Phosphorylation modulates the activity of the ATP-sensitive K channel in the ventromedial hypothalamic nucleus

Regulation of the ATP-sensitive K⁺ (K-ATP) channel was examined in cell-attached and inside-out membrane patches of freshly isolated neurons from the ventromedial hypothalamic nucleus (VMN) of 7–14 day old male Sprague–Dawley rats. When inside-out patches were exposed to symmetrical K⁺, the reversal potential was −2.85±1.65 mV, the single channel conductance 46 pS, and the total conductance varied as a multiple of this value. Glucose (10 mM) reversibly inhibited channel activity in cell-attached preparations by 81%. In the presence of 0.1 mM ADP, 10, 5, and 1 mM ATP reversibly inhibited VMN K-ATP channels in inside-out patches by 88, 83, and 60%, respectively. This inhibition was not dependent on phosphorylation since 5 mM AMPPNP, the non-hydrolyzable analog of ATP, reversibly inhibited channel activity by 67%. Relatively high concentrations of glibenclamide (100 μM) also reversibly inhibited VMN K-ATP channel activity in cell attached and inside-out patches by 67 and 79%, respectively. Finally, the non-specific kinase inhibitor H7 (200 μM) decreased channel activity by 53% while the non-specific phosphatase inhibitor microcystin (250 nM) increased channel activity by 218%. These data suggest that while the inhibitory effect of ATP is not phosphorylation dependent, phosphorylation state is an important regulator of the VMN K-ATP channel.     

Single-channel Activity in Cultured Cortical Neurons of the Rat in the Presence of a Toxic Dose of Glutamate

Rat cortical neurons grown in cell culture were exposed to 500 μM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca²⁺-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K⁺ channel requiring intracellular calcium ([Ca²⁺]_i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 ± 6 pS;‘BK channel'). Another Ca²⁺-dependent channel permeable for Cl^- (conductance for outward currents in cell-attached patches 72±17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K⁺ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca²⁺]_i. Both phases of increasing channel activity required the presence of extracellular Ca²⁺ suggesting that [Ca²⁺]_i was mainly increased by Ca²⁺ influx. The N-methyl-d -aspartate (NMDA) antagonists dizocilpine (MK-801, 10 μM) and dl -2-amino-5-phosphonovaleric acid (AP5; 100 μM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca²⁺ through NMDA receptor channels causes a strong activation of Ca²⁺-dependent K⁺ channels, which is likely to result in pronounced loss of intracellular K⁺. NMDA receptor channels seem to remain active for a long time (>10 min) after the end of glutamate application.     

Properties of Single Calcium-activated Potassium Channels of Large Conductance in Rat Hippocampal Neurons in Culture

Patch-clamp recordings were made on rat hippocampal neurons maintained in culture. In cell-attached and excised inside-out and outside-out patches a large single-channel current was observed. This channel had a conductance of 220 and 100 pS in 140 mM [K⁺]_i/140 mM [K⁺]_o and 140 mM [K⁺]_i/3 mM [K⁺]_o respectively. From the reversal potential the channel was highly selective for K⁺, the P_K+/P_na+ ratio being 50/1. Channel activity was voltage-dependent, the open probability at 100 mM [Ca²⁺]_i increasing by e-fold for a 22 mV depolarization. It was also dependent on [Ca²⁺]_i at both resting and depolarized membrane potentials. Channel open states were best described by the sum of two exponentials with time constants that increased as the membrane potential became more positive. Channel activity was sensitive to both external (500 μM) and internal (5 mM) tetraethylammonium chloride. These data are consistent with the properties of maxi-K⁺ channels described in other preparations, and further suggest a role for maxi-channel activity in regulating neuronal excitability at the resting membrane potential. Channel activity was not altered by 8-chlorophenyl thio cAMP, concanavalin A, pH reduction or neuraminidase. In two of five patches lemakalim (BRL 38227) increased channel activity. Internal ruthenium red (10 μM) blocked the channel by shortening the duration of both open states. This change in channel gating was distinct from the ‘mode switching’ seen in two patches, where a channel switched spontaneously from normal activity typified by two open states to a mode where only short openings were represented.     

Ion channels in cultured adult human Schwann cells.

Single channel currents have been recorded from cultured adult human Schwann cells. In both cell-attached and -excised (inside-out) patches, openings from a high-conductance (360 pS) channel were observed; measurements of the zero-current potential indicated that the channel was predominantly selective for chloride. Depolarizing and hyperpolarizing voltage steps activated the anion channel, which subsequently reverted to a closed state even in the presence of the maintained step. A second channel, with a conductance near 20 pS and with a current amplitude that increased with patch hyperpolarization, passed inward K+ currents in both cell-attached and inside-out patches. The mean open times for this channel were near 20 ms at the cell resting potential and decreased with patch hyperpolarization. The presence of these anion and cation selective channels in the human Schwann cell membrane would be consistent with a role for the cells in the regulation of extracellular K+.     

Single potassium channels in neuropile glial cells of the leech central nervous system

We performed patch-clamp experiments to identify distinct K⁺ channels underlying the high K⁺ conductance and K⁺ uptake mechanism of the neuropile glial cell membrane on the single-channel level. In the soma membrane four different types of K⁺ channels were characterized, which were found to be distributed in clusters. Since no other types of K⁺ channels were observed, these appear to be the complete repertoire of K⁺ channels expressed in the soma region of this cell type. The outward rectifying 42 pS K⁺ channel could markedly contribute to the high K⁺ conductance and the maintenance of the membrane potential, since it shows the highest open probability of all channels. The channel gating occurred in bursts and patch excision decreased the open probability. The outward rectifying 74 pS K⁺ channel was rarely active in the cell-attached configuration; however, patch excision enhanced its open probability considerably. This type of channel may be involved in neuron-glial crosstalk, since it is activated by both depolarizations and increases in the intracellular Ca²⁺ concentration, which are known to be induced by neurotransmitter release following the activation of neurons. The 40 pS and 83 pS K⁺ channels showed inward rectifying properties, suggesting their involvement in the regulation of the extracellular K⁺ content. The 40 pS K⁺ channel could only be observed in the inside-out configuration. The 83 pS channel was activated following patch excision. At membrane potentials more negative than −60 mV, flickering events indicated voltage-dependent gating.     

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca²⁺-activated potassium (BK_Ca) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK_Ca channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K⁺ in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P_o) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P_o was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca²⁺]_i required to half activate BK_Ca channels was only 1 μM in post-ischemic whereas 2 μM in control neurons, indicating an increase in [Ca²⁺]_i sensitivity after ischemia; and (4) BK_Ca channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK_Ca channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.     

Single ion channel currents in neuropile glial cells of the leech central nervous system

The patch-clamp technique was used to investigate the activity of single ion channels in neuropile glial (NG) cells in the central nervous system (CNS) of the medicinal leech, Hirudo medicinalis. We found evidence for two distinct Cl^? channels that could be distinguished by their basic electrical properties and their responses to different inhibitors on single ion channels currents. In the Inside-out configuration in symmetrical Cl solutions, these channels showed current-voltage relationships with slight outward rectification, mean conductances of 70 and 80 pS, and reversal potentials near 0 mV. Significant permeability to Na⁺, K⁺, or SO₄^2? could not be detected. The open-state probability of the 70 pS Cl^? channel increased with membrane depolarization, whereas the open-state probability of the 80 pS Cl^? channel was voltage-independent. The application of the stilbene derivative DIDS (100 μM) to the cytoplasmic side of the glial cell membrane blocked both Cl^? channels. The activity of the 70 pS channel was blocked irreversibly by DIDS, whereas the activity of the 80 pS channel reappeared after wash-out of DIDS. Both channels were blocked reversibly by 1 mM Zn²⁺. K⁺ channels could only be observed occasionally in the soma membrane of the NG cells. We have characterized a 60 pS K⁺ channel with a high selectivity for K⁺ over Na⁺. The low density of K⁺ channels in the soma membrane may indicate a non-uniform distribution of this channel type in NG cells. © 1993 Wiley-Liss, Inc.     

INVOLVEMENT OF Na+-K+-2Cl− COTRANSPORTERS IN HYPERTONICITY-INDUCED RISE IN INTRACELLULAR CALCIUM CONCENTRATION

A hypertonic saline containing propylene glycol facilitates calcium (Ca²⁺) influx through voltage-dependent Ca²⁺ channels. The present study performed experiments to elucidate the mechanism by which Na⁺-K⁺-2Cl^? cotransporters participate in the rise in the intracellular calcium concentration ([Ca²⁺]i) under the hypertonic condition. Both furosemide and ethacryonic acid significantly decreased the [Ca²⁺]i raised by hypertonicity. Similarly, Na⁺-, K⁺-, or Cl^?-free saline also reduced it. Both norepinephrine and dopamine significantly enhanced the rise in [Ca²⁺]i. In conclusion, the findings obtained indicate that the Na⁺-K⁺-2Cl^? cotransporters evoke cell depolarization and that this depolarization raises the [Ca²⁺]i by activating voltage-dependent Ca²⁺ channels.     

Two types of chloride channel in the apical membrane of rat choroid plexus epithelial cells

The patch clamp technique has been used to study ion channel activity in the apical (ventricular) membrane of epithelial cells from the rat choroid plexus. Two different classes of Cl(-)-selective channel were identified. A low conductance (26 pS) channel which was the predominant feature in cell-attached and inside-out patches. The occurrence of this channel appeared to increase in tissue bathed in forskolin. It was activated in inside-out patches by increasing the Ca2+ concentration at the intracellular face of the membrane and by depolarising potentials. The second class of channel was observed infrequently (2% of patches) and appeared to be similar to 'maxi'-Cl- channels which have been described in many other cell types. It had a conductance of 320 pS, opened to sub-conductance levels and displayed a marked voltage dependence in inside-out patches. The possible contribution of these channels to Cl- transport during the production of cerebrospinal fluid (CSF) is discussed.     

Properties of Ca2+-activated K+ channels in erythrocytes from patients with myotonic muscular dystrophy

Using the single-channel patch-clamp technique, Ca²⁺-activated K⁺ channels of erythrocytes from patients with myotonic muscular dystrophy (MyD) were studied. Elementary single-channel properties—conductance, rectification, kinetics, voltage- and calcium-dependence—measured in inside-out patches of MyD erythrocytes, did not differ significantly from those of control cells. The activity of the channels, studied in patches attached to red cells from MyD patients, exhibited mean patch currents which were significantly higher than the controls. The increased mean patch current was due to a higher opening frequency, associated with a reduced mean channel closed time. These results indicate that Ca²⁺-activated K⁺ channels of erythrocytes from patients either detect a higher intracellular calcium concentration and/or express an augmented calcium-sensitivity. Since these channels are targets for phosphorylation, our findings make it possible to identify defective kinase mechanisms, in minimally disturbed cells of the patient, at a molecular level of resolution. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1465–1472, 1998     

A Ca2+ - and pH-Dependent K+ Channel of rat C6 glioma cells and its possible role in acidosis-induced cell swelling

The aim of the present study was to explore whether a change in membrane K⁺ conductance contributes to acidosis-induced swelling of cultured rat C6 glioma cells. Electrophysiological studies were perfomed using whole-cell and single-channel recordings in combination with cell volume measurements in cell suspension by flow cytometry. Whole-cell recordings revealed a voltage-dependent K⁺ conductance. The predominant K⁺ channel in single-channel recordings with symmetrical high K⁺ concentrations was inwardly rectifying and had conductances of 35 and 15 pS, respectively. A raised internal free Ca²⁺ concentration and membrane depolarization increased the open probability of this channel. Internal acidosis (pH 6.4?5.4), on the other hand, reduced open probability and single-channel conductance. Both whole-cell and single-channel K⁺ currents were blocked by quinidine (0.1?1 mM), which was therefor used to analyze the functional consequences of an inhibition of this conductance for volume. Thereby, quinidine (1 mM) produced a small (5%) and transient cell swelling of C6 glioma cells. In contrast, acidosis (pH 5.6) caused a much larger (about 20%) and maintained swelling. Since quinidine produced only a minor swelling of C6 cells, it is unlikely that inhibition of the K⁺ conductance caused acidosis-induced cell swelling. Other mechanisms, such as activation of ion transporters, must therefore be responsible. © 1993 Wiley-Liss, Inc.     

Membrane depolarization in LA-N-1 cells

We investigated the influence of ion compositions on the membrane potential in LA-N-1 human neuroblastoma cells using bisoxonol as a potential-sensitive fluorescent dye. The ability of K⁺, ouabain, veratridine, and maitotoxin to induce membrane depolarization was evaluated. Increasing concentrations of K⁺ ions from 10 to 50 mM caused a dose-dependent increase of bisoxonol fluorescence, which was completely independent on Na⁺ and Ca²⁺. Ouabain (5 mM), an inhibitor of the Na⁺, K⁺-ATPase, failed to induce membrane depolarization. Veratridine (40 and 100 μM), a Na⁺ channel activator, only in the presence of 10 μg of Leiurus scorpion venom reduced the membrane potential. Maitotoxin (MTX) from 3 to 10 ng/mL depolarized LA-N-1 cells in a dose-dependent manner, and produced a rapid and sustained increase of intracellular free calcium monitored by means of fluorescent probe fura-2. The MTX-induced depolarization and the increase in cytosolic free calcium concentration were dependent on extracellular Ca²⁺ ions. On the other hand, Na⁺ ions also seem to be, although only partially, implicated in the MTX effects, since both the blockade of tetrodotoxin (TTX)-sensitive voltage-operated Na⁺ channels and the removal of Na⁺ ions were able to reduce the depolarization. In conclusion, our data indicate that the depolarizing action of MTX on LA-N-1 cells is Ca²⁺- and Na⁺-dependent, although the latter only partially, and that this effect is dependent on Ca²⁺ influx into the cells likely through a voltage-insensitive calcium-entry system.     

Single-channel K+ currents recorded from the somatic and dendritic regions of cerebellar Purkinje neurons in culture

Voltage-sensitive K+ channels were studied in rat cerebellar Purkinje neurons in culture using the single-channel recording technique. Recordings in the cell-attached and outside-out configuration revealed multiple voltage-sensitive K+ channel types in patches from both the somatic and the dendritic regions. K+ channel types were present in all patches studied. The same channel types were observed in somatic and dendritic recordings. Channel types were identified by reversal potential, single-channel conductance, voltage sensitivity, and patterns of activity. In cell-attached patches recorded under physiological conditions, 3 channel types were identified. Mean single-channel conductances were 92, 57, and 12 pS. All 3 channel types were activated by membrane depolarization. Similar channel types were identified in inside-out and outside-out patches recorded under physiological conditions. Two additional channel types were identified in the outside-out patches, with mean single-channel conductances of 41 and 26 pS. In cell-attached recordings under symmetrical K+ conditions, 6 channel types were identified. Mean single-channel conductances were 222, 134, 39, 25, 14, and 15 pS. Channel types with mean conductances of 222, 134, and 39 pS required membrane depolarization for activation. A comparison of channel properties indicated that these channel types correlated with the 3 channel types observed in cell-attached patches under physiological conditions. The 3 smaller-conductance channel types (25, 14, and 15 pS) were active at potentials around rest or at hyperpolarized membrane potentials. Two K+ channel types (39 and 25 pS) were commonly associated with the late phase of extracellularly recorded spontaneous spike events, suggesting a functional role in the repolarizing phase of somatic and dendritic action potentials. These results demonstrate that voltage-sensitive K+ channels are a prominent component of both the somatic and the dendritic membrane of the cerebellar Purkinje neuron and support the view that multiple voltage-sensitive K+ channel types contribute to the membrane functions of both cellular regions in this CNS neuronal type.     

Sarcolemmal K+ channel activity in developing rat skeletal muscle membranes

A method has been adapted to produce membrane vesicles suitable for routine membrane patch clamping from neonate rat skeletal muscle. Single K+ channel activity was recorded from cell-free inside-out patches. Most Ca2(+)-activated voltage sensitive channels had large conductances of up to 300 pS, as determined from their current/voltage relationship, and an open probability (Po) approaching unity at positive membrane potentials. A lower conductance K+ channel, probably responsible for inward rectification, had a lower conductance of about 100 pS. Outward rectifying K+ channels were also observed with the lowest conductance, about 40 pS. 0.1 mM ATP when applied to the inner membrane surface reduced or blocked activity, drastically reducing Po without altering single channel conductance. Such an effect has been reported in other preparations but was different in the neonate preparation in that it blocked channels with conductances as high as 300 pS. The simple preparation described, which we have also used successfully on mature rat and mouse skeletal muscle, has potential in the analysis of channel activities in various conditions and pathologies without the need for tissue culture to produce suitable membrane preparations.     

Calcium-activated potassium channels in rat visceral sensory afferents

The purpose of the present study was to describe, at the single-channel level, the activity of a calcium-sensitive potassium channel in rat visceral-sensory neurons which has been suggested to be involved in sensory neuron excitability. Single-channel recordings in the inside-out configuration identified a 220 pS conductance calcium-activated potassium channel (KCa). From a −20 mV holding potential, increasing [Ca²⁺]_i from 0.01 μM to 1.0 μM increased the open probability of this channel 92% (from 0.12 to 0.23). However, from a +20 mV holding potential, increasing [Ca²⁺]_i from 0.01 to 1.0 μM increased the open probability by 326% (from 0.15 to 0.64). In addition, this large conductance KCa channel was blocked by TEA (1.0 μM) and charybdotoxin (40 μM) when applied to the external surface. These results are the first to characterize a large conductance KCa channel in the sensory afferent neurons of the rat nodose ganglia and should further expand the understanding to the ionic currents involved in the regulation of sensory afferent neuronal activity.     

A hypothalamic digoxin mediated model for conscious and subliminal perception

Summary. The isoprenoid pathway and its metabolites – digoxin, dolichol and ubiquinone were assessed in schizophrenia. There was an upregulation of the isoprenoid pathway as evidenced by elevated HMG CoA reductase activity. Digoxin, an endogenous Na⁺-K⁺ ATPase inhibitor secreted by the hypothalamus was found to be elevated and RBC membrane Na⁺-K⁺ ATPase activity was found to be reduced in schizophrenia. Membrane Na⁺-K⁺ ATPase inhibition can result in increased intracellular Ca²⁺ and reduced magnesium levels. Hypothalamic digoxin can modulate conscious and subliminal perception and its dysfunction may lead on to schizophrenia. Digoxin can also preferentially upregulate tryptophan transport over tyrosine resulting in increased levels of depolarising tryptophan catabolites – serotonin and quinolinic acid (NMDA agonist), and decreased levels of hyperpolarising tyrosine catabolites – dopamine and noradrenaline contributing to membrane Na⁺-K⁺ ATPase inhibition. NMDA excitotoxicity could result from hypomagnesemia induced by membrane Na⁺-K⁺ ATPase inhibition and quinolinic acid, an NMDA agonist acting on the NMDA receptor. Hypomagnesemia and increased dolichol level can affect glycoconjugate metabolism and membranogenesis leading on to disordered synaptic connectivity in the limbic allocortex and defective presentation of viral antigens and neuronal antigens contributing to autoimmunity and viral persistance important in the pathogenesis. Membrane Na⁺-K⁺ ATPase inhibition can produce immune activation, a component of autoimmunity. Mitochondrial dysfunction consequent to altered calcium/magnesium ratios and reduced ubiquinone levels can result in increased free radical generation and reduced free radical scavenging & defective apoptosis leading on to abnormal synaptogenesis. Schizophrenia can thus be considered as a syndrome of hypothalamic digoxin hypersecretion consequent to an upregulated isoprenoid pathway. Received August 14, 2000; accepted February 13, 2001     

Reconstitution of a voltage and calcium dependent potassium channel from rat cerebellum

Membrane vesicles from rat cerebellum were reconstituted into lipid bilayers. The activity of two different potassium channels was recorded: (1) a small conducting voltage dependent potassium channel insensitive to [Ca²⁺]_i, (2) a calcium and voltage dependent potassium channel (K_Ca). K_Ca channels had a conductance of (302±15) pS (n=5) and were activated by [Ca²⁺]_i and membrane depolarizations. They were blocked by tetraethylamonium (TEA) and charybdotoxin (CTX) but insensitive to noxiustoxin (NTX). Finally, we showed the blocking effect of Androctonus australis Hector (AaH) scorpion venom on K_Ca channels from rat cerebellum.     

Ionic channel currents in cultured neurons from human cortex

Ionic channels in human cortical neurons have not been studied extensively. HCN-1 and HCN-1A cells, which recently were established as continuous cultures from human cortical tissue, have been shown by histochemical and immunochemical methods to exhibit a neuronal phenotype, but expression of functional ionic channels was not demonstrated. For the present study, HCN-1 and HCN-1A cells were cultured in Dulbecco's modified Eagle's medium with 15% fetal calf serum, in some cases supplemented with 10 ng/ml nerve growth factor, 10 μM forskolin, and 1 mM dibutyryl cyclic adenosine monophosphate to promote differentiation. Cells or membrane patches were voltage clamped using conventional patch clamp techniques. In HCN-1A cells, we identified a tetrodotoxin-sensitive Na⁺ current, two types of Ca²⁺ channel current, including L-type current and a second type that in some respects resembled N-type current, and four types of K⁺ current, including a delayed outward rectifier that showed voltage-dependent inactivation, two types of noninactivating Ca²⁺-activated K⁺ channels with slope conductances of 146 and 23 pS (K⁺ _iK⁺ _o 145 mM/5 mM), and less frequently, a noninactivating, intermediate conductance channel that was not sensitive to internal Ca²⁺. When HCN-1A cells were examined after 3 days of exposure to differentiating agents, pronounced morphological changes were evident but no differences in ionic currents were apparent. HCN-1 cells also exhibited K⁺ and Ca²⁺ channel currents, but Na⁺ currents were not detected in these cells. Our data provide additional evidence indicating a functional neuronal phenotype for HCN-1A cells, and represent the most comprehensive survey to date of the variety of ionic channels expressed by human cortical neurons. © 1993 Wiley-Liss, Inc.     

Characterization of large conductance Ca2+-activated K+ channels in cerebellar Purkinje neurons

We investigated the role of large conductance, calcium-activated potassium channels (BK channels) in regulation of the excitability of cerebellar Purkinje neurons. Block of BK channels by iberiotoxin reduced the afterhyperpolarization of spontaneous action potentials in Purkinje neurons in acutely prepared cerebellar slices. To establish the conditions required for activation of BK channels in Purkinje neurons, the dependence of BK channel open probability on calcium concentration and membrane voltage were investigated in excised patches from soma of acutely prepared Purkinje cells. Single channel currents were studied under conditions designed to select for potassium currents and in which voltage-activated currents were largely inactivated. Micromolar calcium concentrations activated channels with a mean single channel conductance of 266 pS. BK channels were activated by both calcium and membrane depolarization, and showed no sign of inactivation. At a given calcium concentration, depolarization over a 60-mV range increased the mean open probability (P(O)) from < 0.1 to > 0.8. Increasing the calcium concentration shifted the voltage required for half maximal activation to more hyperpolarized potentials. The apparent affinity of the channels for calcium increased with depolarization. At -60 mV the apparent affinity was approximately 35 micro m decreasing to approximately 3 micro M at +40 mV. These results suggest that BK channels are unlikely to be activated at resting membrane potentials and calcium concentrations. We tested the hypothesis that Purkinje cell BK channels may be activated by calcium entry during individual action potentials. Significant BK channel activation could be detected when brief action potential-like depolarizations were applied to patches under conditions in which the sole source of calcium was flux across the plasma membrane via the endogenous voltage-gated calcium channels. It is proposed that BK channels regulate the excitability of Purkinje cells by contributing to afterhyperpolarizations and perhaps by shaping individual action potentials.     

Potassium channels from normal and denervated mouse skeletal muscle fibers

The properties of singles K⁺ channels in normal and denervated muscles were compared using the “patch-clamp” technique. Single channels were recorded from vesicles obtained by stretching bundles of normal and denervated extensor digitorium longus (EDL) muscles. The most frequently observed channel in normal muscles was a high conductance (266 pS) Ca⁺⁺ activated K⁺ channel. Although channel density, as estimated by patch recording, showed a significant decrease in denervated muscles, no differences were found in conductance and gating properties. Another voltage-dependent K⁺ channel (81 pS) was only recorded from normal muscles, but never from denervated ones. In addition, a 35 pS conductance was recorded from both normal and denervated fibers. This channel displayed neither voltage dependence nor sensitivity to tetraethylammonium (TEA). In contrast, another TEA-insensitive (16 pS) channel was recorded only from denervated muscles. We conclude that denervation induces significant changes in the distribution and expression of K⁺ channels in mammalian skeletal muscles. © 1993 John Wiley & Sons, Inc.