Recombinant Kv1.3 potassium channels stabilize tonic firing of cultured rat hippocampal neurons

We transfected cultured hippocampal neurons with the cDNA of the voltage-gated K+ channel Kv1.3 to investigate the mechanisms by which a specific ion channel influences excitability. In transfected neurons under voltage clamp we observed an additional outward current that was blocked selectively by margatoxin. Under current-clamp conditions, Kv1.3-expressing neurons fired tonically over a wide range of stimulation intensity. In non-transfected neurons, or in Kv1.3-expressing cells blocked with margatoxin, only a few action potentials were elicited before a stationary depolarized state was reached. We attribute the specific effect of Kv1.3 to its particularly slow deactivation near the resting potential. A computational model showed that a continuous outwards current arises in Kv1.3-expressing neurons during the interspike intervals. It expands the dynamic range so that these neurons still fire tonically at stimulus current intensities at which non-transfected cells have already been driven into a stationary depolarized state.     

Excision-activated chloride channels in cultured postnatal rat hippocampal neurons

Chloride permeable intermediate conductance single channel events activated on patch excision were found in outside-out patches from cultured postnatal hippocampal neurons. A majority of the channels had a conductance of 83 ± 2.1 pS when recorded in a symmetrical TEAC1 solution. Two other populations of channels with conductance values of 62 ± 2.1 pS and 145 ± 1.9 pS were also observed. The reversal potentials for these intermediate conductance Cl⁻ channels coincided with that of the GABA activated channels. The channels characteristically appeared 5–15 min after patch excision, suggesting that these channels may be blocked by some diffusible factors under physiological conditions. Based on the measurements of channel burst durations while the channel was partially blocked, and the channel open times after complete relief from the block, the mechanism of blockade does not appear to be a simple open channel blockade. The high prevalence and its potential regulation by cytosolic factors suggest an important physiological role for these Cl⁻ channels coupling neuronal excitability with cellular metabolism.     

Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard "run-down" of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the "high-voltage-activated" Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150-450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)     

Dendritic potassium channels in hippocampal pyramidal neurons

Potassium channels located in the dendrites of hippocampal CA1 pyramidal neurons control the shape and amplitude of back-propagating action potentials, the amplitude of excitatory postsynaptic potentials and dendritic excitability. Non-uniform gradients in the distribution of potassium channels in the dendrites make the dendritic electrical properties markedly different from those found in the soma. For example, the influence of a fast, calcium-dependent potassium current on action potential repolarization is progressively reduced in the first 150 μm of the apical dendrites, so that action potentials recorded farther than 200 μm from the soma have no fast after-hyperpolarization and are wider than those in the soma. The peak amplitude of back-propagating action potentials is also progressively reduced in the dendrites because of the increasing density of a transient potassium channel with distance from the soma. The activation of this channel can be reduced by the activity of a number of protein kinases as well as by prior depolarization. The depolarization from excitatory postsynaptic potentials (EPSPs) can inactivate these A-type K⁺ channels and thus lead to an increase in the amplitude of dendritic action potentials, provided the EPSP and the action potentials occur within the appropriate time window. This time window could be in the order of 15 ms and may play a role in long-term potentiation induced by pairing EPSPs and back-propagating action potentials.     

Dynamic localization and clustering of dendritic Kv2.1 voltage-dependent potassium channels in developing hippocampal neurons.

Dendritic excitability is modulated by the highly variable spatial and temporal expression pattern of voltage-dependent potassium channels. Somatodendritic Kv2.1 channels contribute a major component of delayed rectifier potassium current in cultured hippocampal neurons, where Kv2.1 is localized to large clusters on the soma and proximal dendrites. Here we found that dramatic differences exist in the clustering of endogenous Kv2.1 in cultured rat hippocampal GABAergic interneurons and glutamatergic pyramidal neurons. Studies on neurons developing in culture revealed that while a similar sequence of Kv2.1 localization and clustering occurred in both cell types, the process was temporally delayed in pyramidal cells. Localization and clustering of recombinant green fluorescent protein-tagged Kv2.1 occurred by the same sequence of events, and imaging of GFP-Kv2.1 clustering in living neurons revealed dynamic fusion events that underlie cluster formation. Overexpression of GFP-Kv2.1 accelerated the clustering program in pyramidal neurons such that the observed differences in Kv2.1 clustering in pyramidal neurons and interneurons were eliminated. Confocal imaging showed a preferential association of Kv2.1 with the basal membrane in cultured neurons, and electrophysiological recordings from neurons cultured on transistors revealed that Kv2.1 contributed the bulk of a previously described adherens junction delayed rectifier potassium conductance. Finally, Kv2.1 clusters were found spatially associated with ryanodine receptor intracellular Ca(2+) ([Ca(2+)](i)) release channels.These findings reveal a stepwise assembly of Kv2.1 potassium channels into membrane clusters during development, and an association of these clusters with Ca(2+) signaling apparatus. Together these data suggest that the restricted localization of Kv2.1 may play an important role in the previously observed contribution of this potassium channel in regulating dendritic [Ca(2+)](i) transients.     

Voltage-dependent potassium channels are involved in glutamate-induced apoptosis of rat hippocampal neurons

The role of voltage-dependent potassium channel currents in glutamate-treated rat hippocampal neurons was investigated. Cell viability was evaluated by MTT reduction assay and morphological changes. Apoptosis was detected by Hoechst33342 staining with fluorescent microscopy and propidium iodide staining with flow cytometry. Membrane potassium channel currents were recorded with whole-cell patch clamp recordings. Results showed that after shortly exposed to glutamate, about 25 and 50% cells died in 3 h and 24 h, respectively. Meanwhile, the enhancement of IK was observed within 6 h after the glutamate insult. TEA selectively blocked IK and significantly reduced cell apoptosis. IA did not change in the insult though 4-AP, the blocker of this current, showed a protective effect against the injury. These data were in consistent with the hypothesis that K+ efflux contributed to glutamate-triggered apoptosis and IK channels might have a therapeutic effect on the treatment of cerebral ischemia.     

Muscarine-sensitive voltage-dependent potassium current in cultured murine spinal cord neurons

Muscarine produced membrane depolarization and decreased membrane conductance of mouse spinal cord neurons in dissociated cell culture. When the neurons were voltage clamped, muscarine evoked inward currents which increased with membrane depolarization and decreased with membrane hyperpolarization. However, the muscarine-induced inward currents did not invert at large negative potentials, suggesting that muscarine decreased a voltage-dependent potassium current (m-current) [2]. Using the voltage-jump current-relaxation technique, m-current was demonstrated in spinal cord neurons and shown to be a muscarine-sensitive potassium current.     

Modulation of large conductance calcium-activated potassium channels from rat hippocampal neurons by glutathione

We have investigated the modulation of neuronal large conductance Ca2+-activated K+ channels by glutathione. Single channel recordings were made from cultured neonatal rat hippocampal neurons by using excised inside-out patch clamp method. Glutathione, a physiological sulfhydryl specific reducing reagent, increased channel activities in concentration dependent manner with half activation concentration of 710 microM. Conversely, oxidized form of glutathione inhibited channel activities with half inhibition concentration of 520 microM. Our results provide direct evidence that when neuronal large conductance Ca2+-activated K+ channels are exposed to reducing or oxidizing environments, channel activities are increased or decreased in opposite directions due to the redox modification. This may constitute an important regulatory mechanism of neuronal Ca2+-activated K+ channel activities.     

Calcium influx through N-methyl-D-aspartate channels activates a potassium current in postnatal rat hippocampal neurons

Calcium-activated potassium conductances play important roles in modulating neuronal excitability. Indeed, the effects of some neurotransmitters such as acetylcholine and norepinephrine are, in part, due to actions on these conductances. We have found that the N-methyl-D-aspartate (NMDA) class of excitatory amino acid receptors also is coupled to a calcium activated potassium current. In voltage-clamped postnatal rat hippocampal neurons, NMDA responses consist of an initial inward cationic current followed by a slowly developing outward current carried by potassium ions. The slow outward current always follows the inward current, is associated with an increase in membrane conductance and is dependent on the influx of calcium ions. Similar responses are produced by other agonists active at NMDA receptors, including aspartate, glutamate and ibotenate, but are not activated by kainate, quisqualate or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA). Inhibition of the NMDA gated inward current by a competitive antagonist, 2-amino-5-phosphonovalerate (APV), eliminates the outward current. From these results we conclude that calcium influx through NMDA channels activates a potassium current. The extended time course of this outward current suggests that NMDA receptors may modulate neuronal excitability long after the opening of the NMDA channel.     

Phencyclidine at low concentrations selectively blocks the sustained but not the transient voltage-dependent potassium current in cultured hippocampal neurons

We studied the effects of phencyclidine (PCP) on voltage-dependent K+ currents in cultured embryonic rat hippocampal neurons. Whole cell voltage-clamp recordings were made in the presence of tetrodotoxin to block Na+ current. Depolarizing voltage steps activated two outward current components: (i) a rapidly activating and inactivating ('transient') component, IA, and (ii) a slowly activating, minimally inactivating ('sustained'), component, IK. At low concentrations (less than 50-100 microM), PCP produced a selective, reversible blockade of IK with minimal effect on IA; however, at higher concentrations both currents were suppressed. The IC50's for blockade of IK and IA were 36 and 310 microM, respectively.     

cAMP-mediated long-term modulation of voltage-dependent K+ channels in cultured colliculi neurons

 Previously, we have described prolonged cAMP-induced inhibition of a K⁺ current in cultured colliculi neurons. The aim of the present study was to characterize the channel responsible for this cAMP-dependent effect. We detected the presence of a non-inactivating voltage-dependent 16-pS K⁺ channel that displayed long-lasting inhibition upon a brief application of cAMP and greater sensitivity to tetraethylammonium than to 4-aminopyridine. In addition to this channel, colliculi neurons express two other voltage-sensitive, non-inactivating K⁺ channels (8 and 49 pS) whose activity is facilitated by a brief application of cAMP, the effect of which is also long-lasting. These results suggest the presence of common sustained cAMP-dependent processes responsible for both up- and down-regulation of these channels in the neurons studied. They indicate that the 16-pS, but not the 8-pS or the 49-pS channels, participates in the cAMP-inhibited macroscopic K⁺ current. Received: 21 April 1998 / Received after revision: 10 July 1998 / Accepted: 5 August 1998     

Pharmacological characterization of voltage-dependent calcium currents in rat hippocampal neurons.

The kinetics and pharmacology of voltage-dependent calcium (Ca) currents in primary cultures of hippocampal neurons were studied using the whole cell clamp technique. The low voltage-activated (LVA) Ca current was activated at -50 mV and completely inactivated within 100 ms. This current was insensitive to omega-conotoxin (omega-CgTx) and to the calcium agonist Bay K 8644. The high-voltage-activated (HVA) Ca current was activated at -20 mV and inactivated incompletely during pulses of 200 ms duration. The snail toxin omega-CgTx revealed two pharmacological components of the HVA Ca current, one irreversibly blocked and the other insensitive to the toxin. Bay K 8644 had a clear agonistic action mainly on the omega-CgTx insensitive component of the HVA Ca current.     

Mechanosensitive potassium channels in rat colon sensory neurons

Single-channel recording techniques were used to characterize mechanosensitive channels in identified (1.1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine methanesulfonate labeled) colon sensory neurons dissociated from adult S1 dorsal root ganglia. Channels were found in 30% (7/23) of patches in a cell-attached configuration and in 43% (48/111) of excised inside-out patches. Channels were highly selective for K(+), had a slope conductance of 54 pS in symmetrical solutions, and were blocked by tetraethylammonium, amiloride, and benzamil. Channels were also seen under Ca(2+)-free conditions. Gadolinium (Gd(3+)), a known blocker of mechanosensitive ion channels, did not block channel activity. Tetrodotoxin and 4-aminopyridine were also ineffective. The cytoskeletal disrupters colchicine and cytochalasin D reduced the percentage of patches containing mechanosensitive channels. These results indicate that rat colon sensory neurons contain K(+)-selective mechanosensitive channels that may modulate the membrane excitability induced by colonic distension.     

Differential oxidative modulation of voltage-dependent K+ currents in rat hippocampal neurons

Oxidative stress is enhanced by [Ca2+]i-dependent stimulation of phospholipases and mitochondria and has been implicated in immune defense, ischemia, and excitotoxicity. Using whole cell recording from hippocampal neurons, we show that arachidonic acid (AA) and hydrogen peroxide (H2O2) both reduce the transient K+ current I(A) by -54 and -68%, respectively, and shift steady-state inactivation by -10 and -15 mV, respectively. While AA was effective at an extracellular concentration of 1 microM and an intracellular concentration of 1 pM, extracellular H2O2 was equally effective only at a concentration >800 microM (0.0027%). In contrast to AA, H2O2 decreased the slope of activation and increased the slope of inactivation of I(A) and reduced the sustained delayed rectifier current I(K(V)) by 22% and shifted its activation by -9 mV. Intracellular application of the antioxidant glutathione (GSH, 2-5 mM) blocked all effects of AA and the reduction of I(A) by H2O2. In contrast, intracellular GSH enhanced reduction of I(K(V)) by H2O2. Decrease of the slope of activation and increase of the slope of inactivation of I(A) by hydrogen peroxide was blocked and reversed to a decrease, respectively, by intracellular application of GSH. Intracellular GSH did not prevent H2O2 to shift inactivation and activation of I(A) and activation of I(K(V)) to more negative potentials. We conclude, that AA and H2O2 modulate voltage-activated K currents differentially by oxidation of GSH accessible intracellular and GSH inaccessible extracellular K+-channel domains, thereby presumably affecting neuronal information processing and oxidative damage.     

GABAB agonists modulate a transient potassium current in cultured mammalian hippocampal neurons

Depolarization of voltage-clamped cultured rat hippocampal neurons from holding potentials more negative than -60 mV produced a transient outward current with the characteristics of an A-current: it was 50% inactivated at a holding potential of -85 mV and blocked by 4-aminopyridine (1 mM). In the presence of GABA or baclofen (50-200 microM), with or without bicuculline, inactivation of this current was shifted to more positive potentials so that there was little inactivation at -70 mV. Activation of the A-current was also shifted to more positive potentials by these agonists, but the voltage dependence of activation of the sodium current was unaffected. If A-currents with similar properties can influence the time course of action potentials in presynaptic terminals. GABAB agonists could make action potentials briefer by potentiating the A-current and hence depress transmitter release.     

Desensitization of GABA-induced currents in cultured rat hippocampal neurons.

The basic characteristics of desensitization of the GABAA receptor were investigated in cultured rat hippocampal neurons (three days to four weeks in vitro) using whole cell patch clamp techniques. GABA at 10-500 microM was perfused on to neurons for 30 or 60 s, with 60 s intervals of wash with control bath solution between perfusions. Desensitization, evaluated by peak-to-plateau ratio and time constants of current decay (tau), was dose-dependent and culture age-dependent. Desensitization was observed as early as three days in culture, the earliest time tested. At all ages, higher concentrations of GABA induced both larger and faster desensitization. Desensitization was markedly voltage-dependent and decreased with depolarization; peak-to-plateau ratio went from 6.3 to 1.4 and tau went from 4.6 to 26.8 s when holding potentials were changed from -80 mV to +30 mV. Low concentrations of GABA (1-2 microM) perfused for 2-60 s, which did not induce any current, had no effect on the maximal response nor desensitization produced by a subsequent application of 100 microM GABA. This finding suggests that GABA receptors were not desensitized without first being activated.     

Molecular correlates of the M-current in cultured rat hippocampal neurons

M-type K⁺ currents ( I _K(M)) play a key role in regulating neuronal excitability. In sympathetic neurons, M-channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K⁺ channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of I _K(M) in hippocampal pyramidal neurons cultured from 5- to 7-day-old rats. RT-PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single-cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M-channel blocker, fully inhibited I _K(M) with an IC₅₀ of 3.6 ± 1.5 μM. In 70 % of these cells, TEA fully suppressed I _K(M) with an IC₅₀ of 0.7 ± 0.1 m m . In the remaining cells, TEA maximally reduced I _K(M) by only 59.7 ± 5.2 % with an IC₅₀ of 1.4 ± 0.3 m m ; residual I _K(M) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to I _K(M) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.     

Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV, and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+]i) with a Hill plot giving a half-saturating [Ca2+] (K0.5) of 1.35 microM and slope of approximately equals 3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 microM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+]i <100 nM and the increased channel activity evoked by ionomycin was consistent with a rise in [Ca2+]i to > or =0.3 microM. TEA (0.2-1 mM) increased the action potential duration approximately equals 1.5-fold and reduced the amplitude and duration of the afterhyperpolarization (AHP) by 26%. Charybdotoxin (100 nM) did not significantly alter the action potential duration or AHP amplitude but reduced the AHP duration by approximately equals 40%. Taken together, these data indicate that BK channel activation contributes to the action potential and AHP duration in rat intracardiac neurons.