Calcium-dependent chloride current induced by axotomy in rat sympathetic neurons.

1. Seven to ten days after sectioning their axons, rat sympathetic neurons were studied using intracellular recording techniques in an in vitro preparation of the superior cervical ganglion. 2. In 75% of axotomized cells, an after-depolarization (ADP) was observed following spike firing or depolarization with intracellular current pulses. Discontinuous single-electrode voltage-clamp techniques were employed to study the ADP. When the membrane potential was clamped at the resting level just after an action potential, a slow inward current was recorded in cells that showed an ADP. 3. In the presence of TTX and TEA, inward peaks and outward currents were recorded during depolarizing voltage jumps, followed by slowly decaying inward tail currents accompanied by large increases in membrane conductance. The inward peak and tail currents activated between -10 and -20 mV and reached maximum amplitudes around 0 mV. With depolarizing jumps to between +40 and +50 mV, net outward currents were recorded during the depolarizing jumps but inward tail currents were still activated. 4. In the presence of the Ca2+ channel blocker cadmium, or when Ca2+ was substituted by Mg2+, the ADP disappeared. In voltage-clamped cells, cadmium blocked the inward tail currents. The reversal potential for the inward tail current was approximately -15 mV. Substitution of the extracellular NaCl by sucrose or sodium isethionate increased the amplitude of the inward tail current, and displaced its equilibrium potential to more positive values. Changes in extracellular [K+] did not appreciably affect the inward tail current amplitude or equilibrium potential. Niflumic acid, a blocker of chloride channels activated by Ca2+, almost completely blocked the tail current. 5. No ADPs were observed in non-axotomized neurons, and when depolarizing pulses were applied while in voltage clamp no inward tail currents were evoked in these normal cells. 6. It is concluded that axotomy of sympathetic ganglion cells produces the appearance of a Ca(2+)-dependent chloride current responsible for the ADP observed following spike firing.     

Characterization of an apamin-sensitive potassium current in suprachiasmatic nucleus neurons

In neurons of the suprachiasmatic nucleus, spike frequency adaptation and membrane afterhyperpolarization occur during a train of action potentials. Extracellular Ca2+ may regulate neuronal excitability by several mechanisms, including activation of small conductance and large conductance Ca(2+)-activated K+ channels. The overall goal of this study was to examine the role of Ca(2+)-activated K+ currents in individual suprachiasmatic nucleus neurons. To this end, we used the nystatin-perforated patch technique to record currents from suprachiasmatic nucleus neurons. Iberiotoxin and tetraethylammonium, antagonists of large conductance Ca(2+)-activated K+ channels, had no effect on the membrane afterhyperpolarization. However, antagonists of small conductance Ca(2+)-activated K+ channels, apamin and d-tubocurarine, reduced the amplitude of the membrane afterhyperpolarization and inhibited the spike frequency adaptation that occurred during a train of action potentials. Although there was no significant difference in membrane AHP between different portions of the circadian day, apamin and d-tubocurarine increased the spontaneous firing frequency of suprachiasmatic nucleus neurons during the daytime. In voltage-clamp mode, membrane depolarization-activated currents were followed by an outward tail current reversing near the K+ equilibrium potential. The tail current decayed with a time constant of 220 ms at +20 mV and 149 ms at -40 mV. Apamin irreversibly and d-tubocurarine reversibly inhibited the tail current. The tail current amplitude was also reduced by the GABAA receptor antagonist, bicuculline methiodide, while picrotoxin (another GABAA receptor antagonist) was without effect. Removal of extracellular Ca2+ or the addition of Cd2+ reversibly inhibited the tail current. These results indicate that apamin- and d-tubocurarine-sensitive small conductance Ca(2+)-activated K+ channels have a modulatory function on the action potential firing frequency as well as the membrane afterhyperpolarization that follows a train of action potentials in suprachiasmatic nucleus neurons. Importantly, our data also indicate that a portion of the effects of bicuculline methiodide on suprachiasmatic nucleus neurons may be mediated by inhibition of small conductance Ca(2+)-activated K+ channels.     

Long-lasting reduction of excitability by a sodium-dependent potassium current in cat neocortical neurons

1. The function and ionic mechanism of a slow outward current were studied in large layer V neurons of cat sensorimotor cortex using an in vitro slice preparation and single microelectrode voltage clamp. 2. With Ca2+ influx blocked, a slow relaxation ("tail") of outward current followed either (1) repetitive firing evoked for 1 s or (2) a small 1-s depolarizing voltage clamp step that activated the persistent Na+ current of neocortical neurons, INaP. When a depolarization that activated INaP was maintained, an outward current gradually developed and increased in amplitude over a period of tens of seconds to several minutes. An outward tail current of similar duration followed repolarization. The slow outward current was abolished by TTX, indicating it depended on Na+ influx. 3. With Ca2+ influx blocked, the onset of the slow Na+-dependent outward current caused spike frequency adaptation during current-evoked repetitive firing. Following the firing, the decay of the Na+-dependent current caused a slow afterhyperpolarization (sAHP) and a long-lasting reduction of excitability. It also was responsible for habituation of the response to repeated identical current pulses. 4. The Na+-dependent tail current had properties expected of a K+ current. Membrane chord conductance increased during the tail, and tail amplitude was reduced or reversed by membrane potential hyperpolarization and raised extracellular K+ concentration [( K+]0). 5. The current tail was reduced reversibly by the K+ channel blockers TEA (5-10 mM), muscarine (5-20 microM), and norepinephrine (100 microM). These agents also resulted in a larger, more sustained inward current during the preceding step depolarization. Comparison of current time course before and after the application of blocking agents suggested that, in spite of its capability for slow buildup and decay, the onset of the Na+-dependent outward current occurs within 100 ms of an adequate step depolarization. 6. With Ca2+ influx blocked, extracellular application of dantrolene sodium (30 microM) had no clear effect on the current tail or the corresponding sAHP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Patch-clamp study of the calcium-dependent chloride current in AtT-20 pituitary cells

1. Voltage-clamp recordings were made from cultured AtT-20 pituitary cells using the whole-cell patch-clamp technique. Cells were perfused internally with Cs+ to block K+ currents and bathed externally with either 1 microM tetrodotoxin or with tetraethylammonium (TEA) as a Na+ substitute to block voltage-activated Na+ currents. 2. Depolarizing voltage steps from a holding potential of -80 mV to potentials positive to -30 mV evoked two currents: a fast inward current that activated between -30 and +70 mV and a slowly activating current (designated "slow step current") that was inward between -30 and near 0 mV (the Cl- equilibrium potential) and outward positive to about 0 mV. Repolarization to -80 mV revealed a slowly decaying, inward tail current, whose magnitude with respect to step potential closely matched the current-voltage relationship of the voltage-activated Ca2+ current. 3. Activation of the fast inward current, slow step current, and tail current, was prevented by extracellular application of Cd2+ or removal of extracellular Ca2+. Replacement of extracellular Ca2+ with Ba2+ potentiated the fast inward current but blocked the slow step and tail currents. Intracellular perfusion with greater than 1 mM of the Ca2+ chelators ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) or [1,2-bis(2)aminophenoxy]ethane N,N,N',N'-tetraacetic acid (BAPTA) prevented activation of the slow step and tail currents, but not the fast inward current. 4. The reversal potential of the slow inward current was sensitive to changes in the Cl- equilibrium potential but not to substitution of TEA for Na+. The slow step current, but not the fast inward current, was partially blocked by the Cl- channel blocker, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. 5. These data indicate that both the slow inward tail current and the slowly activating, reversible step current were a Ca2+-dependent Cl- current, similar to that described in other neuronal and nonneuronal cell types. The fast inward current was a voltage-activated Ca2+ current, described previously in these and other cells. 6. In the absence of intracellular EGTA, the tail current decayed with complex kinetics, its time course apparently dependent on the magnitude of the voltage-activated Ca2+ current. In the presence of 200 microM intracellular EGTA, the tail current decayed significantly faster and often decayed exponentially.     

Properties of persistent sodium conductance and calcium conductance of layer V neurons from cat sensorimotor cortex in vitro

Properties of the persistent sodium conductance and the calcium conductance of layer V neurons from cat sensorimotor cortex were examined in an in vitro slice preparation by use of a single microelectrode, somatic voltage clamp, current clamp, intra- and extracellular application of blocking agents, and extracellular ion substitution. The persistent sodium current (INaP) attained its steady level within 2-4 ms of a step change in voltage at every potential where it could be examined directly [to about 40 mV positive to resting potential (RP)]. Because of its fast onset INaP can be activated during a single excitatory postsynaptic potential (EPSP) and can influence the subsequent voltage time course and cell excitability. Application of a depolarizing holding potential greater than or equal to 20 mV positive to RP could inactivate spikes, thus allowing examination of INaP at voltages positive to spike threshold. At every potential where INaP was visible, it was mixed with a slow outward current. After depressing potassium currents with blocking agents, INaP could be observed during depolarizations to about 40 mV positive to RP where it is normally hidden by the larger outward currents. Indirect evidence suggests that INaP is present and large during prolonged depolarizations greater than 50 mV positive to RP. INaP was blocked by intracellular injection of the lidocaine derivative QX-314, as well as by extracellular tetrodotoxin (TTX). INaP was much more sensitive to QX-314 than was the height and rate of rise of the spike. This observation and the results in paragraph 3 above are best explained by separate INaP and spike sodium channels. After blockade of INaP and sodium spikes, Ca2+ spikes could be evoked only if potassium currents were first depressed. The Ca2+-dependent nature of the regenerative potentials was indicated by their disappearance when Co2+ or Mn2+ was substituted for Ca2+ in the perfusate and by the appearance of greatly enhanced potentials of similar form when Ba2+ was substituted for Ca2+. Ba2+ substitution greatly enhanced evoked and spontaneous synaptic potentials. Prolonged-plateau action potentials could be evoked in the presence of TTX and Ba2+. Ca2+ spike threshold was 30-40 mV positive to RP, which is significantly more positive than sodium spike threshold. Results of voltage clamp in the normal perfusate and in the presence of Ca2+-blockers or Ba2+ indicated that little or no Ca2+ conductance is activated in the voltage range 25 mV positive to RP where INaP is the dominant ionic current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacology of a slowly inactivating outward current in hippocampal CA3 pyramidal neurons

The pharmacology of a slowly inactivating outward current was examined using whole cell patch-clamp recordings in CA3 pyramidal cells of guinea pig hippocampal slices. The current had a low activation threshold (about -60 mV) and inactivated slowly (time constant of 3.4 +/- 0.5 s at -50 mV) and completely at membrane voltages depolarized to -50 mV. The slowly inactivating outward current was mainly mediated by K+ with a reversal potential close to the equilibrium potential for K+. The slowly inactivating outward current had distinct pharmacological properties: its time course was not affected by extracellular Cs+ (1 mM) or 4-AP (1-5 mM)-broad spectrum inhibitors of K+ currents and of inactivating K+ currents, respectively. The presence of extracellular Mn2+ (0.5-1 mM), which suppresses several Ca2+ -dependent K+ currents, also did not affect the slowly inactivating outward current. The current was partially suppressed by TEA (50 mM) and was blocked by intracellular Cs+ (134 mM). In addition, intracellular QX-314 (5 mM), a local anesthetic derivative, inhibited this current. The slowly inactivating outward current with its low activation threshold should be operational at the resting potential. Our results suggest that the transient outward current activated at subthreshold membrane potentials in hippocampal pyramidal cells consists of at least three components. In addition to the well-described A- and D-currents, the slowest decaying component reflects the time course of a distinct current, suppressible by QX-314.     

Transient voltage and calcium-dependent outward currents in hippocampal CA3 pyramidal neurons

Membrane currents activated by step changes in membrane potential were studied in hippocampal pyramidal neurons of region CA3 using the single microelectrode voltage-clamp technique. The transient outward current activated by depolarizing steps appeared to be composed of two transient currents that could be distinguished by differences in voltage sensitivity, time course, and pharmacological sensitivity. The more slowly decaying current was activated by voltage steps positive to -60 mV and declined exponentially with a time constant between 200 and 400 ms. This current inactivated as the holding potential was made more positive over the range of -75 to -45 mV and was 50% inactivated near -60 mV. The more slowly decaying transient current was selectively blocked by 0.5 mM 4-aminopyridine (4-AP) but not by 5-10 mM tetraethylammonium (TEA) or 2-5 mM Mn2+. The second transient current had a much faster time course than the 4-AP-sensitive current, having a duration of 5-20 ms. This very fast transient current was observed during potential steps positive to -45 mV. The fast transient current was inactivated when the holding potential was made positive to -45 mV. The amplitude of the fast transient current was greatly reduced by the application of 4 mM Mn2+ or Ca2+-free artificial cerebrospinal fluid (CSF). The fast transient current appeared to be unaffected by 0.5 mM 4-AP but was greatly reduced by 10 mM TEA. These results suggest that the transient outward current observed during depolarizing steps is composed of at least two distinct transient currents. The more slowly decaying current resembles the A-current originally described in molluscan neurons (9, 32, 42) in voltage sensitivity, time course, and pharmacological sensitivity. The faster transient current resembles a fast, Ca2+-dependent transient current previously observed in bull-frog sympathetic neurons (5, 27).     

Rat hippocampal neurons in culture: potassium conductances

Two-electrode voltage-clamp methodology was used to analyze voltage-dependent ionic conductances in 81 rat hippocampal neurons grown in culture for 4-6 wk. Pyramidal and multipolar cells with 15- to 20-micron-diameter cell bodies were impaled with two independent KCl electrodes. The cells had resting potentials of -30 to -60 mV and an average input resistance of about 30 M omega. A depolarizing command applied to a cell maintained in normal medium invariably evoked a fast (2-10 ms) inward current that saturated the current-passing capacity of the system. This was blocked in a reversible manner by application of tetrodotoxin (TTX) (0.1-1.0 microM) near the recorded cell. In the presence of TTX, a depolarizing command evoked a rapidly rising (3-5 ms), rapidly decaying (25 ms) transient outward current reminiscent of "IA" reported in molluscan neurons. This was followed by a more slowly activating (approximately 100 ms) outward current response of greater amplitude that decayed with a time constant of about 2-3 s. These properties resemble those associated with the K+ conductance, IK, underlying delayed rectification described in many excitable membranes. IK was blocked by extracellular application of tetraethylammonium (TEA) but was insensitive to 4-aminopyridine (4-AP) at concentrations that effectively eliminated IA. IA, in turn, was only marginally depressed by TEA. Unlike IK, IA was completely inactivated when the membrane was held at potentials positive to -50 mV. Inactivation was completely removed by conditioning hyperpolarization at -90 mV. A brief hyperpolarizing pulse (10 ms) was sufficient to remove 95% of the inactivation. IA activated on commands to potentials more positive than -50 mV. The inversion potential of the ionic conductance underlying IA and IK was in the range of the K+ equilibrium potential, EK, as measured by the inversion of tail currents; and this potential was shifted in a depolarizing direction by elevated [K+]0. Thus, both current species reflect activation of membrane conductance to K+ ions. Hyperpolarizing commands from resting potentials revealed a time- and voltage-dependent slowly developing inward current in the majority of cells studied. This membrane current was observed in cells exhibiting "anomalous rectification" and was therefore labeled IAR. It was activated at potentials negative to -70 mV with a time constant of 100-200 ms and was not inactivated. A return to resting potential revealed a tail current that disappeared at about EK. IAR was blocked by extracellular CS+ and was enhanced by elevating [K+]0. It thus appears to be carried by inward movement of K+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Current and voltage clamp studies of the spike medium afterhyperpolarization of hypoglossal motoneurons in a rat brain stem slice preparation

Whole-cell patch clamp recordings were performed on hypoglossal motoneurons (HMs) in a brain stem slice preparation from the neonatal rat. The medium afterhyperpolarization (mAHP) was the only afterpotential always present after single or multiple spikes, making it suitable for studying its role in firing behavior. At resting membrane potential (-68.8 +/- 0.7 mV), mAHP (23 +/- 2 ms rise-time and 150 +/- 10 ms decay) had 9.5 +/- 0.7 mV amplitude, was suppressed in Ca(2+)-free medium or by 100 nM apamin, and reversed at -94 mV membrane potential. These observations suggest that mAHP was due to activation of Ca(2+)-dependent, SK-type K(+) channels. Carbachol (10-100 microM) reversibly and dose dependently blocked the mAHP and depolarized HMs (both effects prevented by 10 microM atropine). Similar mAHP block was produced by muscarine (50 microM). In control solution a constant current pulse (1 s) induced HM repetitive firing with small spike frequency adaptation. When the mAHP was blocked by apamin, the same current pulse evoked much higher frequency firing with strong spike frequency adaptation. Carbachol also elicited faster firing and adapting behavior. Voltage clamp experiments demonstrated a slowly deactivating, apamin-sensitive K(+) current (I(AHP)) which could account for the mAHP. I(AHP) reversed at -94 mV membrane potential, was activated by depolarization as short as 1 ms, decayed with a time constant of 154 +/- 9 ms at -50 mV, and was also blocked by 50 microM carbachol. These data suggest that mAHP had an important role in controlling firing behavior as clearly demonstrated after its pharmacological block and was potently modulated by muscarinic receptor activity.     

Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia.

The net outward current in bursting pace-maker neurones of the snail (Helix pomatia) during sustained and repeated voltage clamp pulses was studied. The properties of currents remaining in cobalt-Ringer or after TEA injection were compared with those in untreated cells. 2. With sustained voltage clamp depolarizations the net outward current first increases to a maximum at 150 msec and then declines to 60% or less of its peak intensity. This depression, which is greater during repetition of short pulses (e.g. 100 msec pulses at 0-5 sec intervals), represents a true decrease in the outward flow of K (designated IK) and is not due to a decreased driving force resulting from extracellular K accumulation. The steady-state current-voltage (I-V) relationship for IK is N-shaped (Heyer & Lux, 1976). 3. A component of IK persists when Ca and Mg in the medium are replaced by Co (ICo-res). With voltage clamp depolarizations ICo-res increases rapidly to a maximum and then partially inactivates with voltage dependent time constants of hundredths or tenths of seconds. Repolarization removes the inactivation. Thus, repeated stimulation with short pulses does not increase the depression of ICo-res-ICo-res (e.g. measured during voltage steps from holding potentials of -50 to near 0 mV) is smaller in test pulses preceded by depolarization and larger in pulses preceded by hyperpolarization. The steady state I-V relationship is not N-shaped. ICo-res is blocked by intracellular injection of tetraethylammonium (TEA). 4. Repeated voltage clamp depolarization to near 0 mV with 100 msec pulses for neurones with large Ca currents in normal Ringer produces a long-term depression which is maximal with 300-400 msec repolarizations (to -50 mV) between pulses. This corresponds with stimulus parameters for the maximum Ca current (Heyer & Lux, 1976). Intracellular injection of Ca2+ (also Ba2+ and Co2+) likewise reduces the total net outward current and especially the delayed outward current under voltage clamp. 5. The component of IK which is removed by Co is identified as Ca dependent and designated IK(Ca). With single voltage clamp pulses IK(Ca) follows the approximate time course and voltage dependence of the slow inward Ca current (Iin slow; Heyer & Lux, 1976). Several lines of evidence suggest that Ca ions moving through the membrane activate IK(Ca). 6. Part of IK cannot be blocked by intracellular TEA injection. In different neurones the magnitude of the IK component resistant to TEA (ITEA-res) is approximately proportional to the relative magnitudes of Iin slow.ITEA-res does not inactivate with sustained depolarization and shows pronounced long-term depression with repetitive stimulation at intermediate intervals and an increased outward current at the onset of the second and subsequent pulses following short repolarizations. The steady-state I-V relationship is N-shaped. ITEA-res is abolished by extracellular Co. 7. A net inward current with low depolarizations can be measured after TEA injection...     

Rat hippocampal neurons in culture: Ca2+ and Ca2+-dependent K+ conductances

Rat hippocampal neurons grown in dissociated cell culture were studied in a medium containing 1 microM tetrodotoxin (TTX) and 25 mM tetraethylammonium (TEA), which eliminated the Na+ and K+ conductances normally activated by depolarizing current injections. In this medium depolarizing current pulses evoked depolarizing regenerative potentials and afterhyperpolarizations in most cells. Both of these events were blocked by close application of Co2+ or Cd2+. These events resemble Ca2+ spikes reported previously in hippocampal pyramidal cells. The membrane potential at which these Ca2+ spikes could be triggered and the rheobase current necessary were dependent on the potential at which the cell was conditioned: the more depolarized the holding potential, the more negative the absolute potential at which a spike could be triggered and the less rheobase current required. The duration of these Ca2+ spikes was also sensitive to the holding potential: the more depolarized the holding level, the longer the duration of the triggered spikes. The amplitude and duration of the Ca2+ spikes were enhanced in a reversible manner by 0.5-1.0 mM 4-aminopyridine (4-AP) delivered in the vicinity of the cell. Two-electrode voltage-clamp analysis of cells studied in TTX, TEA-containing medium revealed an inward current response that peaked in 25-50 ms during depolarizing commands. This response first became detectable during commands to -30 mV. It peaked in amplitude during commands to -10 mV and was enhanced in medium containing elevated [Ca2+]0. It was blocked by either 20 mM Mg2+, 0.2 mM Cd2+, 5 mM Co2+, or 5 mM Mn2+. These results have led us to identify this inward current response as ICa2+. 4-AP enhanced the magnitude and duration of ICa2+ independent of the drug's depressant effects on a transient K+ current also observed under these same experimental conditions. In many but not all cells the Ca2+ spike was followed by a long-lasting hyperpolarization associated with an increase in membrane conductance. This was blocked by Co2+. Under voltage clamp ICa2+ was followed by a slowly developing outward current response that was attenuated by Co2+ or Cd2+. These properties observed under current- and voltage-clamp recording conditions are superficially similar to those previously reported for Ca2+-dependent K+ conductance mechanisms (IC) recorded in these and other membranes. Long-lasting tail currents following activation of IC inverted in the membrane potential range for the K+ equilibrium potential found in these cells.     

Calcium channel currents in isolated guinea-pig ventricular cells superfused with Ca-free EGTA solution

Changes in membrane currents seen in Ca-free, EGTA (1 mM)-containing Tyrode solution (EGTA Tyrode), were studied in isolated guinea-pig ventricular cells, under the voltage clamp performed with a "G omega seal" patch electrode. Application of the EGTA Tyrode (calculated [Ca]0 = 1.3 X 10(-9) M) first eliminated the usual calcium current, but induced an extra inward current within 2 min. The reversal potential of this current, as judged by the direction of the current change, was about +25 mV (without correction of a liquid junction potential of -12 mV), but above this voltage a decaying outward current was observed. The decay of these inward and outward currents during depolarization was slow, but a large, nearly time-independent component was evident. These currents, regardless of their polarity and time course, were reduced by application of verapamil (10(-5) M) and Mg (5 mM), and were inactivated by pre-depolarizations. In Na-free EGTA Tyrode, the inward current disappeared but the outward current persisted at high voltages. These results suggest that in ventricular cells, reduction of external Ca concentrations to a nanomolar range induces a Ca channel current composed of an inward current carried by Na, and an outward current, presumably carried by K ions. Because of the persistence of the apparently non-inactivating Ca channel current, the net membrane current evoked at voltages around 0 mV remained close to zero, or even inward, after the decay of the time-dependent component, which was completed within a few hundreds ms. This characteristic I-V relation was considered to be linked to the development of the long-lasting action potentials, with a plateau maintained at around 0 mV, in EGTA Tyrode.     

Currents under voltage clamp of burst-forming neurons of the cardiac ganglion of the lobster (Homarus americanus)

Crustacean cardiac ganglion neuronal somata, although incapable of generating action potentials, produce regenerative, slow (greater than 200 ms) depolarizing potentials reaching -20 mV (from -50 mV) in response to depolarizing stimuli. These potentials initiate a burst of action potentials in the axon and are thus termed driver potentials. The somata of the anterior-most neurons (cells 1 or 2) were isolated by ligaturing for study of their membrane currents with a two-electrode voltage clamp. Inward current is attributed to Ca2+ by reason of dependence of driver potential amplitude on [Ca2+]0, independence of [Na+]0, resistance to tetrodotoxin, and inhibition by Cd (0.2 mM) and Mn (4 mM). Ca-mediated current (ICa) is present at -40 mV. It is optimally activated by a holding potential (Vh) of -50 to -60 mV and by clamps (command potential, Vc) to -10 mV. Time to peak (10-30 ms) and amplitude are strongly voltage dependent. Maximum tail-current amplitudes observed at -70 to -85 mV are ca. 100 nA. Inward tail peaks may not be resolved by our clamp (settling time, 2 ms). Tails relax with a time constant (tau) of approximately equal to 12 ms (at -70 to -85 mV). ICa exhibits inactivation in double pulse regimes. Recovery has a tau of approximately equal to 0.7 s. Tail current analyses indicate an exponential decline (tau approximately equal to 23 ms at -20 mV) toward a maintained amplitude of inward current tails. Analysis of outward currents indicates the presence of three conductance mechanisms having voltage dependences, time courses, and pharmacology similar to those of early outward current (IA), delayed outward current (IK), and outward current (IC) of molluscan neurons. Analysis of tail currents indicates a reversal potential for each of these near -75 mV, indicating that they are K currents. Early outward current, IA, shows a peak at 5 ms followed by rapid decline. Response to a second clamp given within 0.4 s is reduced; recovery is exponential, with a tau of approximately equal to 200 ms (at Vh = -50 mV). The amplitude of IA tested at 0 mV shows activation or deactivation by subthreshold shifts of Vh. The extent and rate of these changes shows voltage dependence (tau approximately equal to 100-500 ms for subthreshold prepulses). At the normal cell resting potential of -50 mV the amplitude of IA is 25% of that tested from -80 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Muscarinic receptor activation induces a prolonged post-stimulus afterdepolarization with a conductance decrease in guinea-pig olfactory cortex neurones in vitro.

The persistent excitation of guinea-pig olfactory cortical neurones in vitro by the muscarinic agonist oxotremorine-M (OXO-M) was investigated. In OXO-M (10-20 microM), a slowly-decaying afterdepolarization (sADP) accompanied by sustained repetitive firing was induced following a long depolarizing stimulus. The corresponding slow inward current (IADP) revealed under voltage clamp behaved like a K(+)-mediated tail current, but was associated with a decreased membrane conductance. IADP was insensitive to tetrodotoxin (TTX), Ba2+, Cs+, or 4-aminopyridine (4-AP), but was blocked by 500 microM TEA or TBA (tetrabutylammonium). The OXO-M response and IADP were also reduced by Cd2+ or Ca(2+)-free solution, suggesting a dependence on Ca(2+)-entry. We propose that OXO-M induces a novel outward K+ current that can be slowly de-activated by Ca(2+)-entry during a depolarizing stimulus. Summation of IADP tail currents could contribute to the sustained muscarinic excitation of mammalian cortical neurones.     

Calcium inward currents in internally perfused giant axons

1. Voltage clamp experiments were carried out on squid axons perfused with an isotonic solution of 25 mM-CsF + sucrose and placed in a Na-free solution of 100 mM-CaCl(2) + sucrose.2. Depolarizing voltage steps produced inward currents of 4-6 muA/cm(2) peak amplitude which decayed slightly during a 60 msec pulse; the inward current disappeared when the internal potential reached +50 to +60 mV and became outward for larger depolarizations.3. Tetrodotoxin completely blocked the inward current and part of the outward current. No inward currents were seen with 100 mM-MgCl(2) + sucrose as the external solution. Substituting acetate for external Cl(-) did not abolish the tetrodotoxin-sensitive outward currents.4. It is concluded that the inward current is carried by Ca and the tetrodotoxin-sensitive outward current by Cs ions, both moving through the Na channel.5. The reversal potential of the tetrodotoxin-sensitive current was in the average +54 mV. Raising the external Ca concentration or adding NaCl to the external solution increased the reversal potential; lowering the external Ca concentration or replacing the internal CsF by a Na salt decreased the reversal potential.6. From the reversal potentials of the tetrodotoxin-sensitive current measured with varying external and internal solutions the relative permeabilities of the Na channel were calculated as P(Ca)/P(Cs) = 1/0.6, P(Ca)/P(Na) = 1/10 to 1/7 and P(Cs)/P(Na) = 1/22 to 1/9 by means of the constant field equation. The permeability ratios suggest that under these experimental conditions the Na channel is still primarily permeable to Na ions, although its selectivity is relatively small.7. The time course of the tetrodotoxin-sensitive Ca inward current was different from the time course of the Na inward current. The Na current consisted of an initial peak followed by a more slowly decaying component, the Ca current showed only the slow component.8. The slowly inactivating tetrodotoxin-sensitive Ca inward currents give rise to the long lasting action potentials which have first been observed by Tasaki and coworkers under similar conditions.     

Activity-dependent initiation of a prolonged depolarization in aplysia bag cell neurons: role for a cation channel

The translation of prior activity into changes in excitability is essential for memory and the initiation of behavior. After brief synaptic input, the bag cell neurons of Aplysia californica undergo a nearly 30-min afterdischarge to release egg-laying hormone. The present study examines a prolonged depolarization in cultured bag cell neurons. A 5-Hz, 10-s action potential train elicited a depolarization of about 10 mV, which lasted 相似文献   

Ca2+-channel current and its modification by the dihydropyridine agonist BAY k 8644 in isolated smooth muscle cells

The electrophysiological properties of single smooth muscle cells isolated from the longitudinal layer of the guinea-pig ileum were studied with the whole-cell patch-clamp technique. The finding of resting potentials between -45 and -50 mV and the occurrence of spontaneous electrical activity when K+ was the predominant intracellular cation indicated that the cells were not leaky or hyperpermeable. The existence of an inward Ca2+ current overlapping in time with an outward rectifying K+ current was demonstrated. The latter could be selectively blocked by replacing internal K+ with Cs+ and external Ca2+ with Ba2+. Depolarizations to potentials between -40 and +50 mV evoked time-dependent inward currents, with a maximum peak value between -20 and 0 mV. For depolarizations beyond +50 mV time-dependent outward currents appeared. These currents were inhibited by 0.1 mM CdCl2. The activation of the inward current showed a sigmoidal time course, and the rate of onset of the current increased at more positive potentials. Inactivation could be described by two exponentials. The threshold for activation was about -40 mV, and full activation was reached at 0 mV. Inactivation was complete near 0 mV, whereas the channels were fully available at -80 mV. The fully-activated Ca2+-channel current was strongly voltage dependent. The conductance decreased for potentials close to the reversal potential, and showed rectification for hyperpolarizing potentials. The Ca2+ agonist BAY k 8644 enhanced the Ca2+-channel current without a significant effect on its kinetics. The fully-activated current and the steady-state activation were enhanced in a rather voltage-independent way.     

HERG-Like potassium current regulates the resting membrane potential in glomus cells of the rabbit carotid body

Direct evidence for a specific K(+) channel underlying the resting membrane potential in glomus cells of the carotid body has been absent. The product of the human ether-a-go-go-related gene (HERG) produces inward rectifier currents that are known to contribute to the resting membrane potential in other neuronal cells. The goal of the present study was to determine whether carotid body glomus cells express HERG-like K(+) current, and if so, to determine whether a HERG-like current regulates the resting membrane potential. Freshly dissociated rabbit glomus cells under whole cell voltage clamp exhibited slowly decaying outward currents that activated 20-30 mV positive to the resting membrane potential. Raising extracellular K(+) revealed a slowly deactivating inward tail current indicative of HERG-like K(+) current. HERG-like currents were not found in cells resembling type II cells. The HERG-like current was blocked by dofetilide (DOF) in a concentration-dependent manner (IC(50) = 13 +/- 4 nM, mean +/- SE) and high concentrations of Ba(2+) (1 and 10 mM). The biophysical and pharmacological characteristics of this inward tail current suggest that it is conducted by a HERG-like channel. The steady-state activation properties of the HERG-like current (V(h) = -44 +/- 2 mV) suggest that it is active at the resting membrane potential in glomus cells. In whole cell, current-clamped glomus cells (average resting membrane potential, - 48 +/- 4 mV), DOF, but not tetraethylammonium, caused a significant (13 mV) depolarizing shift in the resting membrane potential. Using fluorescence imaging, DOF increased [Ca(2+)](i) in isolated glomus cells. In an in-vitro carotid body preparation, DOF increased basal sensory discharge in the carotid sinus nerve in a concentration-dependent manner. These results demonstrate that glomus cells express a HERG-like current that is active at, and responsible for controlling the resting membrane potential.     

Muscarine induces two distinct current responses in adrenal chromaffin cells of the guinea-pig

Muscarine-induced membrane responses were studied in dissociated chromaffin cells of the guinea-pig adrenal medulla, using the whole-cell version of the patch-clamp technique. Bath application of muscarine (1-10 microM) produced two distinct current responses at a holding potential of -40 mV. One is an inward current associated with an increase in current noise. This current response was sustained during stimulation and had a reversal potential of 4.5 +/- 3.4 mV (n = 6) with a negative slope conductance below about -30 mV in 12.5 mM K(+)-containing perfusate. The other is a transient outward current. This was evoked at membrane potentials more positive than -60 mV and completely suppressed by addition of 2 mM TEA to the bath solution, suggesting a possible involvement of the Ca2(+)-dependent K+ channel. Generation of the outward current response was suppressed for at least 60-90 s following 25 s muscarinic stimulation and was facilitated by activation of the nicotinic receptor. The maximum inward current seemed to be produced by 3 microM, whereas the threshold concentration required for generation of the outward current was somewhere between 3 and 10 microM. The outward current was evoked less often in cells treated with 2% collagenase for 1 h than in those treated with 0.2% for 30 min. The results suggest that guinea-pig chromaffin cells have two muscarinic receptors: one is coupled with a cation nonselective channel and the other may be related to a Ca2(+)-dependent K+ channel.     

Outward currents in rabbit pulmonary artery cells dissociated with a new technique.

Single cells from the rabbit pulmonary artery were isolated using a new and convenient procedure. Strips of muscle were incubated overnight in papain at 6 degrees C and dispersed the following morning after warming the tissue for 10 min. This method consistently produced a high yield of relaxed cells, which reversibly responded to vasoconstrictors and remained viable for many hours. The electrophysiological properties of these cells were studied using the patch-clamp technique in the whole-cell configuration. In physiological Ca2+ solution with K(+)-filled pipettes, cells had a high input resistance (approximately 17 G omega) and an average resting potential of -55 mV. In voltage clamp, several components of outward current could be identified. Depolarizing voltage steps revealed a prominent, transient current (Itran), having extremely rapid activation (less than 5 ms) and inactivation (less than 15 ms) kinetics. Itran was followed by a more slowly activating current (IKso) that was sustained over 100 ms. Both currents were essentially abolished by a 4-aminopyridine (4-AP) and sensitive to Ca2+ influx. IKso, but not Itran, was blocked by tetraethylammonium (TEA) and had the properties of a Ca(2+)-activated K+ current. Holding the membrane potential at -40 mV completely inactivated Itran and unmasked a time-independent, background current superimposed on IKso. The background current was also blocked by 4-AP. In addition, when adenosine triphosphate (ATP), but not guanosine triphosphate (GTP), was omitted from the patch-pipette, spontaneous bursts of outward current (SOCs) were superimposed on the voltage-activated currents. However, since SOCs were rarely observed when ATP and GTP were present together, they are unlikely to be active under physiological conditions. Thus at least four types of outward current can be distinguished in isolated rabbit pulmonary artery cells. These include a novel transient current which could be activated from the resting potential. It activates much more rapidly than outward currents previously reported in vascular muscle, and would rapidly oppose action potential firing. This current could therefore be responsible for the inability of large elastic arteries to fire action potentials.