDA receptor activation, suggesting a different location of the Ca 2+ entry during the two conditions in relation to the location of the activated Ca 2+-dependent K + channels.

Vasoactive intestinal peptide (VIP) may reduce the removal rate of cytosolic Ca2+ after transient elevations in clonal rat lactotrophs

The prolactin-producing rat anterior pituitary GH4C1 cells possess Ca2+-activated K channels which are activated by physiological elevations of the cytosolic Ca2+ concentration even at membrane potentials more negative than the normal level of about -50 mV. Whole-cell current recordings showed a marked outward tail current following depolarizing voltage steps to 0 mV from a holding potential close to the normal membrane potential. The half-time of this tail current was about 1.3 s after a 4-s depolarization step. The GH4C1 cells also possess voltage-activated Ca channels, and we conclude that this tail current is a Ca2+-activated K+ current for the following reasons: (1) The reversal potential for the tail current was close to the K+ equilibrium potential for a range of transmembrane K+ gradients. (2) The tail current was blocked by a Ca2+ antagonist, and the voltage dependence of this current closely mirrored the voltage dependence of the isolated Ca2+ current. The time-course of the decline of the tail current thus reflects the removal rate of the Ca2+ entering the cytosol through voltage-dependent Ca channels during the depolarizing voltage step. VIP stimulates prolactin secretion from GH4C1 cells, and this peptide prolonged the half-time of the tail current by about 47% in 63% of the cells. This indicates that VIP may prolong the transient cytosolic Ca2+ elevations following the action potentials in these cells. Such a mechanism might be an important factor for the control of the cytosolic Ca2+ level, and hence hormone secretion.