Single-channel properties of BK-type calcium-activated potassium channels at a cholinergic presynaptic nerve terminal.
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1. A high-conductance calcium-activated potassium channel (BK KCa) was characterized at a cholinergic presynaptic nerve terminal using the calyx synapse isolated from the chick ciliary ganglion. 2. The channel had a conductance of 210 pS in a 150 mM:150 mM K+ gradient, was highly selective for K+ over Na+, and was sensitive to block by external charybdotoxin or tetraethylammonium (TEA) and by internal Ba2+. At +60 mV it was activated by cytoplasmic calcium [Ca2+]i with a Kd of approximately 0.5 microM and a Hill coefficient of approximately 2.0. At 10 microM [Ca2+]i the channel was 50 % activated (V) at -8.0 mV with a voltage dependence (Boltzmann slope-factor) of 32.7 mV. The V values hyperpolarized with an increase in [Ca2+]i while the slope factors decreased. There were no overt differences in conductance or [Ca2+]i sensitivity between BK channels from the transmitter release face and the non-release face. 3. Open and closed times were fitted by two and three exponentials, respectively. The slow time constants were strongly affected by both [Ca2+]i and membrane potential changes. 4. In cell-attached patch recordings BK channel opening was enhanced by a prepulse permissive for calcium influx through the patch, suggesting that the channel can be activated by calcium ion influx through neighbouring calcium channels. 5. The properties of the presynaptic BK channel are well suited for rapid activation during the presynaptic depolarization and Ca2+ influx that are associated with transmitter release. This channel may play an important role in terminating release by rapid repolarization of the action potential. (+info)
Voltage-gated K+ currents in freshly isolated myocytes of the pregnant human myometrium.
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1. Voltage-gated K+ currents in human myometrium are not well characterized, and were therefore investigated, using the whole-cell patch clamp technique, in freshly isolated myometrial smooth muscle cells from pregnant women at term. 2. Three types of voltage-gated K+ currents were identified. IK1 was a 4-aminopyridine-insensitive current with a negative half-inactivation (V0.5 = -61 to -67 mV) and negative activation characteristics (threshold between -60 and -40 mV) and slow kinetics. IK2 was a 4-aminopyridine-sensitive current (half-maximal block at approximately 1 mM) with relatively positive half-inactivation (V0.5 = -30 mV) and activation characteristics (threshold between -40 and -30 mV) and faster kinetics. IK,A was a 4-aminopyridine-sensitive current with a negative inactivation and very fast inactivation kinetics. 3. Both IK1 and IK2 were sensitive to high concentrations of tetraethylammonium (half-maximal block at approximately 3 mM) and low concentrations of clofilium (half-maximal block by 3-10 microM). 4. IK1 and IK2 were unevenly distributed between myometrial cells, most cells possessing either IK1 (30 cells) or IK2 (24 cells) as the predominant current. 5. The characteristics of these currents suggest a possible function in the control of membrane potentials and smooth muscle quiescence in the pregnant human myometrium. (+info)
Basolateral K+ channel involvement in forskolin-activated chloride secretion in human colon.
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1. In this study we investigated the role of basolateral potassium transport in maintaining cAMP-activated chloride secretion in human colonic epithelium. 2. Ion transport was quantified in isolated human colonic epithelium using the short-circuit current technique. Basolateral potassium transport was studied using nystatin permeabilization. Intracellular calcium measurements were obtained from isolated human colonic crypts using fura-2 spectrofluorescence imaging. 3. In intact isolated colonic strips, forskolin and prostaglandin E2 (PGE2) activated an inward transmembrane current (ISC) consistent with anion secretion (for forskolin DeltaISC = 63.8+/-6.2 microA cm(-2), n = 6; for PGE2 DeltaISC = 34.3+/-5.2 microA cm(-2), n = 6). This current was inhibited in chloride-free Krebs solution or by inhibiting basolateral chloride uptake with bumetanide and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid DIDS). 4. The forskolin- and PGE2-induced chloride secretion was inhibited by basolateral exposure to barium (5 mM), tetrapentylammonium (10 microM) and tetraethylammonium (10 mM). 5. The transepithelial current produced under an apical to serosal K+ gradient in nystatin-perforated colon is generated at the basolateral membrane by K+ transport. Forskolin failed to activate this current under conditions of high or low calcium and failed to increase the levels of intracellular calcium in isolated crypts 6. In conclusion, we propose that potassium recycling through basolateral K+ channels is essential for cAMP-activated chloride secretion. (+info)
Myogenic mechanism for peristalsis in the cat esophagus.
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A myogenic control system (MCS) is a fundamental determinant of peristalsis in the stomach, small bowel, and colon. In the esophagus, attention has focused on neuronal control, the potential for a MCS receiving less attention. The myogenic properties of the cat esophagus were studied in vitro with and without nerves blocked by 1 microM TTX. Muscle contraction was recorded, while electrical activity was monitored by suction electrodes. Spontaneous, nonperistaltic, electrical, and mechanical activity was seen in the longitudinal muscle and persisted after TTX. Spontaneous circular muscle activity was minimal, and peristalsis was not observed without pharmacological activation. Direct electrical stimulation (ES) in the presence of bethanechol or tetraethylammonium chloride (TEA) produced slow-wave oscillations and spike potentials accompanying smooth muscle contraction that progressed along the esophagus. Increased concentrations of either drug in the presence of TTX produced slow waves and spike discharges, accompanied by peristalsis in 5 of 8 TEA- and 2 of 11 bethanechol-stimulated preparations without ES. Depolarization of the muscle by increasing K(+) concentration also produced slow waves but no peristalsis. We conclude that the MCS in the esophagus requires specific activation and is manifest by slow-wave oscillations of the membrane potential, which appear to be necessary, but are not sufficient for myogenic peristalsis. In vivo, additional control mechanisms are likely supplied by nerves. (+info)
Ca(2+)-activated nonselective cationic current (I(CAN)) in turtle motoneurons.
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The presence of a calcium-activated nonspecific cationic (CAN) current in turtle motoneurons and its involvement in plateau potentials, bistability, and wind-up was investigated by intracellular recordings in a spinal cord slice preparation. In the presence of tetraethylammonium (TEA) and tetrodotoxin (TTX), calcium action potentials evoked by depolarizing current pulses were always followed by an afterdepolarization associated with a decrease in input resistance. The presence of the afterdepolarization depended on the calcium spike and not on membrane potential. Replacement of extracellular sodium by choline or N-methyl-D-glucamine (NMDG) reduced the afterdepolarization, confirming that it was mediated by a CAN current. Plateau potentials and wind-up were evoked in response to intracellular current pulses in the presence of agonist for different metabotropic receptors. Replacement of extracellular sodium by choline or NMDG did not abolish the generation of plateau potentials, bistability, or wind-up, showing that Na(+) was not the principal charge carrier. It is concluded that plateau potentials, bistability and wind-up in turtle motoneurons do not depend on a CAN current even though its presence can be detected. (+info)
Effects of potassium channel blockers on CO2-induced slowly adapting pulmonary stretch receptor inhibition.
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In anesthetized, artificially ventilated rabbits with vagus nerve section, inhalation of CO(2) gas mixtures (tracheal CO(2) concentration ranging from 8.0 to 10.2%) for 60 s decreased slowly adapting pulmonary stretch receptor (SAR) activity during both inflation and deflation. The magnitude of decreased receptor activity during deflation had a more pronounced effect than that seen during inflation. CO(2) inhalation did not cause any significant change in tracheal pressure (P(T)) as an index of bronchomotor tone. Intravenous administration of 4-aminopyridine (0. 7 and 2.0 mg/kg i.v.), a K(+) channel blocker, which dose-dependently increased SAR activity during deflation and had no effect on P(T), abolished or attenuated the decrease in SAR activities induced by CO(2) inhalation in a dose-dependent manner. The K(+) channel blocker tetraethylammonium (2.0 and 6.0 mg/kg i.v.) that did not significantly alter either basal SAR discharge or P(T) had no effect on the inhibitory responses of receptor activity to CO(2) inhalation. These results suggest that the inhibitory mechanism of CO(2) inhalation on SARs may be involved in the activation of 4-aminopyridine-sensitive K(+) channels in the nerve terminals of SARs. (+info)
Ruthenium red-mediated inhibition of large-conductance Ca2+-activated K+ channels in rat pituitary GH3 cells.
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The ionic mechanism of actions of ruthenium red was examined in rat anterior pituitary GH(3) cells. In whole-cell recording experiments, ruthenium red reversibly caused an inhibition of Ca(2+)-activated K(+) current [I(K(Ca))] in a dose-dependent manner. The IC(50) value of ruthenium red-induced inhibition of I(K(Ca)) was 15 microM. Neither carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 10 microM), an uncoupler of oxidative phosphorylation in mitochondria, nor cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore, affected the amplitude of I(K(Ca)). In inside-out configuration, application of ruthenium red (50 microM) into the bath medium did not change single-channel conductance but significantly suppressed the activity of large-conductance Ca(2+)-activated K(+) channel (BK(Ca)) channels. The ruthenium red-induced decrease in the channel activity of BK(Ca) channels was reversed by an increase in intracellular Ca(2+) concentration. Ruthenium red also shifted the activation curve of BK(Ca) channels to positive membrane potentials. The change in the kinetic behavior of BK(Ca) channels caused by ruthenium red in these cells is due to a decrease in mean open time and an increase in mean closed time. Ruthenium red (50 microM) did not affect the amplitude of voltage-dependent K(+) current but produced a significant reduction of voltage-dependent L-type Ca(2+) current. These results indicate that ruthenium red can directly suppress the activity of BK(Ca) channels in GH(3) cells. This effect is independent on the inhibition of Ca(2+) release from internal stores or mitochondria. (+info)
Functional characteristics and tissue distribution pattern of organic cation transporter 2 (OCTN2), an organic cation/carnitine transporter.
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We have demonstrated in the present study that novel organic cation transporter (OCTN) 2 is a transporter for organic cations as well as carnitine. OCTN2 transports organic cations without involving Na(+), but it transports carnitine only in the presence of Na(+). The ability to transport organic cations and carnitine is demonstrable with human, rat, and mouse OCTN2s. Na(+) does not influence the affinity of OCTN2 for organic cations, but it increases the affinity severalfold for carnitine. The short-chain acyl esters of carnitine are also transported by OCTN2. Two mutations, M352R and P478L, in human OCTN2 are associated with loss of transport function, but the protein expression of these mutants is comparable to that of the wild-type human OCTN2. In situ hybridization in the rat shows that OCTN2 is expressed in the proximal and distal tubules and in the glomeruli in the kidney, in the myocardium, valves, and arterioles in the heart, in the labyrinthine layer of the placenta, and in the cortex, hippocampus, and cerebellum in the brain. This is the first report that OCTN2 is a Na(+)-independent organic cation transporter as well as a Na(+)-dependent carnitine transporter and that OCTN2 is expressed not only in the heart, kidney, and placenta but also in the brain. (+info)