Slow gating charge immobilization in the human potassium channel Kv1.5 and its prevention by 4‐aminopyridine.
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1. The relationship between ionic current inactivation and immobilization of ‘off'‐gating charge in human Kv1.5 channels expressed in human embryonic kidney (HEK293) cells was studied using 4‐aminopyridine (4‐AP) and tetraethylammonium chloride (TEA‐Cl). 2. The charge transferred during short (< 10 ms) depolarizations (Q(on)) was conserved on repolarization (Q(off)) although peak off‐gating current (off‐Ig) was reduced and the time course prolonged (tau decay increased from 0.4 to > 1.2 ms). For +80 mV pulses longer than 50 ms, Q(off) at 20 ms was less than Q(on) (Q(off)/Q(on) ratio was 0.26 +/‐ 0.06 at 450 ms). We attribute this to a relative ‘immobilization’ of gating charge during long depolarizations. 3. 4‐AP (0.1‐1 mM) prevented slowing of off‐Ig, allowing saturation of peak off‐Ig. 4‐AP also completely prevented immobilization of off‐Ig after long depolarizations. In 1 mM 4‐AP, off‐Ig waveforms decayed rapidly and the charge ratio Q(off)/Q(on) remained at 1.0. 4. In addition to its effects on Ig, 1 mM 4‐AP prevented the slow inactivation of ionic current seen during strong depolarizations. An initial block was caused by 4‐AP or 1 mM intracellular TEA internally applied. However, only 4‐AP prevented the slower, later development of C‐type inactivation. 5. We suggest that slow current inactivation is accompanied by a gating charge immobilization in Kv1.5. 4‐AP potently inhibits the changes in Q(off)/Q(on0, off‐Ig, and ionic currents that underlie slow inactivation. Some actions of 4‐AP appear independent of its properties as a blocker of open K+ channels, and are not mimicked by internal TEA.Keywords:
4-Aminopyridine
Tetraethylammonium chloride
Tetraethylammonium
Potassium channel blocker
HEK 293 cells
the present study was undertaken to evaluate [ca 2+ ] i signals that occur in human sperm cells exposed in vitro to three diverse compounds; progesterone, 4-aminopyridine (a highly effective inducer of hyperactivation in human sperm) and tetraethylammonium.the [ca 2+ ] i reached after the extracellular calcium treatment was always higher in normozoospermic samples pretreated with progesterone than in pathozoospermic samples pretreated with progesterone.there were no changes in calcium signal in spermatozoa pretreated with progesterone from patients with oligozoospermia and leucocytospermia compared to control samples (without progesterone).[ca 2+ ] i .was always higher in pathozoospermic samples without 4-aminopyridine and always lower in pathozoospermic samples with 4-aminopyridine compared to these values in normozoospermic men. the 2 mM extracellular calcium administration to spermatozoa pretreated with tetraethylammonium did not result in a detectable increase in [ca 2+ ] i in normo-and pathozoospermic samples.the mechanisms of progesterone-dependent activation of the ca 2+ -entry and the functioning of the voltage gated ca 2+ -channels of plasmalemma are disturbed in pathologies -there was no increase in the ca 2+ level in the conditions of k + -depolarization (in the presence of inhibitors of k + -channels).k e y w o r d s: calcium, spermatozoa, progesterone, inhibitors of k + channels.
4-Aminopyridine
Tetraethylammonium
Potassium channel blocker
2-Aminopyridine
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4-Aminopyridine
Tetraethylammonium
Potassium channel blocker
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Tetraethylammonium
4-Aminopyridine
Afterhyperpolarization
Potassium channel blocker
Hyperpolarization
Tetraethylammonium chloride
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1. The relationship between ionic current inactivation and immobilization of ‘off'‐gating charge in human Kv1.5 channels expressed in human embryonic kidney (HEK293) cells was studied using 4‐aminopyridine (4‐AP) and tetraethylammonium chloride (TEA‐Cl). 2. The charge transferred during short (< 10 ms) depolarizations (Q(on)) was conserved on repolarization (Q(off)) although peak off‐gating current (off‐Ig) was reduced and the time course prolonged (tau decay increased from 0.4 to > 1.2 ms). For +80 mV pulses longer than 50 ms, Q(off) at 20 ms was less than Q(on) (Q(off)/Q(on) ratio was 0.26 +/‐ 0.06 at 450 ms). We attribute this to a relative ‘immobilization’ of gating charge during long depolarizations. 3. 4‐AP (0.1‐1 mM) prevented slowing of off‐Ig, allowing saturation of peak off‐Ig. 4‐AP also completely prevented immobilization of off‐Ig after long depolarizations. In 1 mM 4‐AP, off‐Ig waveforms decayed rapidly and the charge ratio Q(off)/Q(on) remained at 1.0. 4. In addition to its effects on Ig, 1 mM 4‐AP prevented the slow inactivation of ionic current seen during strong depolarizations. An initial block was caused by 4‐AP or 1 mM intracellular TEA internally applied. However, only 4‐AP prevented the slower, later development of C‐type inactivation. 5. We suggest that slow current inactivation is accompanied by a gating charge immobilization in Kv1.5. 4‐AP potently inhibits the changes in Q(off)/Q(on0, off‐Ig, and ionic currents that underlie slow inactivation. Some actions of 4‐AP appear independent of its properties as a blocker of open K+ channels, and are not mimicked by internal TEA.
4-Aminopyridine
Tetraethylammonium chloride
Tetraethylammonium
Potassium channel blocker
HEK 293 cells
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Tetraethylammonium chloride
Potassium channel blocker
Channel blocker
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Effects of extracellular pH (pHo) were examined on the changes in atrial repolarization induced by 4-aminopyridine (4AP), which is a selective blocker of the transient outward potassium channel, I(to). Action potential parameters were measured, using the conventional microelectrode technique, in the absence and presence of 4AP (0.1-3.0 mM) at pHo 6.5, 7.25, and 8.0. Phase 1 amplitude served as an index of I(to). The onset and recovery kinetics of phase 1 amplitude were assessed at a basic cycle length of 0.5 s, and time constants (tau on and tau r) were computed. Both onset and recovery kinetics had monoexponential functions. Tonic blockade was influenced by external pH, and Kd for half block was 0.19, 0.44, and 2.43 mM for pHo 8.0, 7.25, and 6.5, respectively. Phasic block was defined and exhibited cycle length dependence. Phasic block was also modified by external pH with the greatest effect at pHo 8.0. 4AP (0.3 mM) accelerated tau on, 0.62 +/- 0.2, 0.55 +/- 0.1, and 2.0 +/- 0.8 beats for pHo 8.0, 7.25, and 6.5 compared with control 6.8 +/- 1.9, 6.3 +/- 1.9, and 5.1 +/- 1.4 beats. In contrast, 4AP slowed tau r by about 1 s from control value to 1.5 +/- 0.5 s at pHo 6.5, 4.8 +/- 1.5 s at pHo 7.25 (p < 0.05), and 5.7 +/- 2.0 s at pHo 8.0. We conclude that an increase in extracellular pH enhances block of Ito induced by 4AP, whereas low pHo attenuates the block.
4-Aminopyridine
Potassium channel blocker
Tonic (physiology)
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The effects of 4‐aminopyridine (4‐AP) and tetraethylammonium (TEA) on the outward potassium currents in the rapidly and slowly adapting stretch receptor neurons (SRNs) of the crayfish ( Pacifastacus leniusculus ) were studied using a two micro‐electrode voltage‐clamp technique. The leakage current was not affected by either 4‐AP or TEA. External 4‐AP blocked the peak outward current in a dose‐dependent manner (1:1 stoichiometry) with an apparent dissociation constant ( K d ) of 2.3 ± 0.2 mm (mean ± SEM) in the slowly and 1.4 ± 0.2 mm in the rapidly adapting SRN, the block being voltage dependent. External application of TEA resulted in a block of the steady state current enhancing the transient characteristics of the current response. The block appeared to deviate from a 1: 1 stoichiometry and the apparent K d for TEA was 9.6 ± 3.4 mm with a cooperativity factor n = 0.43 ± 0.03 in the slowly adapting SRN and 34.5 ± 9.2 mm and 0.37 ± 0.03 respectively in the rapidly adapting SRN. Low Ca 2+ , apamin and charybdotoxin, which are known to block Ca 2+ ‐dependent K‐currents, had no effects on the outward current as was also the case with catechol. It is concluded that the different effects of TEA and 4‐AP on the outward current in the two types of SRNs can be explained by the presence of at least two, probably heteromultimeric, channel populations having similar sensitivity to 4‐AP but different sensitivity to TEA. One channel has a high affinity ( K d = 0.8–1.6 mm) for TEA and the other a low affinity ( K d = 173–213 mm) for TEA. The low‐affinity channel seems to dominate in the slowly adapting SRN while both channels are equally common in the rapidly adapting SRN. Further, the present results do not support the existence of a macroscopic Ca 2+ ‐dependent K + current in the SRNs.
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Apamin
Tetraethylammonium chloride
Potassium channel blocker
Tetrodotoxin
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1 Cat splenic slices prelabelled with [3H]-noradrenaline were incubated in oxygenated Krebs-bicarbonate solution at 37°C, and the spontaneous total 3H release into different incubation media monitored. In normal Krebs bicarbonate solution, the spontaneous tritium fractional release amounted to 3.7% of the tissue radiactivity content per 5 min collection period. 2 Tetraethylammonium (TEA) increased spontaneous transmitter release in a concentration-dependent manner; the release was maximal at 30 mm and was 3.5 times the basal release. 3 4-Aminopyridine (4-AP) also enhanced the spontaneous release of tritium. The response increased linearly with 4-AP concentration (1–10 mm). With 10 mm 4-AP, the release was as much as 6 times the basal transmitter release. Guanidine was much less potent than either TEA or 4-AP. 4 The secretory response to TEA or 4-AP was little affected by changes in external Ca2+, Mg2+, Na+, Cl−, H2PO4− or by tetrodotoxin. 5 However, transmitter release evoked by TEA or 4-AP strongly depended upon the concentration of HCO3− of the incubation solution; in fact, the secretory response varied almost linearly between 1 and 25 mm HCO3−. 6 The mechanisms underlying these effects are probably related to the well-known ability of TEA and 4-AP to block K+ conductance that would cause depolarization of the splenic sympathetic nerve terminals. The HCO3− requirements for the secretory response are probably related to the ability of CO2/HCO3− solutions to mobilize and release Ca2+ from intracellular organelles.
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Tetrodotoxin
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