Apamin,a selective blocker of SKCa channels,inhibits posthypoxic hyperexcitability but does not affect rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro

The aim of this study was to investigate the effects of apamin, a selective blocker of SK_Ca channels, on the repeated brief hypoxia-induced posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro. The method of field potentials measurement in CA1 region of the rat hippocampal slices was used. Application of apamin (50 nM) to the hippocampal slices during hypoxic episodes significantly abolished posthypoxic hyperexcitability induced by brief hypoxic episodes. However, in contrast to our previous results with iberiotoxin, a selective blocker of BK_Ca channels, apamin significantly enhanced the depressive effect of brief hypoxia on the PS amplitude during hypoxic episode and did not abolish the rapid hypoxic preconditioning in CA1 pyramidal neurons. Present results indicate that SK_Ca channels, along with previously implicated BK_Ca channels, play an important role in the development of posthypoxic hyperexcitability induced by brief hypoxic episodes in CA1 pyramidal neurons. However, SK_Ca channels, in contrast to the BK_Ca channels, are not involved in the rapid hypoxic preconditioning in CA1 hippocampal region in vitro.     

The Role of High-Conductivity Ca<Superscript>2+</Superscript>-Activated Potassium Channels in the Preconditioning Effects of Hypoxia and Posthypoxic Hyperexcitability of Neurons in Hippocampal Field CA1 in Vitro

The effects of the specific high-conductivity Ca²⁺-activated potassium channel blocker iberiotoxin on processes induced in rat hippocampal field CA1 slices by transient episodes of hypoxia were studied, i.e., 1) the suppressing effect of hypoxia on pyramidal neuron activity during hypoxic episodes, 2) the preconditioning action of hypoxia, and 3) posthypoxic neuron hyperexcitability. These experiments showed that iberiotoxin (10–20 nM) produced a tendency to decreases in the effectiveness of hypoxic episodes in depressing the amplitudes of population spikes recorded in hippocampal field CA1 during hypoxia. The high-conductivity Ca²⁺-activated potassium channel blocker significantly decreased the preconditioning effects of the first two episodes of hypoxia on the ability of the third episode of hypoxia to suppress neuron activity. Iberiotoxin also suppressed the posthypoxic hyperexcitability of pyramidal neurons. It is suggested that high-conductivity Ca²⁺-activated potassium channels play an important role in the mechanisms of development of types of neuroplasticity induced by transient episodes of hypoxia such as rapid hypoxic preconditioning and posthypoxic hyperexcitability.     

Impaired dopamine release and uptake in R6/1 Huntington's disease model mice

The aim of this study was to evaluate the intracellular cytosolic calcium concentration ([Ca(2+)](i)) changes induced by activation of ionotropic glutamate receptors in cultured hippocampal neurons after repeated brief episodes of hypoxia. To investigate what kinds of ionotropic glutamate receptors are involved we used specific agonists for AMPA- and NMDA-type glutamate receptors. Measurements of [Ca(2+)](i) in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. In the rat hippocampal slice method, field potential measurements in CA1 pyramidal neurons were used. The main result of our study is that brief hypoxic episodes progressively depress the [Ca(2+)](i) increases induced by agonists of AMPA and NMDA glutamate receptors in cultured hippocampal neurons. An effectiveness of this depression is increased from the first hypoxic episode to the third one. Hypoxic preconditioning effect is observed during 10-20 min after termination of hypoxic episode and depends on [Ca(2+)](i) response amplitudes to agonists before hypoxia. In contrast to AMPA receptor activation, NMDA receptor activation before hypoxia induce the spontaneous [Ca(2+)](i) increase about 3 min after each hypoxic episode. These spontaneous [Ca(2+)](i) increases may be an indicator of the development of posthypoxic hyperexcitability in hippocampal neurons. Our results suggest that brief hypoxia-induced depression of the glutamate receptor-mediated [Ca(2+)](i) responses contributes to the development of rapid hypoxic preconditioning in hippocampal CA1 neurons.     

ATP maintains ATP-inhibited K+ channels in an operational state

In patch-clamp records of K⁺ _ATP channels in an insulin-secreting cell line (RINm5F) inhibition evoked by exposing the internal surface of the membrane to ATP is followed not just by the recovery of K⁺ _ATP channel activity when the ATP is removed but by a marked activation of K⁺ _ATP channels. This phenomenon is not a direct consequence of channel closure as inhibition induced by quinidine and quinine is followed upon the removal of the drug only by the recovery of K⁺ _ATP channel activity and not by post-inhibitory activation. If ATP is applied to the exposed internal surface of a membrane patch when all of its K⁺ _ATP channel have run down subsequent removal of the ATP causes their activation. The magnitude and duration of the reactivation of K⁺ _ATP channels is shown to depend upon both the concentration of ATP and the length of time for which the membrane is exposed to ATP. We therefore have a paradoxical situation in that K⁺ channels which are inhibited by intracellular ATP require intracellular ATP to retain the ability to open.     

CGRP-induced activation of KATP channels in follicular Xenopus oocytes

The two-microelectrode voltage-clamp technique was used to monitor K⁺ channel activity in Xenopus oocyte follicular cells, which are electrically coupled to the oocyte itself by gap junctions. Endogenous vasodilators such as calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), prostaglandin E₂ (PGE₂) and adenosine activate glibenclamide-ATP-sensitive K⁺ (K_ATP) channels in Xenopus oocyte follicular cells. The mechanism of action of CGRP was studied in detail. CGRP effects undergo a rapid desensitization. CGRP acts via CGRP_I receptors. Its effects are antagonized by the amino-truncated CGRP analog hCGRP(8–37). The second messenger for CGRP activation of K_ATP channels is cAMP. Phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine enhances the CGRP response while adenyl cyclase inhibition by either 2,5-dideoxyadenosine or progesterone nearly completely depresses the CGRP response. Vasoconstrictors such as ACh and angiotensin II also have receptors in follicular cells. ACh strongly inhibits the CGRP activation of K⁺ channels as it inhibits the activation of K_ATP channels by P1060, but angiotensin II does not. It is concluded that as in vascular smooth muscle cells, CGRP and probably other hyperpolarizing vasodilators open K_ATP channels in follicular cells by protein kinase A activation.Thanks are due to C. Roulinat and F. Aguila for expert technical assistance. This work was supported by the Centre National de la Recherche Scientifique (CNRS).     

Neuroprotective effects of interleukin-10 and tumor necrosis factor-alpha against hypoxia-induced hyperexcitability in hippocampal slice neurons

In our previous experiments we have demonstrated that repeated exposures of rat hippocampal slices to brief episodes of hypoxia induce a sustained decrease in the threshold of stimulus-evoked epileptiform discharges in CA1 pyramidal neurons. The aim of this study was to investigate the comparative effects of interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) on the hyperexcitability of CA1 pyramidal neurons induced by brief episodes of hypoxia in the rat hippocampal slices. The method of field potentials measurement in CA1 region of hippocampal slices have been described in our previous work [O. Godukhin, A. Savin, S. Kalemenev, S. Levin, Neuronal hyperexcitability induced by repeated brief episodes of hypoxia in rat hippocampal slices: involvement of ionotropic glutamate receptors and L-type Ca2+ channels, Neuropharmacology 42 (2002) 459-466]. The principal results of our work are summarized as follow. Pro-inflammatory cytokine TNF-alpha (0.8, 4 and 20 ng/ml) and anti-inflammatory cytokine IL-10 (1 and 10 ng/ml) significantly reduced the hyperexcitability in CA1 pyramidal neurons induced by brief episodes of hypoxia in the rat hippocampal slices. The neuroprotective effects of IL-10 and TNF-alpha against the hypoxia-induced hyperexcitability were mediated by anti-hypoxic actions of these cytokines through, possibly, mechanism of preconditioning.     

Developmental changes in hyperexcitability of CA1 pyramidal neurons induced by repeated brief episodes of hypoxia in the rat hippocampal slices

We have previously demonstrated that repeated exposure of adult rat hippocampal slices to brief episodes of hypoxia induce a sustained decrease in the threshold of stimulus-evoked population spike discharges in CA1 pyramidal neurons [O. Godukhin, A. Savin, S. Kalemenev, S. Levin, Neuronal hyperexcitability induced by repeated brief episodes of hypoxia in rat hippocampal slices: involvement of ionotropic glutamate receptors and L-type Ca2+ channels, Neuropharmacology 42 (2002) 459-466, S.V. Kalemenev, A.V. Savin, S.G. Levin, O.V. Godukhin, Long-term potentiation and epileptiform activity induced by brief hypoxic episodes in CA1 area of the rat hippocampal slices. Russ. Physiol. J. 86 (2000) 1676-1681]. In the present study, using the above-mentioned in vitro model of epileptogenesis, we compared the developmental changes in hypoxia-induced hyperexcitability of CA1 neuronal network in the rat hippocampal slices prepared from three age rat groups: postnatal days (P) 13-14 (young), P60-70 (adult) and P600-650 (old). Furthermore, we were interested in learning about an age dependence of the hypoxia-induced changes in the efficacies of glutamatergic transmission and paired-pulse inhibition in CA3-CA1 synapses that may underlie ontogenetic differences in seizure susceptibility in hippocampal network. The principal results of this work are summarized as follow. In comparison with P60-70 hippocampal slices, CA1 pyramidal neurons in P13-14 and P600-650 slices showed intrinsically (without repeated brief hypoxa) an increased propensity to generate epileptiform stimulus-evoked population spike discharges. However, in contrast to adult and old animals, repeated brief episodes of hypoxia are incapable to induce a sustained decrease in the threshold of stimulus-evoked population spike discharges in CA1 pyramidal neurons of hippocampal slices prepared from of P13-14 rats, though they transform paired-pulse inhibition to paired-pulse facilitation and induce hypoxic LTP in CA3-CA1 synapses. The role of some other factors in the developmental changes in hyperexcitability of CA1 pyramidal neurons in response to repeated brief episodes of hypoxia is discussed.     

Biophysical,pharmacological and developmental properties of ATP-sensitive K+ channels in cultured myotomal muscle cells from Xenopus embryos

Unlike mammalian muscle cells in culture, cultured myotomal muscle cells of Xenopus embryos express ATP-sensitive K⁺ (K_ATP) channels. The K_ATP channels are blocked by internal ATP (half-maximal inhibition K _0.5 = 16 M) and to a lesser extent by internal ADP, are voltage independent, have an inward rectification at positive potentials and are inhibited by glibenclamide (K _0.5 = 2 M). Surprisingly, these K_ATP channels are not sensitive to K⁺ channel openers such as cromakalim. Opening of these K_ATP channels does not occur under normal physiological conditions. It is elicited by metabolic exhaustion of the muscle cell and it precedes the development of an irreversible rigor state. Neither intracellular acidosis nor an increase of intracellular Ca²⁺ are involved in K_ATP channel opening. Different types of K⁺ channels are successively expressed after plating of myotomal muscle cells: (1) sustained delayed-rectifier K⁺ channels; (2) K_ATP channels; (3) inward-rectifier K⁺ channels; (4) transient delayed-rectifier K⁺ channels. The current density associated with K_ATP channels far exceeds that of voltage-dependent K⁺ channels. Innervation controls the expression of these K_ATP channels. Co-culture of muscle cells with neurons from the neural tube decreases the number of active K_ATP channels per patch. Similarly, in situ innervated submaxillaris muscle of tadpoles at stage 50–55 has a very low density of K_ATP channels.     

Diadenosine polyphosphate-induced inhibition of cardiac KATP channels: Operative state-dependent regulation by a nucleoside diphosphate

It has been proposed that the regulatory action of mononucleotides, such as ATP and UDP, on cardiac ATP-sensitive K⁺ (K_ATP) channels is determined by the state of the channel. Recently, dinucleotides, such as diadenosine tetraphosphate (Ap₄A) and diadenosine pentaphosphate (Ap₅A), have been recognized as novel intracellular ligands of cardiac K_ATP channels. However, it is not known whether the state of K_ATP channels also determines the response of the channel to dinucleotides. Therefore, we examined the action of diadenosine polyphosphates on K_ATP channel activity during different operative channel states, using the inside-out patch clamp technique applied to patches excised from guinea-pig ventricular myocytes. Spontaneous openings of K_ATP channels (or operative condition 1) were inhibited by Ap₄A and Ap₅A. Addition of UDP, which on its own did not affect spontaneous openings of K_ATP channels, prevented Ap₄A and Ap₅A to inhibit spontaneous K_ATP channel activity. In contrast, following run-down of spontaneous channel activity, UDP induced K_ATP channel openings (or operative condition 2), but could no longer antagonize the inhibitory effect of Ap₄A and Ap₅A. We conclude that the inhibitory action of diadenosine polyphosphates on K_ATP channels can be blocked by UDP only when K_ATP channels are in operative condition 1, but not in operative condition 2. Thus, the operative condition of KATp channels determines the UDP-mediated regulation of the diadenosine polyphosphate-dependent inhibitory channel gating. This finding further suggests that the operative state of the cardiac K_ATP channel protein is a critical determinant of the regulatory action of an intracellular ligand.     

Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH₃ cells. In whole-cell configuration, di-8-ANEPPS (10 μM) reversibly increased the amplitude of Ca²⁺-activated K⁺ current. In inside-out configuration, di-8-ANEPPS (10 μM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with an EC₅₀ value of 7.5 μM. This compound caused a left shift in the activation curve of BK_Ca channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK_Ca channels. However, neither voltage-gated K⁺ nor ether-à-go-go-related gene (erg)-mediated K⁺ currents in GH₃ cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 μM) decreased the firing of action potentials in GH₃ cells. In pancreatic βTC-6 cells, di-8-APPNES (10 μM) also increased BK_Ca-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK_Ca channels could be one of potential mechanisms through which it may affect cell excitability.     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

The 2P-domain K+ channels: role in apoptosis and tumorigenesis

Two-pore (2P)-domain K⁺ channels have been shown recently to play a critical role in both cell apoptosis and tumorigenesis. The activity of two-pore, (TWIK)-related acid-sensitive-3 (TASK-3) K⁺ channels, is responsible for K⁺-dependent apoptosis of cultured cerebellar granule neurons. Neuron death can be prevented by conditions that specifically reduce K⁺ efflux through the TASK-3 channels. Moreover, genetic transfer of TASK subunits into hippocampal neurons that lack TASK-3, induces apoptosis. These results indicate a direct link between TASK K⁺ channel activity and the physiological process of programmed cell death. The TASK-3 K⁺ channel gene has also been shown to be amplified genomically and over-expressed in a significant number of breast tumours. TASK-3 has a potent oncogenic potential that appears to be related directly to its K⁺ channel function. In the present review, we will examine the pro-apoptotic and oncogenic properties of TASK-3. We will discuss: (1) the molecular and functional properties of the novel family of mammalian 2P domain K⁺ channels; (2) the role of TASK-3 in cerebellar granule neuron apoptosis and (3) the role of TASK-3 in breast tumorigenesis.     

Identification of an ATP-sensitive K+ channel in rat cultured cortical neurons

To determine whether membranes of mammalian central neurons contain an ATP-sensitive K⁺ (K_ATP) channel similar to that present in pancreatic cells, the patch-clamp technique was applied to cultured neurons prepared from the neonatal rat cerebral cortex and hippocampus. In whole-cell experiments with hippocampal neurons, extracellular application of 0.5 mM diazoxide (a K_ATP channel activator) elicited a hyperpolarization concomitant with an increase in membrane conductance, whereas application of 0.5 mM tolbutamide (a K_ATP channel blocker) induced a depolarization with a decrease in conductance. Similar results were obtained with cortical neurons. In outside-out patch experiments with cortical neurons, a K⁺ channel sensitive to these drugs was found. The channel was completely blocked by 0.5 mM tolbutamide and activated by 0.5 mM diazoxide. The single-channel conductance was 65 pS under symmetrical 145 mM K⁺ conditions and 24 pS in a physiological K⁺ gradient. In inside-out patch experiments, this channel was demonstrated to be inhibited by an application of 0.2–1 mM ATP to the cytoplasmic surface of the patch membrane. These results indicate that the membranes of rat cortical neurons contain a K_ATP channel that is quite similar to that found in pancreatic cells. It is also suggested that the same or a similar K⁺ channel may exist in membranes of hippocampal neurons.     

Physiological role of inward rectifier K+ channels in vascular smooth muscle cells

K⁺ channels play indispensable roles in establishing the membrane potential and in regulating the contractile tone of arterial smooth muscle cells. There are four types of K⁺ channels in arterial smooth muscle: voltage-dependent K⁺ (K_V), Ca²⁺-dependent K⁺ (BK_Ca), ATP-dependent K⁺ (K_ATP), and inward rectifier K⁺ (Kir2) channels. Comparatively few physiological studies have focused on Kir2 channels because they are present only in certain small-diameter cerebral and submucosal arterioles and in coronary arterial smooth muscle. Here, we review the characteristics and regulation of Kir2 channels in vascular arterial smooth muscle. Current knowledge of the predominant Kir2 channel subtype is Kir2.1, not Kir2.2 and 2.3. Electrophysiological measurements to determine the current–voltage relationship in arterial smooth muscle revealed inward rectification with a single-channel conductance of 21 pS. Kir2 channels were found to influence the resting tone of cerebral and coronary arteries based on the fact that barium (Ba²⁺) induces the constriction of these arteries at resting tone. Kir2 channels are also highly responsive to vasoconstrictors and vasodilators. For example, the vasoconstrictors endothelin-1 and angiotensin II inhibit Kir2 channel function by activating protein kinase C (PKC), and the vasodilator adenosine stimulates Kir2 channel function by increasing the level of cAMP, which subsequently activates protein kinase A (PKA). Certain pathological conditions such as left ventricular hypertrophy are associated with a decrease in Kir2 channel expression. Although our understanding of the physiological role and regulation of Kir2 channels is incomplete, it is believed that Kir2 channels contribute to the control of vascular tone in small-diameter vessels via various intracellular signalling pathways that regulate cell membrane potential.     

Polarized targeting of ion channels in neurons

Since the time of Cajal it has been understood that axons and dendrites perform distinct electrophysiological functions that require unique sets of proteins [Cajal SR Histology of the nervous system, Oxford University Press, New York, (1995)]. To establish and maintain functional polarity, neurons localize many proteins specifically to either the axonal or the somatodendritic compartment. In particular, ion channels, which are the major regulators of electrical activity in neurons, are often distributed in a polarized fashion. Recently, the ability to introduce tagged proteins into neurons in culture has allowed the molecular mechanisms underlying axon- and dendrite-specific targeting of ion channels to be explored. These investigations have identified peptide signals from voltage-gated Na⁺ and K⁺ channels that direct trafficking to either axonal or dendritic compartments. In this article we will discuss the molecular mechanisms underlying polarized targeting of voltage-gated ion channels from the Kv4, Kv1, and Na_v1 families.     

K channels in O2 sensing and postnatal development of carotid body glomus cell response to hypoxia

The sensitivity of carotid body chemoreceptors to hypoxia is low just after birth and increases over the first few weeks of the postnatal period. At present, it is believed that the hypoxia-induced excitation of carotid body glomus cells begins with the inhibition of the outward K⁺ current via one or more O₂ sensors. Although the nature of the O₂ sensors and their signals that inhibit the K⁺ current are not well defined, studies suggest that the postnatal maturation of the glomus cell response to hypoxia is largely due to the increased sensitivity of K⁺ channels to hypoxia. As K_V, BK and TASK channels that are O₂-sensitive contribute to the K⁺ current, it is important to identify the O₂ sensor and the signaling molecule for each of these K⁺ channels. Various O₂ sensors (mitochondrial hemeprotein, hemeoxygenase-2, NADPH oxidase) and associated signals have been proposed to mediate the inhibition of K⁺ channels by hypoxia. Studies suggest that a mitochondrial hemeprotein is likely to serve as an O₂ sensor for K⁺ channels, particularly for TASK, and that multiple signals may be involved. Thus, changes in the sensitivity of the mitochondrial O₂ sensor to hypoxia, the sensitivity of K⁺ channels to signals generated by mitochondria, and/or the expression levels of K⁺ channels are likely to account for the postnatal maturation of O₂ sensing by glomus cells.     

A potassium current activated by lemakalim and metabolic inhibition in rabbit mesenteric artery

K⁺ channels which are inhibited by intracellular ATP (ATP_i) (K_ATP channels) are thought to be the physiological target site of the K⁺ channel opening drugs (2) and to underlie a variety of physiological phenomena including hypoxia induced vasodilation (3). However, electrophysiological evidence for ATP_i-regulated K⁺ currents in smooth muscle is scarce. We, therefore, investigated the effects of one K⁺ channel opener, lemakalim, and metabolic inhibition on the membrane conductance of freshly dissociated rabbit mesenteric artery smooth muscle cells, using the perforated-patch whole cell recording technique (6). The cells were metabolically inhibited with 1 mM iodoacetic acid and 50 M dinitrophenol. Both lemakalim (0.1–3 M) and metabolic inhibition activated a time-independent and glyburide sensitive K⁺ current at physiological membrane potentials. The similarities between the lemakalim and metabolic inhibition activated currents suggest that a single class of channels underlies both currents. These results are the first whole-cell current recordings to demonstrate the activation of a smooth muscle membrane conductance by metabolic inhibition, lending support to the view that hypoxia induced vasodilation arises from the activation of K_ATP channels.     

K+-dependent stability and ion conduction of Shab K+ channels: a comparison with Shaker channels

K⁺ depletion exerts dramatically variable effects on different potassium channels. Here we report that Shab channels are rather stable in the absence of either internal or external K⁺ alone; however, its stability is greater with K⁺ outside the cell. In contrast, with 0 K⁺ (non-added) solutions on both sides of the membrane, the conductance (G_K) is rapidly and irreversibly lost. G_K is lost with the channels closed and regardless of the composition of the 0 K⁺ solutions. In comparison, it is known that the Shaker B G_K collapses only if the channels are gated in 0 K⁺, Na⁺-containing solutions. In order to compare the behavior of Shab to that of Shaker, we show that after extensively gating the channels in 0 K⁺ N-methyl-D-glucamine solutions, most Shaker channels remain stable, and in a conformation where G_K collapses as soon as there is Na⁺ in the solutions. Regarding ion conduction, in contrast to Kv2.1 and Shaker A463C that have a sizable G_Na in 0 K⁺, Shab, which shares a 463-cysteine and an identical signature sequence with these channels, does not appreciably conduct Na⁺, although it presents a significant Cs⁺ conductance. The observations suggest that there are at least two sites where K⁺ binds and thus maintains Shab G_K stable, one internal and the other(s) most likely located outside the selectivity filter.     

Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na+ channels in rat hippocampal neurones

Actions of the new antiepileptic drug lamotrigine (LTG, Lamictal) were characterised using recombinant rat brain type IIA Na⁺ channels expressed in Chinese hamster ovary (CHO) cells and native Na⁺ channels in rat hippocampal pyramidal neurones, using whole-cell recording and intracellular recording techniques. In CHO cells, LTG caused a tonic inhibition of Na⁺ currents in a concentration-dependent and voltage-dependent manner. The half-maximal inhibitory concentration (IC₅₀) of approximately 500 M was obtained at a holding potential (V _h) of –90 mV compared with an IC₅₀ of 100 M at a V _h of –60 mV. LTG (50 M) caused a 10–mV negative shift in the slow, steady-state inactivation curve and delayed considerably the recovery from inactivation, but had no significant effects on the voltage dependence of activation or fast inactivation, suggesting that LTG acts mainly on the slow inactivated state. The affinity for the inactivated channels was estimated at 12 M. The tonic inhibition was augmented by a use-dependent action in which a further inhibition by the drug developed during rapid repetitive stimulation using a train of 20-ms duration pulses (11 Hz). These results were consistent with the drug action being on firing properties of pyramidal neurones. Only in those epileptiform bursts which caused cumulative inactivation of Na⁺ spikes did LTG produce a potent inhibition. Our data suggest that the inactivated channel is a primary target for LTG action at therapeutic concentrations.     

Roles of K+ channels in regulating tumour cell proliferation and apoptosis

K⁺ channels are a most diverse class of ion channels in the cytoplasmic membrane and are distributed widely in a variety of cells including cancer cells. Cell proliferation and apoptosis (programmed cell death or cell suicide) are two counterparts that share the responsibility for maintaining normal tissue homeostasis. Evidence has been accumulating from fundamental studies indicating that tumour cells possess various types of K⁺ channels, and that these K⁺ channels play important roles in regulating tumour cell proliferation and apoptosis, i.e. facilitating unlimited growth and promoting apoptotic death of tumour cells. The potential implications of K⁺ channels as a pharmacological target for cancer therapy and a biomarker for diagnosis of carcinogenesis are attracting increasing interest. This review aims to provide a comprehensive overview of current status of research on K⁺ channels/currents in tumour cells. Focus is placed on the roles of K⁺ channels/currents in regulating tumour cell proliferation and apoptosis. The possible mechanisms by which K⁺ channels affect tumour cell growth and death are discussed. Speculations are also made on the potential implications of regulation of tumour cell proliferation and apoptosis by K⁺ channels.