Modulation of glutamate-induced outward current by prostaglandin E(2) in rat dissociated preoptic neurons

The preoptic/anterior hypothalamus (POA) is one of the major brain regions where cytokines and their related mediators (i.e., prostaglandins) exert diverse actions. In the present study, the modulatory effects of prostaglandin E(2) (PGE(2)) on the glutamate-induced membrane currents were examined using perforated-patch clamp method in rat POA neurons that had been mechanically dissociated by vibration without enzyme treatment. Application of glutamate through U-tube induced a slow outward current following fast inward ionotroic current at a holding membrane potential of -30 mV. The slow outward current was also induced by N-methyl-d-aspartate (NMDA), accompanied by an increased membrane conductance, and inhibited by perfusion with Ca(2+)-free solution, tetraethylammonium chloride (TEA), and apamin, suggesting a Ca(2+)-dependent K(+) current (KCa) activated by Ca(2+) entry through NMDA channels. Perfusion with PGE(2) at 0.1-10 microM, a principal mediator of fever and neuroendocrine control at the POA, did not produce apparent current by itself, but selectively potentiated the glutamate- or NMDA-induced KCa without affecting inward currents. The KCa induced by activation of NMDA receptors may serve as a feedback mechanism and the modulatory effects of PGE(2) on the KCa may have an important physiological significance.     

Cobalt-dependent potentiation of net inward current density in Helix aspersa neurons.

A low concentration of transition metal ions Co2+ and Ni2+ increases the inward current density in neurons from the land snail Helix aspersa. The currents were measured using a single electrode voltage-clamp/internal perfusion method under conditions in which the external Na+ was replaced by Tris+, the predominant external current carrying cation was Ca2+, and the internal perfusate contained 120 mM Cs+/0 K+; 30 mM tetraethylammonium (TEA) was added externally to block K+ current. In the presence of Co2+ (3 mM) or Ni2+ (0.5 mM) inward Ca2+ currents were stimulated normally by voltage-dependent activation of Ca2+ channels. There was a 5-10% decrease in the rate of rise of the inward current. The principal effect of Co2+ and Ni2+ in increasing the current density seems to be a decrease in the rate at which the inward currents decline during a depolarizing voltage pulse. The results may be due to a decrease in a voltage-dependent or Ca(2+)-dependent outward current and/or an inhibition of Ca2+ channel inactivation. Outward current under these conditions (zero internal K+) was significant and most likely due to Cs+ efflux through the voltage-activated or Ca(2+)-activated nonspecific cation channels. Co2+ is an extremely effective blocker of this outward current. These results are not an artifact of internal perfusion or the special ionic conditions. Intracellular recording of unperfused neurons in normal Helix Ringer's solution showed that the Ca(2+)-dependent action potential duration was increased significantly by low concentrations of Co2+. This result is consistant with the Co(2+)-dependent increase in inward (depolarizing) current seen in voltage-clamp experiments.     

Calcium-dependent subthreshold oscillations determine bursting activity induced by N-methyl-D-aspartate in rat subthalamic neurons in vitro

We used whole-cell patch recordings in current clamp to investigate the ionic dependence of burst firing induced by N-methyl-d-aspartate (NMDA) in neurons of the subthalamic nucleus (STN) in slices of rat brain. NMDA (20 microm) converted single-spike firing to burst firing in 87% of STN neurons tested. NMDA-induced bursting was blocked by AP5 (50 microm), and was not mimicked by the non-NMDA receptor agonist AMPA (0.6 microm). Tetrodotoxin (1 microm) converted bursts to oscillations of membrane potential, which were most robust when oscillations ranged between -50 and -70 mV. The NMDA bursts were blocked by an elevated extracellular concentration of Mg(2+), but superfusate containing no added Mg(2+) either reduced or increased burst firing, depending upon the amount of intracellular current injection. Block of K(+) conductances by apamin and tetraethylammonium prolonged burst duration, but iberiotoxin had no effect. NMDA-induced burst firing and membrane oscillations were completely blocked by superfusate containing no added Ca(2+), and they were significantly reduced when patch pipettes contained BAPTA. Selective antagonists for T-type (mibefradil, 10 microm), L-type (nifedipine, 3 microm), and N-type (omega-conotoxin GVIA, 1 micro m) Ca(2+) channels had no effect on NMDA burst firing. Superfusate containing a low concentration of Na(+) (20 mm) completely abolished NMDA-induced burst firing. Flufenamic acid (10 microm), which blocks current mediated by Ca(2+)-activated nonselective cation channels (I(CAN)), reversibly abolished NMDA-depended bursting. These results are consistent with the hypothesis that NMDA-induced burst firing in STN neurons requires activation of either an I(CAN) or a Na(+)-Ca(2+) exchanger.     

Cellular mechanisms underlying the rhythmic bursts induced by NMDA microiontophoresis at the apical dendrites of CA1 pyramidal neurons

This article reports the cellular mechanisms underlying a form of intracellular "theta-like" (theta-like) rhythm evoked in vitro by microiontophoresis of N-methyl-D-aspartate (NMDA) at the apical dendrites of CA1 pyramidal neurons. Rhythmic membrane potential (Vm) oscillations and action potential (AP) bursts (approximately 6 Hz; approximately 20 mV; approximately 2-5 APs) were evoked in all cells. The response lasted approximately 2 s, and the initial oscillations were usually small (< 20 mV) and below AP threshold. Rhythmic bursts were never evoked by imposed depolarization in the absence of NMDA. Block of Na+ conductance with tetrodotoxin (TTX) (1.5 microM), of non-NMDA receptors with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 microM) and of synaptic inhibition by bicuculline (50 microM) and picrotoxin (50 microM) did not prevent NMDA oscillation. Inhibition of the voltage dependence of the NMDA conductance in Mg2+-free Ringer's solution blocked oscillations. Preventing Ca2+ influx with Ca2+-free and Co2+ (2-mM) solutions and block of the slow Ca2+-dependent afterhyperpolarization (sAHP) by carbamilcholine (5 microM), isoproterenol (10 microM), and intracellular BAPTA blocked NMDA oscillations. Inhibition of L-type Ca2+ conductance with nifedipine (30 microM) reduced oscillation amplitude. Block of tetraethylammonium (TEA) (10 mM) and 4AP (10 mM)-sensitive K+ conductance increased the duration and amplitude, but not the frequency, of oscillations. In conclusion, theta-like bursts relied on the voltage dependence of the NMDA conductance and on high-threshold Ca2+ spikes to initiate and boost the depolarizing phase of oscillations. The repolarization is initiated by TEA-sensitive K+ conductance and is controlled by the sAHP. These results suggest a role of interactions between NMDA conductance and intrinsic membrane properties in generating the CA1 theta-rhythm.     

Norepinephrine and cyclic adenosine 3':5'-cyclic monophosphate enhance a nifedipine-sensitive calcium current in cultured rat astrocytes

We employed two microelectrode current-clamp and voltage-clamp methods to examine the modulation of Ca++ channels by norepinephrine and cyclic AMP (cAMP) in cultured astrocytes from the rat cerebral cortex. Currents owing to Ca++ channels were maximized by replacing Ca++ with Ba++ in the extracellular solution and pharmacologically blocking K+ and Na+ currents. In current-clamp experiments, we observed that norepinephrine, isoproterenol (an agonist of beta-receptors for norepinephrine), or dibutyryl cAMP (dbcAMP, a membrane permeant analogue of cAMP) induced or enhanced slow Ba++-dependent action potentials in the cells. In voltage-clamp experiments, we confirmed that the slow action potentials were generated by a voltage-activated and Ba++-dependent inward current. This current was mediated by channels that resembled L-type calcium channels (cf. McCleskey et al., Journal of Experimental Biology 124:177-190, 1986) in their voltage-activation range, slow inactivation, and sensitivity to blockage by Co++, Cd++, and nifedipine. DbcAMP, or isoproterenol, enhanced the Ba++ current. Modulation of Ca++ channel function in glial cells could have functional implications.     

Intracellular Ca2+ suppressed a transient potassium current in hippocampal neurons

The effects of intracellular Ca2+ (Ca2+i) on K+ currents in hippocampal cells were examined using acutely isolated cells obtained from adult guinea pigs. Whole-cell voltage-clamp recordings were carried out in a configuration that allowed a continuous perfusion of the intracellular medium. Recording media were made to block inward currents and allowed selective activation of K(+)-dependent outward currents. Voltage-dependent outward currents consisted of an initial rapidly decaying component followed by a sustained component. The time constant of decay of the transient current was about 25 msec, and previous studies (Numann et al., 1987) showed that the kinetic and pharmacological properties of this current closely resembled the A current recorded in invertebrate neurons (Connor and Stevens, 1971; Thompson, 1982). Intracellular perfusion of hippocampal cells with a solution containing elevated Ca2+ (about 4.5 x 10(-4) M) elicited outward currents at the holding potential (-45 to -55 mV) and produced changes in voltage-dependent K+ currents. The transient outward current (IA) activated by depolarization was suppressed with increases in Ca2+i. Delayed, sustained K+ currents were greatly potentiated. Data also showed that, among the 3 effects elicited by Ca2+i, suppression of IA was most sensitive to Ca2+i elevation. Previous results (Numann et al., 1987) showed that IA had a lower threshold (about -45 mV) than sustained currents (about -40 mV). By using low levels of depolarization (-40 mV), IA can be selectively activated, and the suppressive effect of Ca2+i on IA was confirmed on the kinetically isolated IA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemokine modulation of high-conductance Ca(2+)-sensitive K(+) currents in microglia from human hippocampi

During acute pathological processes, microglia transform into an activated state characterized by a defined morphology and current profile, and are recruited to injury sites by chemokines. No information is available on the ion channels and the mode of action of chemokines in microglia in brain slices from humans with a chronic pathology. Thus, patch-clamp recordings of microglia were performed in hippocampal slices from seven patients who underwent surgery for pharmaco-resistant epilepsy. Cells were identified as microglia by positive labelling with fluorescein-conjugated tomato lectin before recording. All the recorded cells had an ameboid morphology characteristic of activated microglia. However, they had a high input resistance (3.6 G omega), a zero-current resting potential of -16 mV, and lacked Na+ currents, inwardly rectifying and delayed rectifying K+ currents such as non-activated microglia. Importantly, recorded cells expressed Ca2+-sensitive outward currents that activated at 0 mV with non-buffered intracellular Ca2+ and were sensitive to 1 mm tetraethylammonium (TEA). The estimated single-channel conductances were 187 pS in cell-attached and 149 pS in outside-out patches, similar to those of high-conductance Ca2+-dependent K+ channels. The chemokine MIP1-alpha increased whole-cell outward current amplitudes measured at +60 mV by a factor of 3.3. Thus, microglia in hippocampi from epileptic patients express high-conductance Ca2+-dependent K+ channels that are modulated by the chemokine MIP1-alpha. This modulation may contribute to the migratory effect of MIP1-alpha on microglia.     

N-methyl-D-aspartate receptor-induced, inherent oscillatory activity in neurons active during fictive locomotion in the lamprey

Bath application of N-methyl-aspartate induces fictive locomotor activity in the isolated spinal cord preparation of the lamprey, as well as TTX-resistant membrane potential oscillations in many individual neurons. This inherent oscillatory activity is shown to depend on a specific activation of N-methyl-D-aspartate (NMDA) receptors. This activation initiates voltage-dependent, magnesium-requiring membrane potential bistability, presumably due to a development of a region of negative slope conductance in the current-voltage relation of the neuron. When sodium ions were removed from the bathing solution, oscillations disappeared, and the membrane potential was maintained at a hyperpolarized level, suggesting that the depolarizing current during the oscillatory cycle is mainly carried by sodium ions. Replacing Ca2+ with Ba2+ also leads to a cessation of oscillatory activity, with the membrane potential remaining at the more depolarized level. This indicates an involvement of a Ca2+-dependent K+ current during the repolarization phase. These findings, together with the voltage dependence, can account for the main characteristics of the NMDA receptor-induced, TTX-resistant membrane potential oscillations. This oscillatory behavior has been demonstrated in motoneurons and in several interneurons including CC interneurons but has not been found in edge cells, dorsal cells, or lateral interneurons. The possibility that inherent oscillatory membrane properties may contribute to the activity pattern during fictive locomotion was investigated in experiments with intracellular current injection in the absence of TTX. The stimulation effects obtained required the presence of magnesium ions and were analogous to the stimulation effects seen during oscillations after TTX blockade. Together with similarities in, for instance, frequency and amplitude between the locomotor oscillatory activity and the TTX-resistant oscillations, the results are compatible with an involvement of inherent, oscillatory membrane properties during fictive locomotion in the lamprey spinal cord.     

Calcium action on the membrane currents possessing the properties of mechano-electric transducer currents in inner hair cells of the Guinea-pig cochlea

In free-standing hair bundle, depolarization to +80 mV evoked a stable outward current and repolarization to -80 mV evoked a transient inward current attributable to the opening of mechano-electric transducer channels. The study investigated the Ca2+ dependence of this transducer-like membrane current in isolated inner hair cells of guinea-pig cochlea. The amplitude of outward currents increased and the rate of inward current decay, corresponding to adaptation kinetics, decreasing as the extracellular Ca2+ concentrations lessened, whereas the amplitude of outward current decreased and an adaptation accelerated as the extracellular Ca2+ elevated. Treatment with the cAMP agonist, 8-bromo-cAMP, induced an effect similar to that caused by elevating the extracellular Ca2+.     

N-methyl-D-aspartate enhancement of the glycine response in the rat sacral dorsal commissural neurons

The effect of N-methyl-D-aspartate (NMDA) on the glycine (Gly) response was examined in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The application of 100 microM NMDA to SDCN neurons reversibly potentiated Gly-activated Cl- currents (IGly) without affecting the Gly binding affinity and the reversal potential of IGly. A selective NMDA receptor antagonist, APV (100 microM), blocked the NMDA-induced potentiation of IGly, whereas 50 microM CNQX, a non-NMDA receptor antagonist, did not. The potentiation effect was reduced when NMDA was applied in a Ca2+-free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage-dependent Ca2+ channels. Pretreatment with KN-62, a selective Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the NMDA action. Inhibition of calcineurin (CaN) further enhanced the NMDA-induced potentiation of IGly. In addition, the GABAA receptor-mediated currents were suppressed by NMDA receptor activation in the SDCN neurons. The present results show that Ca2+ entry through NMDA receptors modulates the Gly receptor function via coactivation of CaMKII and CaN in the rat SDCN neurons. This interaction may represent one of the important regulatory mechanisms of spinal nociception. The results also suggest that GABAA and Gly receptors may be subject to different intracellular modulatory pathways.     

ATP-sensitive and Ca2+-activated K+ channel activities in the rat locus coeruleus neurons during metabolic inhibition.

Locus coeruleus (LC) is the significant nucleus for consciousness and it is sensitive to metabolic inhibition. We investigated the effects of a metabolic inhibitor sodium cyanide (NaCN) on the rat dissociated LC neurons using nystatin-perforated patch recordings. Under voltage-clamp (VH=-40 mV), application of NaCN evoked outward currents composed of ATP-sensitive and Ca2+-dependent K+ channel currents (IKATP and IKCa2+). Onset of IKATP was faster than that of IKCa2+. Prolonged application of NaCN brought IKATP rundown but not IKCa2+ rundown. Okadaic acid prevented IKATP rundown, indicating that KATP channels are deactivated by dephosphorylation with protein phosphatase.     

Differential NMDA-dependent activation of glial cells in mouse hippocampus

In the hippocampus, the NMDA receptor is thought to be an important glutamate receptor involved in synaptic plasticity and in memory processes. Until recently, NMDA receptors have been considered solely as neuronal components, but some evidence suggests that glial cells in the hippocampus, and in particular astrocytes, also could be activated by NMDA applications. On the basis of their shape and electrophysiological properties (linear and rectified I/V curve), we describe two different populations of glial cells from GFAP-GFP transgenic mice that are activated differentially by NMDA. We found that linear glial cells were depolarized by NMDA that was not dependent on Ca2+ rise but partially involved a Ca2+ entry. Additionally, NMDA-induced depolarization of linear glial cells involved both a TTX-independent pathway likely through a direct activation, and a TTX-dependent pathway that required neuronal activity. The NMDA-induced depolarization in these cells was in part due to the activation of glutamate transporters and GABA B receptors. Furthermore, TTX-dependent NMDA-induced activation regulates the level of gap junction coupling between linear glial cells. In contrast, NMDA-induced depolarization in outward rectifying cells do not require a Ca2+ rise but are mediated directly by Ca2+ entry and are independent of glutamate transporters, GABA B and GABA A receptors. Our findings reveal that NMDA differentially activates hippocampal glial cells and the glial network through heterogeneous mechanisms in a cell-type specific manner.     

Multiple Ca2+ currents elicited by action potential waveforms in acutely isolated adult rat dorsal root ganglion neurons.

Ca2+ entry into different diameter cell bodies of dorsal root ganglion (DRG) neurons depolarized with action potential (AP) waveform commands was studied using the whole-cell patch-clamp technique and pharmacological probes. We have previously shown that Ca2+ current expression in DRG neuron cell bodies depends on cell diameter. In small diameter DRG neurons, L- and N-type Ca2+ currents usually accounted for most Ca2+ entry during APs as determined by blockade with nimodipine and omega-conotoxin GVIA (omega-CgTx). In medium- diameter DRG neurons, T-type Ca2+ currents accounted for 29% or 54% of Ca2+ entry in cells held at -60 mV or -80 mV, respectively, based on blockade by amiloride. T-type Ca2+ currents did not usually contribute to Ca2+ entry in large diameter DRG neurons. An amiloride/omega-CgTx/nimodipine-resistant Ca2+ current was prominent in medium diameter DRG neurons, while L- and N-type Ca2+ currents played a relatively small role in Ca2+ entry. In all DRG neuron sizes, AP-generated currents were large in amplitude, resulting in significant Ca2+ entry. APs with slower rates of repolarization increased Ca2+ entry. In DRG neurons that expressed T-type Ca2+ currents, the duration of Ca2+ current entry during APs was prolonged, and this prolongation was reduced by amiloride. Thus, antagonists selective for different Ca2+ channels produced different patterns of blockade of AP-generated Ca2+ entry in different diameter DRG cell bodies. Selective Ca2+ channel modulation by neurotransmitters might be expected to have similar effects.     

Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using arsenazo III

Spinal cord neurons is dissociated cell culture were loaded with the calcium indicator arsenazo III using the whole-cell patch-clamp recording technique. Under voltage-clamp, depolarizing voltage steps evoked transient increases in absorbance at 660 nm, with no change at 570 nm, the isosbestic wavelength for calcium-arsenazo III complexes. The optical response occurred with a threshold depolarization to -30 mV, peaked at +10 mV, and decreased with further depolarization, consistent with an elevation of cytoplasmic free calcium resulting from Ca2+ flux through voltage-dependent calcium channels. Inward current responses to the excitatory amino acids N-methyl-D-aspartic acid (NMDA) and L-glutamate were also accompanied by calcium transients; these were dose-dependent, varied with the driving force for inward current, and were blocked by extracellular Mg2+ in a voltage-dependent manner, suggesting Ca2+ flux through NMDA-receptor channels. Responses to kainate, quisqualate, and GABA were not accompanied by comparable calcium transients. [Ca2+]i transients evoked by depolarizing voltage steps were of maximal amplitude at the start of recording and declined with time, reflecting rundown of voltage-dependent calcium channels. In contrast, [Ca2+]i transients evoked by NMDA gradually increased in amplitude during periods of whole-cell recording lasting 1-2 hr. Procedures resulting in loading of the neuron with Ca2+ accelerated the increase in amplitude of [Ca2+]i transients evoked by NMDA, but slowed the decay of [Ca2+]i transients evoked by voltage steps. Our results provide evidence for 2 independent sources of transmembrane Ca2+ flux in vertebrate neurons, through voltage-gated calcium channels and through NMDA-receptor channels. The Ca2+ flux gated by NMDA-receptor-specific agonists may play a role in synaptic plasticity, in regulating excitability, and in the excitotoxic response to excitatory amino acids.     

Expression of ion channels and mutational effects in giant Drosophila neurons differentiated from cell division-arrested embryonic neuroblasts.

A culture system of "giant" Drosophila neurons derived from cytokinesis-arrested embryonic neuroblasts was developed to overcome the technical difficulties usually encountered in studying small Drosophila neurons. Cytochalasin B-treated neuroblasts differentiated into giant multinucleated cells that displayed neuronal morphology and neuron-specific markers (Wu et al., 1990). Here, we report that these giant neurons express different excitability patterns and membrane channels similar to those reported in excitable tissues of Drosophila. Individual neurons exhibited distinct all-or-none or graded voltage responses upon current injection. Both current- and voltage-clamp recordings could be performed on the same neuron because of the large cell size, thus making it possible to elucidate the functional role of the individual types of channels. By using pharmacological agents and ion substitution, the following currents were identified in these giant neurons: inward Na+ and Ca2+ currents and outward voltage-activated (the A-type and delayed rectifier) and Ca(2+)-activated K+ currents. In addition, we found a tetrodotoxin (TTX)-sensitive, Na(+)-dependent outward K+ current and a persistent component of an inward Na+ current, which have not been reported in Drosophila previously. This culture system can be used to analyze the mutational perturbations in ion channels and the resultant alterations in membrane excitability. Neurons from the mutant slowpoke (slo), which is known to lack a component of the Ca(2+)-activated K+ currents in muscles, exhibited prolonged action potentials associated with defects in the Ca(2+)-activated K+ current. This abnormality appeared to be more severe in the neurites than in the soma.     

Serotonin Induces a Cyclic AMP-Mediated Outwardly Rectifying Slow K+ Current in a Single Identified Snail Neuron

In the F2 neuron of the parietal ganglion of the snail Helix aspersa either bath or iontophoretic application of serotonin (5-HT) induces an outward current. This current has a long latency (10 - 60 s) and a slow time course, a 500 ms iontophoretic application of 5-HT evoking a response lasting 3 - 5 min. This slow outward current reverses at -80 mV, a value equal to EK. After doubling the extracellular K+ concentration the reversal potential of the 5-HT response is shifted by 19 mV, as predicted by the Nernst equation. The I-V curves reveal that the 5-HT-induced slow outward current is outwardly rectifying. This 5-HT response is blocked by intracellular Cs+ and tetraethylammonium (TEA+) and by extracellular TEA+ and Ba2+, but is not affected by the removal of extracellular Ca2+ or the intracellular injection of ethyleneglycol-bis-(beta-amino-ethylether)-N,N,N',N'-tetra-acetic acid (EGTA). These results indicate that the outwardly rectifying slow outward current induced by 5-HT in the F2 neuron is carried by K+ and is Ca2+-independent. In the single isolated F2 neuron, 5-HT induces a 2.5-fold stimulation of the adenylate cyclase activity. In addition, both the intracellular injection of 3',5'-adenosine monophosphate (cAMP) and the application of forskolin mimic the effect of 5-HT on the F2 neuron. The phosphodiesterase inhibitor isobutylmethylxanthine also induces a slow outward current and potentiates the 5-HT response. The intracellular injection of a synthetic 20-residue peptide inhibitor of the cAMP-dependent protein kinase blocks the slow outward K+ current induced by 5-HT. These results show that in the F2 neuron, 5-HT elicits a slow K+ current via the stimulation of adenylate cyclase, an increase in intracellular cAMP and the activation of the cAMP-dependent kinase which probably phosphorylates a population of outwardly rectifying K+ channels or some protein/s associated with these channels.     

Repolarization of the plasma membrane shapes NMDA-induced cytosolic [Ca2+ ] transients.

After inactivation of NMDA receptors, restoration of basal cytosolic [Ca2+] ([Ca2+]c) is delayed. This may be caused by Ca2+ influx via reverse Na/Ca exchange or voltage-gated Ca2+ channels, and/or by Ca2+ efflux from internal stores. Monitoring of [Na+]c, [Ca2+]c, and plasma membrane potential in cultured cerebellar granule cells showed that repolarization of the plasma membrane and inactivation of voltage-gated Ca channels plays the most critical role in restoration of low [Ca2+]c following NMDA receptor inactivation. During NMDA receptor activation, however, an Na-dependent mechanism enhanced NMDA-induced elevation in [Ca2+]c. This mechanism did not involve Na,K-ATPase activation by Na+, because it operated even when Na,K-ATPase was inhibited.     

Blocking of cloned and native delayed rectifier K channels from visceral smooth muscles by phencyclidine.

We investigated the effect of phencyclidine (PCP) on three native delayed rectifier K+ currents and three channels cloned from canine and human circular colonic myocytes using voltage-clamp techniques. Native delayed rectifier K+ current in canine circular colon is composed of at least three components: (i) a rapidly activating, 4-aminopyridine-sensitive component (termed IdK(f)); (ii) a slowly activating, tetraethylammonium (TEA)-sensitive component (IdK(s)); and (iii) a rapidly activating, TEA-sensitive component, which has a steady-state inactivation curve shifted towards more negative potentials (IdK(n)). PCP blocked all three components with EC50 values of 45, 27 and 59 micromol L-1, respectively. Blocking was neither use-dependent nor voltage-dependent. Delayed rectifier K+ channels cloned from canine (Kv1.2, Kv1.5) and from human (Kv2.2) colon were expressed in Xenopus oocytes. PCP blocked all three currents with similar potency. In contrast, PCP (up to 10-4 mol L-1) did not reduce the magnitude of Ca2+-dependent outward current of large conductance Ca2+-activated K+ channels (BK channels).     

Voltage-gated ionic currents in an identified modulatory cell type controlling molluscan feeding

An important modulatory cell type, found in all molluscan feeding networks, was investigated using two-electrode voltage- and current-clamp methods. In the cerebral giant cells of Lymnaea, a transient inward Na+ current was identified with activation at -58 +/- 2 mV. It was sensitive to tetrodotoxin only in high concentrations (approximately 50% block at 100 microm), a characteristic of Na+ channels in many molluscan neurons. A much smaller low-threshold persistent Na+ current (activation at < -90 mV) was also identified. Two purely voltage-sensitive outward K+ currents were also found: (i) a transient A-current type which was activated at -59 +/- 4 mV and blocked by 4-aminopyridine; (ii) a sustained tetraethylammonium-sensitive delayed rectifier current which was activated at -47 +/- 2 mV. There was also evidence that a third, Ca2+-activated, K+ channel made a contribution to the total outward current. No inwardly rectifying currents were found. Two Ca2+ currents were characterized: (i) a transient low-voltage (-65 +/- 2 mV) activated T-type current, which was blocked in NiCl2 (2 mm) and was completely inactivated at approximately -50 mV; (ii) A sustained high voltage (-40 +/- 1 mV) activated current, which was blocked in CdCl2 (100 microm) but not in omega-conotoxin GVIA (10 microm), omega-agatoxin IVA (500 nm) or nifedipine (10 microm). This current was enhanced in Ba2+ saline. Current-clamp experiments revealed how these different current types could define the membrane potential and firing properties of the cerebral giant cells, which are important in shaping the wide-acting modulatory influence of this neuron on the rest of the feeding network.     

Intracellular injection of Ca2+ chelator does not affect spike repolarization of cat spinal motoneurons

Both intra- and extracellular injections of tetraethylammonium (TEA) prolonged the spike repolarization of motoneurons in the spinal cord of cats under pentabarbitone anaesthesia, but did not depress the afterhyperpolarization (AHP). Intracellular injections of EGTA and the fast-acting Ca2+ chelator, BAPTA, greatly depressed the AHP, but the spike shape remained unchanged. Extracellular applications of Cd2+ had similar effects. These observations suggest that a Ca2+-dependent K+ outward current is not a major mechanism of spike repolarization in motoneurons.