Depolarization-activated K+ currents of the bushy neurones of the rat cochlear nucleus in a thin brain slice preparation

Depolarization-activated outward currents of bushy neurones of 6-14-day-old Wistar rats have been investigated in a brain slice preparation. Under current-clamp, the cells produced a single action potential at the beginning of suprathreshold depolarizing current steps. On voltage-clamp depolarizations, the cells produced a mixed outward K+ current that included a component with rapid activation and rapid inactivation, little TEA+ sensitivity, a half-inactivation voltage of -77 +/- 2 mV (T = 25 degrees C; n = 7; Mean +/- S.E.M.) and single-exponential recovery from inactivation (taurecovery= 12 +/- 1 ms at -100 mV; n=3). This transient component was identified as an A-type K+ current. Bushy cells developed a high-threshold TEA-sensitive K+ current that exhibited less prominent inactivation. These characteristics suggested that this current was associated with the activation of delayed rectifier K+ channels. Bushy neurones also possessed a low-threshold outward K+ current that showed partial inactivation and high 4-aminopyridine sensitivity. Part of this current component was blocked by 200 nmol/l dendrotoxin-I. Application of 100 micromol/l 4-aminopyridine changed the firing behaviour of the bushy neurones from the primary-like pattern to a much less rapidly adapting one, suggesting that the low-threshold current might have important roles in maintaining the physiological function of the cells.     

Ion-dependency of the GABA-potentiating effects of benzodiazepine tranquilizers and harmane

Experiments on an isolated spinal cord of 8-15-day-old rats have shown that one of the possible mechanisms of the GABA-potentiating action of the benzodiazepine tranquilizer, chlorodiazepoxide, may be a decrease in the intraneuronal concentration of Ca2+. This is evidenced by the enhancement of the GABA-potentiating action of chlorodiazepoxide under Ca2+ deficiency in the medium and in the presence of the blockers of the voltage-dependent Ca2+ ionic channels--Mn2+ and Co2+, and by the reduction of the effect in question under Ca2+ excess in the medium and in the presence of the K+ channels blockers--tetraethylammonium and 4-aminopyridine. The GABA-potentiating action of harmane is likely to be related to the blockade of the voltage-dependent K+ channels and elevation of the intracellular concentration of Ca2+.     

The effects of 4-aminopyridine and tetraethylammonium on the kinetics of transmitter release at the mammalian neuromuscular synapse

The effect of 4-aminopyridine and tetraethylammonium on the time course of neurotransmitter release was examined at the neuromuscular junction using a computer-aided method which directly measured the time of occurrence of individual quanta. It is apparent that the action of 4-aminopyridine, at concentrations of 0.1 to 1 mM, when examined in isolation from other experimental manipulations, is to cause a greatly enhanced probability of release at times subsequent to the time over which the initial phase is essentially unchanged, i.e., there is no evidence of an increased latency of release caused by 4-aminopyridine. Similar results were obtained with tetraethylammonium, although the prolongation of release was much less, even at a concentration of 1 mM. The results are consistent with the view that the predominant action of 4-aminopyridine is to block the potassium conductance responsible for repolarization of the action potential and hence cause a prolonged Ca2+ current. The action of tetraethylammonium is consistent with the block of a different K+ conductance, with consequent enhancement of action potential effectiveness, but with little prolongation of release. The observation of multiple peaks, or oscillations in the release probability function at high (ca. 1 mM) concentrations of 4-aminopyridine, may be related, as is suggested, to oscillations of presynaptic membrane potential, or perhaps to changes in the electrochemical gradient for Ca2+ influx.     

Properties of Xenopus Kv1.10 channels expressed in HEK293 cells

Voltage-gated K+ channels play important roles in shaping the characteristics of action potentials and electrical activity. In a previous study, we isolated cDNAs encoding several distinct K+ channel isoforms, including a novel isoform (XKv1.10) expressed in Xenopus laevis spinal cord neurons and myocytes. Here, we report the biophysical characterization of XKv1.10 expressed in transiently transfected HEK293 cells. Whole cell patch clamp recordings revealed a voltage-gated, rapidly activating and inactivating K+ current. Interestingly, the rate of inactivation of XKv1.10 channels showed apparent voltage dependence, with time constants between 77.7-213.3 ms. The predicted protein sequence of XKv1.10 does not appear to encode an N-terminal inactivating "ball and chain" domain, and instead these channels may inactivate via a C/P-type mechanism. Consistent with this, either increasing the external concentration of K+ or external application of tetraethylammonium caused a decrease in the rate of inactivation. Pharmacologically, XKv1.10 K+ channels were sensitive to 4-aminopyridine and tetraethylammonium with apparent IC50 values of 68.5 microM and 17.1 mM, respectively. When simulated action potentials were used as a voltage command, XKv1.10 was similar to XKv1.4 in that it carried more repolarizing current during the action potential than XKv1.2. However, while XKv1.4 was active during the interspike interval, XKv1.10 and XKv1.2 were not. Overall, the data suggest that XKv1.10 channels make a unique contribution to the developmental maturation of electrical signaling in Xenopus laevis.     

Mutational analysis of ion conduction and drug binding sites in the inner mouth of voltage-gated K+ channels.

Pore properties that distinguish two cloned, voltage-gated K+ channels, Kv2.1 and Kv3.1, include single-channel conductance, block by external and internal tetraethylammonium, and block by 4-aminopyridine. To define the inner mouth of voltage-gated K+ channels, segmental exchanges and point mutations of nonconserved residues were used. Transplanting the cytoplasmic half of either transmembrane segments S5 or S6 from Kv3.1 into Kv2.1 reduced sensitivity to block by internal tetraethylammonium, increased sensitivity to 4-aminopyridine, and reduced single-channel conductance. In S6, changes in single-channel conductance and internal tetraethylammonium sensitivity were associated with point mutations V400T and L403 M, respectively. Although individual residues in both S5 and S6 were found to affect 4-aminopyridine blockade, the most effective change was L327F in S5. Thus, both S5 and S6 contribute to the inner mouth of the pore but different residues regulate ion conduction and blockade by internal tetraethylammonium and 4-aminopyridine.     

Dendrotoxin, 4-Aminopyridine, and β-Bungarotoxin Act at Common Loci but by Two Distinct Mechanisms to Induce Ca2+-Dependent Release of Glutamate from Guinea-Pig Cerebrocortical Synaptosomes

The release of endogenous glutamate from guinea-pig cerebrocortical synaptosomes evoked by dendrotoxin, beta-bungarotoxin, and 4-aminopyridine is compared. Dendrotoxin and 4-aminopyridine cause Ca2+-dependent release, representing a partial depletion of the KCl-releasable transmitter pool. The decrease in the plasma membrane potential caused by 4-aminopyridine or dendrotoxin and the evoked release of glutamate from a transmitter pool accord with the inhibitory action of these agents on certain K+ conductances. In contrast, the massive release of glutamate evoked by beta-bungarotoxin is produced in the presence of Ca2+ but not of Sr2+, a result consistent with a generalised permeabilisation of synaptosomal plasma membranes. Although dendrotoxin inhibits the binding of beta-bungarotoxin and the resultant synaptosomal lysis, demonstration of a direct effect of beta-bungarotoxin binding per se on K+ permeability is impractical owing to its phospholipase A2 activity.     

An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+)-dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.     

The mechanism of the rate-dependent changes of the conducted action potential in rabbit ventricle

Blockers of the transient outward current (4-aminopyridine) and the Ca current (Co2+) as well as injection of polarizing current during the plateau were used to assess the role of these current systems as determinants of action potential duration at different pacing rates. Papillary muscles and ventricular trabecula were superfused with oxygenated Krebs solution at 33 degrees C and driven at a basic rate of 1 Hz. The effects of varying the frequency of stimulation between 0.1 and 4 Hz on action potential parameters were determined under control conditions and during exposure to 2 mM 4-aminopyridine, 1-3 mM CoCl2, or a mixture of 4-aminopyridine and CoCl2. The control relationship between action potential duration and pacing rate showed a maximum between 1 and 2 Hz. Under 4-aminopyridine, the plateau height and the action potential duration increased. The rate-dependent shortening of the action potential at frequencies below 1 Hz was reduced or abolished, and enhanced shortening was observed at rates above 1 Hz. Exposure to Co2+ reduced the action potential shortening at rates higher than 1 Hz. Both blockers, 4-aminopyridine and Co2+ were necessary to eliminate the rate-dependent changes of the action potential duration. Our results indicated that both the transient outward current and the inward calcium current determine the plateau height and duration for frequencies less than or equal to 2 Hz, whereas at higher rates, the Ca current plays a dominant role.     

K+ channels involved in contractility of rabbit small intestine

Most excitable cells, including gastrointestinal smooth muscle cells, express several types of K+ channels. The aim of this study was to examine the types of K' channels involved in the contractility of longitudinal smooth muscle of rabbit small intestine in vitro. Spontaneous contractions and KCl-stimulated contractions were reduced by atropine, phentolamine, propranolol, suramin, tetrodotoxin and indomethacin. The amplitude and tone of spontaneous contractions were increased by apamin, charybdotoxin, iberiotoxin, E4031, tetraetylammonium (TEA) and BaCl2. The frequency of contractions was reduced in the presence of apamin and TEA and increased by charybdotoxin. It was found that 4-aminopyridine increased the tone of spontaneous contractions and reduced the amplitude and frequency of contractions. Glibenclamide did not modify the amplitude, frequency or tone of contractions. KCl-stimulated contractions were increased by E4031, were not modified by apamin, glibenclamide, NS1619 or diazoxide, and were reduced by charybdotoxin, TEA, 4-aminopyridine or BaCl2. These results suggest that both Ca2+-activated K+ channels of small and high conductance, and HERG K+ channels and inward rectifier K+ channels participate in spontaneous contractions of small intestine. On the other hand, voltage-dependent K+ channels, HERG K+ channels, inward rectifier K+ channels and high conductance Ca2+-activated K+ channels are involved in KCl-stimulated contractions.     

Repetitive Action Potentials in Isolated Nerve Terminals in the Presence of 4-Aminopyridine: Effects on Cytosolic Free Ca2+ and Glutamate Release

The mechanisms by which an elevated KCl level and the K+-channel inhibitor 4-aminopyridine induce release of transmitter glutamate from guinea-pig cerebral cortical synaptosomes are contrasted. KCl at 30 mM caused an initial spike in the cytosolic free Ca2+ concentration ([Ca2+]c), followed by a partial recovery to a plateau 112 +/- 13 nM above the polarized control. The Ca2+-dependent release of endogenous glutamate, determined by continuous fluorimetry, was largely complete by 3 min, by which time 1.70 +/- 0.35 nmol/mg was released. [Ca2+]c elevation and glutamate release were both insensitive to tetrodotoxin. KCl-induced elevation in [Ca2+]c could be observed in both low-Na+ medium and in the presence of low concentrations of veratridine. 4-Aminopyridine at 1 mM increased [Ca2+]c by 143 +/- 18 nM to a plateau similar to that following 30 mM KCl. The initial rate of increase in [Ca2+]c following 4-aminopyridine administration was slower than that following 30 mM KCl, and a transient spike was less apparent. Consistent with this, the 4-aminopyridine-induced net uptake of 45Ca2+ is much lower than that following an elevated KCl level. 4-Aminopyridine induced the Ca2+-dependent release of glutamate, although with somewhat slower kinetics than that for KCl. The measured release was 0.81 nmol of glutamate/mg in the first 3 min of 4-aminopyridine action. In contrast to KCl, glutamate release and the increase in [Ca2+]c with 4-aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na+ channels. Basal [Ca2+]c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium and potassium channels in regenerating and developing mammalian myelinated nerves

A study has been made of how the normal complementary distribution of sodium and potassium channels in mammalian myelinated nerve fibres (all the sodium channels being in the node with all the potassium channels in the internode) is altered in regenerating and in developing rabbit sciatic nerves. In regenerating nerve fibres, where a marked increase in the number of nodes per unit length occurs, there is a corresponding increase in the sodium channel content (determined from the maximum saturable binding of labelled saxitoxin), consistent with the idea that the number of sodium channels per node remains roughly constant. The use of 4-aminopyridine, which by blocking potassium channels prolongs the action potential, has shown that both in regenerating nerve fibres and in developing nerve fibres potassium currents contribute to the mammalian action potential. In both cases, with the passage of time, the sensitivity to 4-aminopyridine progressively decreases.     

Effects of abscisic acid on K+ channels in Vicia faba guard cell protoplasts

Potassium channels were resolved in Vicia faba guard cell protoplasts by patch voltage-clamp. Whole-cell currents and single K+ channels had linear instantaneous current-voltage relations, reversing at the calculated Nernst potential for K+. Whole cell K+ currents activated exponentially during step depolarizations, with half-activation times of 400-450 msec at +80 mV and 90-110 msec at +150 mV. Single K+ channel conductance was 65 +/- 5 pS with a mean open time of 1.25 +/- 0.30 msec at 150 mV. Potassium channels were blocked by internal Cs+ and by external TEA+, but they were insensitive to external 4-aminopyridine. Application of 10 microM abscisic acid increased mean open time and caused long-lasting bursts of channel openings. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology.     

Inhibition of the acetylcholine-induced relaxation of canine isolated basilar artery by potassium-conductance blockers

Canine basilar artery rings precontracted with 5-hydroxytryptamine (0.1-0.5 microM) relaxed in the presence of acetylcholine (25-100 microM), sodium nitroprusside (0.1 microM), or stimulation of the electrogenic sodium pump by restoration of extracellular K+ (4.5 mM) after K(+)-deprivation. Acetylcholine-induced relaxation is believed to be caused by the release of endothelium-derived relaxing factor (EDRF) and is prevented by mechanical removal of the endothelium, while relaxations induced by sodium nitroprusside or restarting of the sodium pump are endothelium-independent. Acetylcholine-induced relaxation was selectively blocked by pretreatment of the tissue with the nonselective K+ conductance inhibitors, 4-aminopyridine (4-AP, 3 mM), Ba2+ (1 mM), and tetraethylammonium (20 mM), 4-AP also blocked ACh-mediated relaxation in muscles contracted with elevated external K+. Relaxation of 5-hydroxytryptamine-induced contraction by sodium nitroprusside, or by addition of K+ to K(+)-deprived muscle, was not affected by 4-AP. Relaxation of basilar artery with acidified sodium nitrite solution (containing nitric oxide) was reduced by 4-AP. These results suggest that 4-AP and possibly Ba2+ inhibit acetylcholine-induced endothelium-dependent relaxation by inhibition of the action of EDRF on the smooth muscle rather than through inhibition of release of EDRF. The increase in K+ conductance involved in acetylcholine-induced relaxation is not due to ATP-inhibited K+ channels, as it is not blocked by glyburide (10(-6) M). Endothelium-derived relaxant factor(s) may relax smooth muscle by mode(s) of action different from that of sodium nitroprusside or by hyperpolarization due to the electrogenic sodium pumping.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

The effect of Lambert-Eaton myasthenic syndrome antibody on slow action potentials in mouse cardiac ventricle

Immunoglobulin G (IgG) from Lambert-Eaton myasthenic syndrome (LEMS) patients acts at motor nerve terminal Ca2+ channels. It was injected into mice to investigate effects on cardiac Ca2+ channels. Intracellular recordings were made of slow action potentials in right ventricular muscle cells in the presence of high K+ concentrations and isoprenaline (1 microM). Reduction in Ca2+ concentration reduced the rate of rise and amplitude, but not the duration, of slow action potentials whereas verapamil (1 microM) blocked them. They were not blocked by tetrodotoxin (10 microM), and 4-aminopyridine (1 mM) prolonged the decay phase without affecting the rate of rise and amplitude. The rate of rise, amplitude and duration of slow action potentials were not affected by LEMS IgG. These results show that LEMS IgG does not act on Ca2+ channel currents that underlie slow action potentials in mouse ventricles, suggesting antigenic differences between Ca2+ channels at motor nerve terminals and heart.     

Diversity of K+ channels in circular smooth muscle of opossum lower esophageal sphincter

We previously demonstrated that a balance of K+ and Ca2+-activated Cl- channel activity maintained the basal tone of circular smooth muscle of opossum lower esophageal sphincter (LES). In the current studies, the contribution of major K+ channels to the LES basal tone was investigated in circular smooth muscle of opossum LES in vitro. K+ channel activity was recorded in dispersed single cells at room temperature using patch-clamp recordings. Whole-cell patch-clamp recordings displayed an outward current beginning to activate at -60 mV by step test pulses lasting 400 ms (-120 mV to +100 mV) with increments of 20 mV from holding potential of -80 mV ([K+]I = 150 mM, [K+]o = 2.5 mM). However, no inward rectification was observed. The outward current peaked within 50 ms and showed little or no inactivation. It was significantly decreased by bath application of nifedipine, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and iberiotoxin (IBTN). Further combination of TEA with 4-AP, nifedipine with 4-AP, and IBTN with TEA, or vice versa, blocked more than 90% of the outward current. Ca2+-sensitive single channels were recorded at asymetrical K+ gradients in cell-attached patch-clamp configurations (100.8+/-3.2 pS, n = 8). Open probability of the single channels recorded in inside-out patch-clamp configurations were greatly decreased by bath application of IBTN (100 nM) (Vh = -14.4+/-4.8 mV in control vs. 27.3+/-0.1 mV, n = 3, P < 0.05). These data suggest that large conductance Ca2+-activated K+ and delayed rectifier K+ channels contribute to the membrane potential, and thereby regulate the basal tone of opossum LES circular smooth muscle.     

Adsorption of aminopyridines to phosphatidylserine membranes

Aminopyridines belong to the class of compounds which facilitate synaptic transmission at low calcium concentration, an effect associated with the block of K+ channels, enhanced entry of calcium into presynaptic terminals and greater release of transmitter. We have measured the zeta-potential of phosphatidylserine vesicles in the presence of aminopyridines and some related compounds in order to relate the strength of association of the aminopyridines with their biological effectiveness. The dependence of zeta-potential on the concentration of aminopyridines was analyzed in terms of the Langmuir-Stern-Grahame adsorption model. The rank order of the association constants (in M-1) obtained in the study was as follows: 3,4-diaminopyridine (6.5), 4,5-diaminopyrimidine (3.8), 4-aminopyridine (2.6), 3-aminopyridine (1.8), 2-aminopyridine (1.6), 4-dimethylaminopyridine (0.5), 4-aminopyridine methiodide (0.2), and, as control, calcium (12.1). The comparison of association constants with published results of the electric potential maps obtained by the CNDO/2 method suggests that binding to phosphatidylserine membrane increases with the density of excess charge on the protonated aminopyridine ring. We find that the sequence of potencies of aminopyridines in blocking K+ channels, in releasing transmitter, and in the shifts of calcium concentration dependence of synaptic transmission are about the same as the sequence of association constants with the phosphatidylserine membrane. Assuming that the binding domain for aminopyridines in the presynaptic terminal has similar adsorption properties as the phosphatidylserine membrane, we estimate the electric potential difference between the domain and the external solution to be between -300 and -340 mV.     

Functional expression of a GFP-tagged Kv1.5 alpha-subunit in mouse ventricle

The experiments here were undertaken to determine the feasibility of increasing the cell surface expression of voltage-gated ion channels in cardiac cells in vivo and to explore the functional consequences of ectopic channel expression. Transgenic mice expressing a green fluorescent protein (GFP)-tagged, voltage-gated K+ (Kv) channel alpha-subunit, Kv1.5-GFP, driven by the cardiac-specific alpha-MHC promoter, were generated. In recent studies, Kv1.5 has been shown to encode the micromolar 4-aminopyridine (4-AP)-sensitive delayed rectifier K+ current (I(K,slow)) in mouse myocardium. Unexpectedly, Kv1.5-GFP expression is heterogeneous in the ventricles of these animals. Although no electrocardiographic abnormalities were evident, expression of Kv1.5-GFP results in marked decreases in action potential durations in GFP-positive ventricular myocytes. In voltage-clamp recordings from GFP-positive ventricular myocytes, peak outward K+ currents are significantly higher, and their waveforms are distinct from those recorded from wild-type cells. Pharmacological experiments revealed a selective increase in a micromolar 4-AP-sensitive current, similar to the 4-AP-sensitive component of I(K,slow) in wild-type cells. The inactivation rate of the "overexpressed" current, however, is significantly slower than the Kv1.5-encoded component of I(K,slow) in wild-type cells, suggesting differences in association with accessory subunits and/or posttranslational processing.     

Differences in presynaptic action of 4-aminopyridine and tetraethylammonium at frog neuromuscular junction

4-Aminopyridine markedly potentiates transmitter release at the frog pectoris neuromuscular junction by increasing the quantal content even when applied at low concentrations (5-20 microM). This enhancement of transmitter release is associated with greater minimum synaptic latency, but the dispersion of the synaptic latencies does not appear much affected. This is in contrast with the action of tetraethylammonium (0.2-0.5 mM) in which case similar enhancement of transmitter release results not only in larger minimum synaptic latency but also in greater dispersion of the synaptic latencies. The time course of transmitter release associated with enhanced transmitter output is hence much more prolonged in the presence of tetraethylammonium than 4-aminopyridine, at least for low concentrations of 4-aminopyridine (5-20 microM). This indicates that their presynaptic actions differ significantly. This conclusion is further strengthened by the finding that unlike tetraethylammonium, 4-aminopyridine induces bursts of release, presumably by producing multiple action potentials in the nerve terminal. Tetraethylammonium probably acts by blocking the delayed potassium conductance, but the blockade of Ca2+-activated K+ conductance cannot be excluded. 4-Aminopyridine, however, probably blocks the fast inactivating (IA) K+ current, but it also may be acting directly on the voltage-dependent Ca2+ conductance or on the intracellular Ca2+ buffering.