Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons

A single-electrode voltage-clamp technique was employed on in vitro hippocampal slices to examine the membrane current responsible for the slow afterhyperpolarization (AHP) in CA1 pyramidal cells. This was achieved by using conventional procedures to evoke an AHP in current clamp, followed rapidly by a switch into voltage clamp (hybrid clamp). The AHP current showed a dependence on extracellular K+, which was close to that predicted for a K+ current by the Nernst equation. The AHP current could be blocked by Cd2+ or norepinephrine. Although the AHP current showed a requirement for voltage-dependent Ca2+ entry, the current did not show any clear intrinsic voltage dependence. Once activated, AHP current is not turned off by hyperpolarizing the membrane potential. The effects of norepinephrine, Cd2+, and tetraethylammonium (TEA) were used to identify an AHP current component to the outward current evoked by depolarizing voltage commands from holding potentials that approximate to the resting potential for these cells. The AHP current can contribute significantly to the outward current during the depolarizing command. Upon repolarization it is evident as a slow outward tail current. This slow tail current had the same time constant as AHP currents evoked by hybrid clamp. Fast components to the tail currents were also observed. These were sensitive to Cd2+ and TEA. They probably represent a voltage-sensitive gKCa, sometimes termed C-current. The strong sensitivity to voltage and TEA displayed by the conventionally described gKCa (IC) are properties inconsistent with the AHP. It seems likely that the AHP current (IAHP) represents a Ca2+-activated K+ current separate from IC and that these two currents coexist in the same cell.     

Characteristics of potassium and calcium currents of hepatic stellate cells (ito) in rat

Hepatic stellate cells (HSCs) are known to play a role in the pathogenesis of the increased intrahepatic vascular resistance found in chronic liver diseases. The aim of this study was to evaluate the K+ and Ca2+ currents in cultured HSCs from rat liver, through the patch-clamp technique. Most cells were positive for desmin immunostain after isolation and in alpha-smooth muscle actin immunostain after 10 - 14 days of culturing. Outward and inward rectifying K+ currents were confirmed. Two different types of K+ currents were distinguished: one with the inward rectifying current and the other without. The outward K+ currents consisted of at least four components: tetraethylammonium (TEA)-sensitive current, 4-aminopyridine (4-AP)-sensitive current, pimozide-sensitive current and three blocker-resistant current. The peaks of the outward K+ currents evoked by a depolarizing pulse were decreased to 32.0 +/- 3.0, 62.8 +/- 3.7 and 32.8 +/- 3.5% by 5 mM TEA, 2 mM 4-AP and 15 micro M pimozide, respectively. Moreover, the combined application of three blockers caused 86.6 +/- 4.8% suppression. The inward currents evoked hyperpolarizing pulses were inwardly rectifying and almost blocked by Ba2+. Elevation of external K+ increased the inward current amplitude and positively shifted its reversal potential. Voltage- dependent Ca2+ currents which were completely abolished by Cd2+ and nimodipine were detected in 14 day cultured HSCs. In this study, the cultured HSCs were found to express outward K+ currents composed of multiple pharmacological components, Ba2+-sensitive inward rectifying K+ current and L-type Ca2+ current.     

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.     

Plateau potentials in developing antennal-lobe neurons of the moth, Manduca sexta

Using whole cell recordings from antennal-lobe (AL) neurons in vitro and in situ, in semi-intact brain preparations, we examined membrane properties that contribute to electrical activity exhibited by developing neurons in primary olfactory centers of the brain of the sphinx moth, Manduca sexta. This activity is characterized by prolonged periods of membrane depolarization that resemble plateau potentials. The presence of plateau potential-generating mechanisms was confirmed using a series of tests established earlier. Brief depolarizing current pulses could be used to trigger a plateau state. Once triggered, plateau potentials could be terminated by brief pulses of hyperpolarizing current. Both triggering and terminating of firing states were threshold phenomena, and both conditions resulted in all-or-none responses. Rebound excitation from prolonged hyperpolarizing pulses could also be used to generate plateau potentials in some cells. These neurons were found to express a hyperpolarization-activated inward current. Neither the generation nor the maintenance of plateau potentials was affected by removal of Na+ ions from the extracellular medium or by blockade of Na+ currents with TTX. However, blocking of Ca2+ currents with Cd2+ (5 x 10(-4) M) inhibited the generation of plateau potentials, indicating that, in Manduca AL neurons, plateau potentials depend on Ca2+. Examining Ca2+ currents in isolation revealed that activation of these currents occurs in the absence of experimentally applied depolarizing stimuli. Our results suggest that this activity underlies the generation of plateau potentials and characteristic bursts of electrical activity in developing AL neurons of M. sexta.     

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)     

The effect of pentylenetetrazol on the levels of dopamine-beta-hydroxylase in the cerebrospinal fluid of rabbits: a dose-dependent relationship

The soma of olfactory cortex neurones in vitro was voltage-clamped by means of a single microelectrode sample-and-hold technique. In most neurones, hyperpolarizing voltage commands from relatively positive holding potentials (-40 to -50 mV) elicited a slow inward current relaxation with voltage-dependent and kinetic properties similar to the non-inactivating K+-current (M-current; IM), first described in amphibian sympathetic neurons. Deactivation of IM at negative potentials probably accounts for the slow sag of the hyperpolarizing electrotonic potential measured during current-clamp experiments. IM was inhibited by the cholinergic agonist muscarine or barium ions.     

Properties of subthreshold response and action potential recorded in layer V neurons from cat sensorimotor cortex in vitro

Properties of the action potential and subthreshold response were studied in large layer V neurons in in vitro slices of cat sensorimotor cortex using intracellular recording and stimulation, application of agents that block active conductances, and a single-microelectrode voltage clamp (SEVC). A variety of measured parameters, including action-potential duration, afterpotentials, input resistance, rheobase, and membrane time constant, were similar to the same parameters reported for large neurons from this region of cortex in vivo. Action-potential amplitudes and resting potentials were greater in vitro. Most measured parameters were distributed unimodally, suggesting that these parameters are similar in all large layer V neurons irrespective of their axonal termination. The voltage response to subthreshold constant-current pulses exhibited both time and voltage dependence in the great majority of cells. Current pulses in either the hyperpolarizing or subthreshold depolarizing direction cause the membrane potential to attain an early peak and then decay (sag) to a steady level. On termination of the pulse, the membrane response transiently overshoots resting potential. Plots of current-voltage relations demonstrate inward rectification during polarization on either side of resting potential. Subthreshold inward rectification in the depolarizing direction is abolished by tetrodotoxin (TTX). The ionic currents responsible for subthreshold rectification and sag were examined using the SEVC. Steady inward rectification in the depolarizing direction is caused by a persistent, subthreshold sodium current (INaP) (54). Sag observed in response to a depolarizing current pulse is due to activation of a slow outward current, which superimposes on and partially counters the persistent sodium current. Both sag in response to hyperpolarizing current pulses and rectification in the hyperpolarizing direction are caused by a slow inward "sag current" that is activated by hyperpolarizing voltage steps. The sag current is unaltered by TTX, tetraethylammonium, (TEA), Co2+, Ba2+, or 4-aminopyridine. Fast-rising, short-duration action potentials can be elicited by an intracellular current pulse or by orthodromic or antidromic stimulation. Spikes are blocked by TTX. The form of the afterpotential following a directly evoked spike varies among cells with similar resting potentials. Biphasic afterhyperpolarizations (AHPs) with fast and slow components were most frequently seen. About 30% of the cells displayed a depolarizing afterpotential (DAP), which was often followed by an AHP. Other cells displayed a purely monophasic AHP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different discharge properties of rat facial nucleus motoneurons

In this paper, we describe two types of putative facial motoneuron based on their electrophysiological properties and on their firing frequency adaptation as recorded in rat brainstem slices. Type I motoneurons (n = 33, 61%) were characterized by a sustained spike firing during depolarizing current injections and a marked depolarizing sag (inward rectification) during hyperpolarizing pulses. The time-course and voltage-dependence of the inward rectification together with the finding that it was blocked by Cs+ are consistent with the involvement of a Na+ -and K+ -mediated Q current. Type II motoneurons (n = 21, 39%) were identified by a fast spike firing adaptation. Type II cells showed a less pronounced inward rectification with hyperpolarizing current pulses and a higher discharge rate than type I cells during depolarizing current pulses. These distinct discharge properties imply the activation of a Ca2+ -dependent K+ current, because when carbachol was added to the bath, or the slice was exposed to a Ca2+ -free solution, a decrease was noticed in the firing frequency adaptation. The two types of motoneuron were further differentiated by the initial delay of the first spike, observed only in type I cells, which was blocked by bath application of 4-aminopyridine, indicating the presence of a K+ -mediated A current. The addition of 4-aminopyridine to the bath also increased the firing rate due to a decrease of the post-spike afterhyperpolarization. However, the two types of motoneuron were not morphologically differentiated. Facial motoneurons exhibited rhythmic membrane potential oscillations (8-20 Hz) at depolarized membrane potentials or during the silence following spike frequency adaptation. It is suggested that the intrinsic properties of these two types of facial motoneuron may be relevant in the government of distinct facial muscle activities. The fact that their discharge rate and the level of spike frequency adaptation were modified by altering some K+ currents suggests a potential plasticity in the modulation of motoneuron firing activities depending upon functional motor needs.     

Electrophysiology of the mammalian cerebellar cortex in organ culture

A direct comparison was made between the electrical properties of rat Purkinje cells in cerebellar organotype cultures and those in acute slices from age-matched animals. Cultures were prepared from 9-11-day-old animals. Intracellular recordings were made 5-12 days later, at which time the folia architecture of the cerebellum was still well preserved. The resting membrane potentials and input resistances of Purkinje cells in cultured and acute slice preparations from young animals were comparable to those of mature Purkinje cells in slices. Neurons from animals younger than 14 days differed from mature Purkinje cells in that they fired at low frequencies in response to outward current pulses. The latter property was found in all cultured neurons studied, independent of their time in culture. These action potentials were generated by Na+ and Ca2+ conductances as shown by the application of selective channel blockers. Cultured or acute slice preparations from animals younger than 11 days shared other immature electroresponsive features. In both groups, Na+-dependent plateau depolarizations were observed in less than 10% of Purkinje cells unless K-conductances were blocked, and considerable membrane depolarization was often required to elicit Ca2+-dependent action potentials. These findings are compatible with the relative prominence of voltage-dependent outward currents in immature Purkinje cells, a property which may be enhanced in culture. The injection of hyperpolarizing current pulses revealed a marked time-dependent anomalous rectification in all Purkinje cells. At the breaks of such pulses, several events were observed. In all cells, a rebound conductance was identified which could generate post-anodal spike bursts. In cultured neurons, however, hyperpolarizing pulses were also followed by a slow return to resting potential. This membrane potential profile was similar to that produced by the activation of an A conductance. Experiments on acute slices from animals of different ages (P9-P17) showed that this A-like conductance was expressed only during a brief period in Purkinje cell development. A higher level of spontaneous synaptic activity was observed in cultured than in acute slice preparations. Both unitary excitatory postsynaptic potentials and inhibitory postsynaptic potentials could be elicited in the former by parallel fiber stimulation, and could be fully reversed by outward or inward transmembrane current injections, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrotonic properties of motor nerve terminals

To obtain information about the electric membrane properties of frog motor nerve terminals we examined how depolarizing or hyperpolarizing current pulses of 2–8 ms duration to the preterminal, by electrotonic spread of potential, affected depolarization induced transmitter release. Sodium channels were blocked by tetrodotoxin. Under this condition a hyperpolarizing current pulse produced inhibition of release, followed by poten-tiation of release. Inhibition lasted more than 100 ms with a time constant of %I50 ms. When, in addition, potassium channels were blocked by 3,4-diaminopyridine or tetra-ethylammonium a depolarizing current pulse potentiated transmitter release for a period up to 50 ms. The results imply that inward currents in the nerve terminal are carried mainly by sodium and calcium ions and outward currents by potassium ions while “leak” conductances are negligible. A low “leak” conductance and therefore a high specific membrane resistance facilitates the spread of electrotonic potentials and thereby explains the long-lasting effects on transmitter release of brief current pulses to the preterminal.     

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 early afterhyperpolarization after a brief train of action potentials in rat hippocampal neurons in vitro

1. In rat hippocampal pyramidal cells in vitro, a brief train of action potentials elicited by direct depolarizing current pulses injected through an intracellular recording electrode is followed by a medium-duration afterhyperpolarization (mAHP) and a longer, slow AHP. We studied the mAHP with the use of current-clamp techniques in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) to block the slow AHP and isolate the mAHP. 2. The mAHP evoked at hyperpolarized membrane potentials was complicated by a potential generated by the anomalous rectifier current, IQ. The mAHP is insensitive to chloride ions (Cl-), whereas it is sensitive to the extracellular potassium concentration ([K+]o). 3. At slightly depolarized levels, the mAHP is partially Ca2+ dependent, being enhanced by increased [Ca2+]o and BAY K 8644 and depressed by decreased [Ca2+]o, nifedipine, and Cd2+. The Ca2(+)-dependent component of the mAHP was also reduced by 100 microM tetraethylammonium (TEA) and charybdotoxin (CTX), suggesting it is mediated by the voltage- and Ca2(+)-dependent K+ current, IC. 4. Most of the Ca2(+)-independent mAHP was blocked by carbachol, implying that IM plays a major role. In a few cells, a small Ca2(+)- and carbachol-insensitive mAHP component was detectable, and this component was blocked by 10 mM TEA, suggesting it was mediated by the delayed rectifier current, IK. The K+ channel antagonist 4-aminopyridine (4-AP, 500 microM) did not reduce the mAHP. 5. We infer that the mAHP is a complex potential due either to IQ or to the combined effects of IM and IC. The contributions of each current depend on the recording conditions, with IC playing a role when the cells are activated from depolarized potentials and IM dominating at the usual resting potential. IQ is principally responsible for the mAHP recorded at hyperpolarized membrane potentials.     

Mechanosensitive channel currents recorded in rat microvascular endothelial cells with whole-cell mode.

Using the whole-cell patch-clamp technique, we recorded delayed outward currents in rat brain microvascular endothelial cells (BRMECs), which were nearly completely inhibited by 20 mmol/L extracellular TEA-Cl and 5 mM extracellular CsCl. Whole-cell currents were elicited under voltage clamp condition by 2100 ms depolarizing voltage pulses applied every 7 s between -100 to 90 mV in 10 mV increment from a holding potential of -100 mV. The currents were defined as delayed rectified K+ currents (IKv), which were inhibited in a concentration-dependent manner by bath application of TEA-Cl, with an IC50 approximately 2.0 mM, similar to that reported on IKv in other preparations. In the present of mechanical force, outward currents were increased in amplitude as compared with controls. These mechanical force induced currents were also defined as IKv, which are different from previous described mechanosensitive currents with characteristic of inward rectifier.     

Effects of flufenamic acid on smooth muscle of the carotid artery isolated from spontaneously hypertensive rats.

Endothelium-removed carotid artery strips from stroke-prone spontaneously hypertensive rats spontaneously developed a tonic myogenic contraction. Flufenamic acid reduced the resting tone observed during superfusion with Tyrode's solution, in a concentration-dependent manner. Flufenamic acid also inhibited contractions produced by high-K solutions in a concentration-dependent manner. The resting membrane potential of smooth muscle cells in the artery was around -32 mV, with occasional oscillatory potentials. Flufenamic acid hyperpolarized the membrane in a concentration-dependent manner. The voltage-dependent outward currents recorded in isolated cells with micropipettes filled with high-K+ solution (holding potential, -60 mV) were enhanced by flufenamic acid and inhibited by tetraethylammonium. When the recording micropipette was filled with high Cs to inhibit the K+-current, depolarizing step pulses evoked nifedipine-sensitive inward currents. Flufenamic acid inhibited the inward currents. These results indicate that flufenamic acid inhibits the spontaneous active tone of the carotid artery by inhibiting L-type Ca2+-channels and possibly by membrane hyperpolarization through activation of the voltage-dependent K+-channels.     

An inward calcium current underlying regenerative calcium potentials in rat striatal neurons in vitro enhanced by Bay K 8644

The single electrode voltage clamp technique was used to characterize the currents underlying the calcium potentials in rat caudate neurons in vitro. In current clamp experiments, long depolarizing current pulses evoked repetitive firing of fast somatic action potentials. These were abolished by tetrodotoxin (1 microM) and replaced by slow graded depolarizing potentials. These were preceded by a transient hyperpolarizing notch. Addition of 4-aminopyridine (100 microM) abolished the hyperpolarizing notch, enhanced the slow graded depolarizing response and induced the appearance of a slow all-or-nothing action potential. Both the slow graded response and the all-or-nothing action potential were abolished by cobalt (2 mM), suggesting the involvement of voltage-dependent calcium conductances. When the neurons were loaded intracellularly with caesium the action potential duration increased. Substitution of the extracellular calcium by barium (1-3 mM) or external addition of tetraethylammonium (5 mM) further prolonged spike duration and induced the appearance of long-lasting plateau potentials. These were insensitive to tetrodotoxin and were reversibly blocked by the calcium antagonists cobalt (2 mM), manganese (2 mM) or cadmium (500 microM). The calcium potentials were enhanced by the calcium 'agonist' BAY K 8644 (1-5 microM). In voltage clamp experiments when intracellular caesium was used to reduce outward currents and tetrodotoxin to block fast regenerative sodium currents, depolarizing voltage steps from a holding potential of -50, -40 mV activated an inward current. This current peaked in 50-80 ms and inactivated in two phases: an initial one at 150-200 ms followed by a second one after several hundred ms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetrabutylammonium: a selective blocker of the somatostatin-activated hyperpolarizing current in mouse AtT-20 corticotrophs

To obtain a clearer understanding of the mechanisms by which somatostatin modulates stimulus-secretion coupling in neuroendocrine cells, we investigated the pharmacology of the somatostatin-activated inward rectifier in mouse pituitary tumour cells (AtT-20 corticotrophs). Individual AtT-20 cells displayed spontaneous, long-lasting action potentials that caused transient spikes in cytosolic [Ca2+] ([Ca]i). Application of 1-10 nM somatostatin led to membrane hyperpolarization and loss of [Ca]i spiking activity. Voltage-clamp recordings revealed that the somatostatin-induced hyperpolarization was due to an inwardly rectifying K+ current. Tetrabutyl-ammonium (TBA+) inhibited both outward and inward currents through the inward rectifier, whereas Cs+ blocked only inward current and tetraethylammonium (TEA+) was completely ineffective in blocking somatostatin-activated currents. However TEA+, but neither TBA+ nor Cs+, blocked voltage-gated outward currents. Correspondingly, TBA+ abolished the hyperpolarizing effects of somatostatin and, of the three K+ channel blockers, only TBA+ prevented the somatostatin-induced inhibition of [Ca]i spiking. TBA+ may thus prove a useful tool in elucidating the underlying mechanisms by which somatostatin affects the secretory activity of neuroendocrine cells.     

Properties of the inwardly rectifying K+ conductance in the toad retinal pigment epithelium.

An inwardly rectifying K+ current was analysed in isolated toad retinal pigment epithelial (RPE) cells using the perforated-patch clamp technique. The zero-current potential (Vo) of RPE cells averaged -71 mV when the extracellular K+ concentration ([K+]o) was 2 mM. Increasing [K+]o from 0.5 to 5 mM shifted V0 by +43 mV, indicating a relative K+ conductance (TK) of 0.74. At [K+]o greater than 5 mM, TK decreased to 0.53. Currents were larger in response to hyperpolarizing voltage pulses than depolarizing pulses, indicating an inwardly rectifying conductance. Currents were time independent except in response to voltage pulses to potentials positive to 0 mV, where the outward current decayed with an exponential time course. Both the inwardly rectifying current and the transient outward current were eliminated by the addition of 0.5 mM Ba2+, 5 mM Cs+ or 2 mM Rb+ to the extracellular solution. The current blocked by these ions reversed near the K+ equilibrium potential (EK) over a wide range of [K+]o, indicating a highly selective K+ channel. The current-voltage relationship of the isolated K+ current exhibited mild inward rectification at voltages negative to -20 mV and a negative slope conductance at voltages positive to -20 mV. The Cs(+)- and Ba(2+)-induced blocks of the K+ current were concentration dependent but voltage independent. The apparent dissociation constants were 0.8 mM for Cs+ and 40 microM for Ba2+. The K+ conductance decreased when extracellular Na+ was removed. Increasing [K+]o decreased the K+ chord conductance (gK) at negative membrane potentials. In the physiological voltage range, increasing [K+]o from 2 to 5 mM caused gK to decrease by approximately 25%. We conclude that the inwardly rectifying K+ conductance represents the resting K+ conductance of the toad RPE apical membrane. The unusual properties of this conductance may enhance the ability of the RPE to buffer [K+]o changes that take place in the subretinal space at the transition between dark and light.     

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.     

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.     

Changes in membrane currents of hippocampal neurons evoked by brief anoxia

1. Effects of anoxia (2-4 min of 95% N2-5% CO2) on membrane currents of CA1 neurons were studied by single-electrode voltage clamp in hippocampal slices (from Sprague-Dawley rats) kept in an interface-type chamber at 33.5 degree. 2. When recording with KCl electrodes at a holding potential (VH) near-70 mV, anoxia evoked a slow outward current [0.18 +/- 0.06 (SE) nA], accompanied by a conductance increase ( + 46 +/- 20%, mean +/- SE). The difference current evoked by N2 had a reversal potential near-100 mV. It was much smaller in presence of 2-4 mM extracellular Cs, and any remaining outward current was abolished by 10 mM tetraethylammonium (TEA). Only inward currents were observed when recording with CsCl electrodes. 3. Inward relaxations evoked by large hyperpolarizing pulses from VH less than or equal to - 70 mV (Q-type) were not significantly depressed by anoxia (-1.5 +/- 6.0%). 4. Some voltage-dependent outward currents (evoked by 200-ms depolarizing pulses) were depressed during anoxia: 1) a fast-inactivating (A-like) current, obtained at VH less than or equal to -70 mV and suppressed by 200 microM 4-AP, was reduced by 25.6 +/- 7.3% (n = 5); 2) a slower, noninactivating (C-like) current, suppressed by TEA, was reduced by 52 +/- 7.2% (n = 16). Neither of these currents (1 or 2) was observed when recording with 2- to 3-M CsCl electrodes; and 3) small (M-like) inward relaxations, observed at VH approximately -40 mV 5. Net inward currents could be evoked after blockage of GK with 10 mM TEA when recording with KCl electrodes or by recording with CsCl electrodes. At VH less than or equal to -70 mV, large, transient, and incompletely controlled currents were evoked by depolarizing pulses; at VH less than or equal to -50 mV, smaller and more persistent currents were evoked by depolarizing pulses (L-like), and transient currents (T-like?) were seen immediately after hyperpolarizing pulses. 6.L-type currents (at VH less than or equal to -50 mV) were nearly abolished after 1-2 min anoxia (by approximately 90%). This was equally true of the currents evoked by constant pulses or peak currents in I-V plots. After reoxygenation, recovery was biphasic, with a quick early phase (to 50-80% in 2 min) and then a much slower one (to 60-90% by 10-15 min).(ABSTRACT TRUNCATED AT 400 WORDS)