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.     

Potassium channels in crustacean glial cells.

Unitary currents through single ion channels in the glial cells, which ensheath the abdominal stretch receptor neurons of the crayfish, were characterized with respect to their basic kinetic properties. In cell-attached and excised patches two types of Ca(++)-independent K+ channels were observed with slope conductances of 57 pS and 96 pS in symmetrical K+ solution. The 57 pS K+ channel was weakly voltage-dependent with a slope of the Po vs. membrane potential relationship of +95 mV for an e-fold change in Po. In addition to the main conductance level, the channel displayed conductance levels of 80 and 109 pS. In excised patches, channel activity of this "subconductance" K+ channel showed "rundown" that could be prevented with 2 mM ATP-Mg on the cytoplasmic side of the membrane. The 96 pS K+ channel was strongly voltage-dependent with a slope of +12 mV for an e-fold change in Po. Averaged single-channel currents elicited by voltage jumps proved the channel to be of the delayed rectifying type. Channel activity persisted in excised patches with minimal salt solution and in virtually Ca(++)-free saline. Because of its dependence on intracellular ATP-Mg, the subconductance K+ channel is discussed as a target of modulation by transmitters or peptides via phosphorylation of the channel.     

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.     

Ionic currents of cultured olfactory receptor neurons from antennae of male Manduca sexta.

Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the ionic currents of insect olfactory receptor neurons (ORNs) in vitro. The cells were isolated from the antennae of male Manduca sexta pupae at stages 3-5 of adult development and maintained in primary cell culture. After 2-3 weeks in vitro, the presumptive ORNs had resting potentials of -62 +/- 12 mV (n = 18) and expressed at least 1 type of Na+ channel and at least 3 types of K+ channels. Na+ currents, recorded in the whole-cell mode, were reversibly blocked by 0.1 microM tetrodotoxin. The predominant type of K+ channel observed was a voltage-activated K+ channel (gamma = 30 pS) with characteristics similar to those of the delayed rectifier. The activity of the 30-pS K+ channel could be inhibited by the application of nucleotides to the cytoplasmic face of inside-out patches of membrane. The nucleotides had relative potencies as follows: ATP greater than cGMP greater than cAMP, with an inhibition constant for ATP of Ki = 0.18 mM. Raising the intracellular Ca2+ concentration from 0.1 to 5 microM induced the opening of a Ca2(+)-activated K+ channel (gamma = 66 pS at 0 mV) that had a low voltage sensitivity. A third, transient type of K+ channel (gamma = 12-18 pS) could be activated by depolarizing voltage steps from very negative resting potentials. Properties of this channel were similar to those of the "A-channel." These results support the conclusion that M. sexta ORNs differentiate in vitro and provide the basis for studying primary mechanisms of olfactory transduction.     

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.     

BK channels in human glioma cells have enhanced calcium sensitivity

We have previously demonstrated the expression of large-conductance, calcium-activated potassium (BK) channels in human glioma cells. In the present study, we characterized the calcium sensitivity of glioma BK channels in excised membrane patches. Channels in inside-out patches were activated at -60 mV by 2.1 x 10(-6) M cytosolic Ca(2+), were highly K(+)-selective, and had a slope conductance of approximately iqual 210 pS. We characterized the Ca(2+) sensitivity of these channels in detail by isolating BK currents in outside-out patches with different free [Ca(2+)](i). The half-maximal voltage for channel activation, V(0.5), of glioma BK currents in outside-out patches was +138 mV with 0 Ca(2+)/10 EGTA. V(0.5) was shifted to +81 mV and -14 mV with free [Ca(2+)](i) of 1.5 x 10(-7) M and 2.1 x 10(-6) M, respectively. These results suggest that glioma BK channels have a higher Ca(2+) sensitivity than that described in many other human preparations. Data obtained from a cloned BK channel (hbr5) expressed in HEK cells support the conclusion that glioma BK channels have an unusually high sensitivity to calcium. In addition, the sensitivity of glioma BK channels to the BK inhibitor tetrandrine suggests the expression of BK channel auxiliary beta-subunits by glioma cells. Expression of the auxiliary beta-subunit of BK channels by glioma cells may relate to the high Ca(2+) sensitivity of glioma BK channels.     

Role of Ca(2+)-dependent K+ channels in erythrocyte lysate-induced contraction of rabbit cerebral artery.

K+ channel openers may be useful in the treatment of cerebral vasospasm following subarachnoid hemorrhage. However, the role of Ca(2+)-dependent K+ channel (KCa) openers in cerebral vasospasm remain unclear. This study was undertaken to examine the role of KCa in hemolysate-induced contraction of rabbit cerebral and peripheral arteries: 1. Iberiotoxin (IBX), a selective KCa channel blocker, produced more pronounced contraction in basilar than in those of carotid or femoral arteries, indicating KCa channels are important regulating factors in cerebral arteries; 2. NS1619, a selective KCa channel opener, abolished the contraction of basilar artery to erythrocyte lysate, a causative agent for cerebral vasospasm; 3. In rabbit basilar artery, NS1619 relaxed the contractions to IBX, erythrocyte lysate and KCl (20 and 60 mM), indicating that NS1619, besides opening KCa channels, possesses other vasodilating actions. We conclude that KCa channels are important factors in the regulation of cerebral vascular tension and KCa channel opener NS1619 may have dual relaxant actions in cerebral arteries.     

Single K(+)-channel currents under steady-state potential conditions in small hippocampal neurons.

Small cultured hippocampal neurons from rat embryos were studied with the patch-clamp technique. Single-channel currents from outside-out membrane patches were recorded under steady-state potential conditions. The most frequently found channel types were selective to K+ and showed conductances of about 30 and 80 pS in the range -20 to 0 mV. Two basic kinetic patterns were observed for the 80 pS channels. In one type, the fraction of time spent in open state increased with potential, and in the other type it decreased. For both types of 80 pS channel, the distribution of dwell times in the open state was well described by the sum of two exponentials while three exponentials sometimes were required for dwell times in the closed state. The time constants of the fitted exponentials could vary considerably during an experiment.     

Single-channel recordings of three K+-selective currents in cultured chick ciliary ganglion neurons

Multiple distinct K+-selective channels may contribute to action potential repolarization and afterpotential generation in chick ciliary neurons. The channel types are difficult to distinguish by traditional voltage-clamp methods, primarily because of coactivation during depolarization. I have used the extracellular patch-clamp technique to resolve single-channel K+ currents in cultured chick ciliary ganglion (CG) neurons. Three unit currents selective for K+ ions were observed. The channels varied with respect to unit conductance, sensitivity to Ca2+ ions and voltage, and steady-state gating parameters. The first channel, GK1, was characterized by a unit conductance of 14 pico-Siemens (pS) under physiological recording conditions, gating that was relatively independent of membrane potential and intracellular Ca2+ ions, and single-component open-time distributions with time constants of approximately 9 msec. The second channel, GK2, was characterized by a unit conductance of 64 pS under physiological recording conditions and gating that was affected by membrane potential but was not dependent on the activity of intracellular Ca2+ ions. Open-time distributions indicated 2 open states, with open-time constants of 0.09 (61%) and 0.35 (39%) msec, at +40 mV membrane potential. The third channel, GKCa2+, was identified in isolated patch recordings in which the concentration of internal Ca2+ was 10(-7) M or greater, which was an absolute prerequisite for channel opening. GKCa2+ was characterized by a unit conductance of 193 pS in symmetrical 0.15 M KCl solutions, an open-state probability that was a function not only of [Ca2+]i, but also of membrane potential, and single-component open-time distribution with a time constant of 1.11 msec at -10 mV patch potential. These results suggest the presence of at least 3 distinct K+ channel populations in the membrane of cultured chick CG neurons.     

Cationic channels in normal and dystrophic human myotubes

Human skeletal muscle cells obtained from normal and Duchenne muscular dystrophy patients were cocultured with explants of rat dorsal root ganglions. Single-channel recordings were performed with the cell-attached configuration of the patch-clamp technique and negative pressure was applied via the patch-pipette in order to mechanically stimulate the membrane patch. Inward elementary current activity was recorded under control or negative pressure conditions. Its occurrence and mean open probability were higher in Duchenne muscular dystrophy. Amplitude histograms reveal that these channels have a small unitary conductance of around 10 pS in 110 mM Ca2+ and could be inhibited in a dose-dependent manner by gadolinium. Results show that the membrane stress favoured calcium permeation through these channels. Taken together these data provide arguments for the involvement of such channels in calcium overload previously observed in cocultured dystrophic human (Duchenne muscular dystrophy) muscle cells.     

Calcium activates two types of potassium channels in rat hippocampal neurons in culture

Several calcium-dependent potassium currents can contribute to the electrophysiological properties of neurons. In hippocampal pyramidal cells, 2 afterhyperpolarizations (AHPs) are mediated by different calcium-activated potassium currents. First, a rapidly activated current contributes to action-potential repolarization and the fast AHP following individual action potentials. In addition, a slowly developing current underlies the slow AHP, which occurs after a burst of action potentials and contributes substantially to the spike-frequency accommodation observed in these cells during a prolonged depolarizing current pulse. In order to investigate the single Ca2(+)-dependent channels that might underlie these currents, we performed patch-clamp experiments on hippocampal neurons in primary culture. When excised inside-out patches were exposed to 1 microM Ca2+, 2 types of channel activity were observed. In symmetrical bathing solutions containing 140 mM K+, the channels had conductances of 19 pS and 220 pS, and both were permeable mainly to potassium ions. The properties of these 2 channels differed in a number of ways. At negative membrane potentials, the small-conductance channels were more sensitive to Ca2+ than the large channels. At positive potentials, the small-conductance channels displayed a flickery block by Mg2+ ions on the cytoplasmic face of the membrane. Low concentrations of tetraethylammonium (TEA) on the extracellular face of the membrane specifically caused an apparent reduction of the large-channel conductance. The properties of the large- and small-conductance channels are in accord with those of the fast and slow AHP, respectively.     

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+.     

Peptide cotransmitter potentiates calcium channel activity in crayfish skeletal muscle.

The activity of 2 types of Ca2+ channels (38 and 14 pS in 137 mM Ba2+) in the plasma membrane of the crayfish tonic flexor muscle is modulated by the peptide proctolin. This peptide serves as a cotransmitter in 3 of the 5 excitatory tonic flexor motoneurons and greatly enhances tension after depolarization by the conventional neurotransmitter. Proctolin alone has no effect on these channels, but renders them capable of sustained activity following depolarization. After depolarization induces activity, 5 x 10(-9) M proctolin increases the open probability of the larger channel up to 50-fold due to a marked decrease in the mean channel closed time. There is also at least a 4-fold increase in the percentage of patches with active channels for the large channel and a 2-fold increase for the small channel. Proctolin modulation appears to occur via an intracellular messenger, possibly cAMP. The peptide's effect on channel activity is dose dependent in a manner that parallels its effect on tension. These results indicate that the activation of these channels and the resulting influx of Ca2+ into the muscle fiber play a role in the potentiation of tension in this muscle.     

Characterisation of the L- and N-type calcium channels in differentiated SH-SY5Y neuroblastoma cells: calcium imaging and single channel recording.

We have used single cell imaging of [Ca2+]i and single channel cell-attached patch clamp recording to characterise the Ca2+ channels present on the plasma membrane of retinoic acid-differentiated human neuroblastoma (SH-SY5Y) cells. Exposure to raised K+ (45 or 60 mM) for 1 min resulted in a transient rise in [Ca2+]i which was abolished by cadmium (100 microM). The amplitude of the evoked rise varied from cell to cell. Both omega-Conus toxin (500 nM) and nifedipine (10 microM) reduced, but did not abolish, the rise in [Ca2+]i whereas Bay K 8644 (3 microM) potentiated it. In single channel records both L- and N-type Ca2+ channel openings were observed during membrane depolarisations from a holding potential of -90 mV. L-type channel openings (unitary conductance 22.5 pS) were prolonged by S(+)-PN 202-791 (500 nM) and could still be evoked from a depolarised holding potential (-40 mV). N-type channel openings (unitary conductance 12.5 pS) were unaffected by the dihydropyridine agonist but were inactivated at a holding potential of -40 mV. These results indicate that, in contrast to previous observations using whole cell recording, retinoic acid-differentiated SH-SY5Y cells express both L- and N-type Ca2+ channels.     

Potassium and caffeine contractures in limb muscles of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice

The strength of contractures, produced by 15 to 146 mM [K]0 (as L-glutamate), was measured in isolated small bundles of muscle fibers from the fast-twitch extensor digitorum longus and from the slow-twitch soleus of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice. The analysis of the relation between the maximal amplitude of the contracture vs the membrane potential and the time constant of relaxation of the K-contractures has shown that dystrophy induced an attenuation of the differences between fast- and slow-twitch muscles. The repriming of K-contractures was more affected by changes in [Ca]0 in normal soleus than in normal extensor digitorum longus and this difference was unaffected by dystrophy. For both types of muscles, the ability of caffeine to produce contractures was reduced in dystrophic muscle and this modification was not related to a change in the fiber typing.     

Activity-dependent neurotransmitter release kinetics: correlation with changes in morphological distributions of small and large vesicles in central nerve terminals

In central nerve terminals transmitter release is tightly regulated and thought to occur in a number of steps. These steps include vesicle mobilization and docking prior to neurotransmitter release. Intrasynaptic changes in vesicle distribution were determined by electron microscopical analysis and neurotransmitter release was monitored by biochemical measurements. We correlated K + -induced changes in distribution of small and large vesicles with the release of their transmitters. For small synaptic vesicles, amino acid release as well as recruitment to and docking at the active zone were activated within 1 s of depolarization. In contrast, the disappearance of large dense-cored vesicles and the release of the neuropeptide cholecystokinin were much slower, and no docking was observed. Studies with diverse Ca2 + channel blockers indicated that mobilization and neurotransmitter release from both vesicle types were regulated by multiple Ca2 + channels, although in different ways. Neurotransmitter release from small synaptic vesicles was predominantly regulated by P-type Ca2 + channels, whereas primarily Q-type Ca2 + channels regulated neurotransmitter release from large dense-cored vesicles. The different Ca2 + channnel types directly regulated mobilization of and neurotransmitter release from small synaptic vesicles whereas, by their cooperativity in raising the intracellular Ca2 + concentration above release threshold, they more indirectly regulated large dense-cored vesicle exocytosis.     

Improvement of dy/dy dystrophic diaphragm by 3,4-diaminopyridine

Concerns have been raised that inotropic agents may worsen function of dystrophic muscle due to structural fragility. Studies tested the hypothesis that force increments elicited by potassium (K(+)) channel blockade can be maintained during the course of repetitive stimulation. In vitro twitch force of dy/dy dystrophic mouse diaphragm was significantly lower than normal (796 versus 1271 g/cm(2)). 3,4-Diaminopyridine (DAP) increased twitch force of dystrophic diaphragm by 111 +/- 12% (P <.0001) and increased force at stimulation frequencies of 5-50 Hz by 41-77%. During fatigue-inducing stimulation, force augmentation by DAP was well maintained in dystrophic muscle throughout 25 Hz (P =.0047) and 50 Hz (P =.0059) stimulation. These findings indicate that the K(+) channel blocker DAP augments the force of dystrophic muscle to values close to that of normal muscle over a range of stimulation frequencies. Furthermore, these functional increments can be achieved without causing force to eventually deteriorate below that of untreated dystrophic muscle during fatiguing stimulation. It is possible that DAP may be useful for the clinical management of a variety of disorders causing muscle weakness.     

Voltage-gated potassium channels in larval CNS neurons of Drosophila

The availability of genetic, molecular, and biophysical techniques makes Drosophila an ideal system for the study of ion channel function. We have used the patch-clamp technique to characterize voltage-gated K+ channels in cultured larval Drosophila CNS neurons. Whole-cell currents from different cells vary in current kinetics and magnitude. Most of the cells contain a transient A-type 4-AP-sensitive current. In addition, many cells also have a more slowly inactivating TEA-sensitive component and/or a sustained component. No clear correlation between cell morphology and whole-cell current kinetics was observed. Single-channel analysis in cell-free patches revealed that 3 types of channels, named A2, KD, and K1 can account for the whole-cell currents. None of these channels requires elevated intracellular calcium concentration for activation. The A2 channels have a conductance of 6-8 pS and underlie the whole-cell A current. They turn on rapidly, inactivate in response to depolarizing voltage steps, and are completely inactivated by prepulses to -50 mV. The KD (delayed) channels have a conductance of 10-16 pS and can account, in part, for the more slowly inactivating component of whole-cell current. They have longer open times and activate and inactivate more slowly than the A2 channels. The K1 channels have a slope conductance, measured between 0 and +40 mV, of 20-40 pS. These channels do not inactivate during 500 msec voltage steps and thus can contribute to the sustained component of current. They exhibit complex gating behavior with increased probability of being open at higher voltages. Although the K1 channels are sufficient to account for the noninactivating component of whole-cell current, we have observed several other channel types that have a similar voltage dependence and average kinetics.     

Variation in the pattern of [Ca2+]i change induced by acetylcholine in cultured hippocampal neurons

Acetylcholine (ACh) caused various patterns of change in the intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal neurons. We studied the underlying mechanisms of the [Ca2+]i changes with simultaneous recording of [Ca2+]i and membrane potential/current. In most cases, [Ca2+]i rise was accompanied by a membrane depolarization. The [Ca2+]i change was significantly reduced when the membrane was voltage clamped, which implies that most of the [Ca2+]i rise results from the Ca2+ influx through the voltage-gated Ca2+ channel activated by the membrane depolarization. The membrane depolarizations were classified into two types, one associated with membrane conductance decrease and the other associated with membrane conductance increase. The former results from potassium conductance ((gK+) decrease, and the latter may result from the activation of a Na(+)-permeable channel. However, [Ca2+]i elevation was also observed in some neurons showing membrane hyperpolarization in response to ACh. This seems to show that ACh liberates Ca2+ from the intracellular Ca2+ store, resulting in the activation of a calcium-dependent K+ channel (KCa). The variations of ACh response in the hippocampal neurons seem to result from a variety of muscarinic acetylcholine receptors and various species of ion channels governed by those receptors.