IS3 peptide-formed ion channels in rat skeletal muscle cell membranes.

A 22-mer peptide, identical to the primary sequence of domain I segment 3 (IS3) of rat brain sodium channel I, was synthesized. With the patch clamp cell-attached technique, single channel currents could be recorded from the patches of cultured rat myotube membranes when the patches were held at hyperpolarized potentials and the electrode solution contained NaCl and 1 microM IS3, indicating that IS3 incorporated into the membranes and formed ion channels. The single channel conductances of IS3 channels were distributed heterogeneously, but mainly in the range of 10-25 pS. There was a tendency that the mean open time and open probability of IS3 channels increased and the mean close time decreased with the increasing of hyperpolarized membrane potentials. IS3 channels are highly selective for Na+ and Li+ but not for Cl- and K+, similar to the authentic Na+ channels.

Blockade of U50488H on potassium currents of acutely isolated mouse hippocampal CA3 pyramidal neurons.

The actions of the opioid agonist U50488H on IA and IK were examined in acutely isolated mouse hippocampal CA3 pyramidal neurons using the whole-cell patch clamp technique. U50488H caused a concentration dependent, rapidly developing and reversible inhibition of voltage-activated IA and IK. The inhibitory actions were still observed in the presence of 30 microM naloxone or 5 microM nor-binaltorphimine dihydrochloride. The IC50 values for the blockade of IA and IK were calculated as 20.1.9 and 3.7 microM, respectively. In the presence of 3.3 microM U50488H, repetitive stimulation induced use-dependent inhibition of IA and IK. A 10 microM concentration of U50488H positively shifted the half-activation membrane potential of IA by +11 mV, but negatively shifted IK by -14 mV. These results demonstrate that U50488H can directly inhibit neuronal IA and IK without involvement of the activation of kappa-opioid receptors.

Effects of Buthus martensii (Karsch) scorpion venom on sodium currents in rat anterior pituitary (GH3/B6) cells.

The effects of two fractions (II, containing anti-insect toxins, and III, containing eight anti-mammal toxins) isolated from the venom of the Old World scorpion Buthus martensii (Karsch) on Na+ currents of rat anterior pituitary cells (GH3/B6 cells) were investigated using the whole-cell configuration of the patch clamp technique. Fraction II induced a temporary, and fraction III a permanent increase of the Na+ current amplitude. Application of each of the venom fractions resulted in a flattening of the curve relating steady state Na+ inactivation to membrane potential. In addition, the two fractions had specific effects. Fraction II shifted the voltage dependence of Na+ current activation by -42 mV, and the voltage dependence of Na+ inactivation by -25 mV in the absence of a conditioning depolarizing pre-pulse. Slowing of Na+ inactivation was most prominent at negative membrane potentials, resulting in a steady Na+ inward current at the holding potential of -80 mV. Fraction III induced a pronounced slowing of Na+ inactivation leading to an increase of peak Na+ currents and to incomplete steady state Na+ inactivation even at positive membrane potentials.

Properties and amino acid sequence of huwentoxin-I, a neurotoxin purified from the venom of the Chinese bird spider Selenocosmia huwena.

By means of reverse phase and ion-exchange high performance liquid chromatography, a neurotoxic peptide named huwentoxin-I was purified from the venom of the Chinese bird spider Selenocosmia huwena. The intraperitoneal and intracisternal LD50 in mice of the toxin were 0.70 mg/kg and 9.40 micrograms/kg, respectively. This toxin at the concentration of 1 x 10(-5) g/ml can irreversibly block the neuromuscular transmission of the isolated mouse phrenic nerve-diaphragm preparation in 13.4 +/- 1.3 min (mean +/- S.D., n = 5). The isoelectric point is 8.95 determined by isoelectric focusing electrophoresis. It consists of 33 amino acids including 6 Cys and 6 Lys determined by amino acid analysis. The complete amino sequence of huwentoxin-I was determined. The N-terminal and C-terminal residues were Ala and Leu, respectively. The primary structure showed partial homology with that of mu-agatoxins from the funnel-web spider Agelenopsis aperta.

Neural electrophysiological effect of crude venom of conus textile from the south China Sea.

In the present study, we characterized effects of the crude venom from Conus textile, a marine molluscivorous snail collected from the South China Sea, on neural electrophysiological activity in insect, molluscan and mammalian species. Our results demonstrate that the venom reversibly blocks the cholinergic synaptic transmission of cockroach Periplaneta americana central nervous system, partially blocks Na(+) currents in rat hippocampal CA(1) pyramidal neurons, and enhances the excitability and spontaneous activity of the giant neurons of garden snail Achatina fulica.

Blockade of neuromuscular transmission by huwentoxin-I, purified from the venom of the Chinese bird spider Selenocosmia huwena.

Huwentoxin-I (HWTX-I) is a neurotoxic peptide purified from the venom of the Chinese bird spider Selenocosmia huwena. The effects of HWTX-I on neuromuscular transmission of vertebrate skeletal muscle have been investigated by means of twitch tension and electrophysiological techniques. On isolated mouse phrenic nerve-hemidiaphragm preparations, HWTX-I blocked the twitch responses to indirect, but not to direct, muscle stimulation. The time needed for complete block of the neuromuscular transmission was dose dependent. The transmission could be mostly restored by prolonged repeated washing with Tyrode's solution. If the preparation was pretreated with D-tubocurarine and then immersed in a mixed solution of D-tubocurarine and HWTX-I, the washout time necessary to restore the neuromuscular transmission was significantly decreased. Intracellular recording at the end-plate region of frog sartorius muscle revealed that HWTX-I could synchronously reduce the amplitude of the acetylcholine potential induced by ionophoretic application of acetylcholine as well as the amplitude of the end-plate potential evoked by nerve stimulation. Both of these effects eventually disappeared; however, both could be restored by prolonged washing. Experiments on Xenopus embryonic myocytes indicated that HWTX-I reduced the open probability of acetylcholine-induced channel activity, and finally blocked the channel. All of these results demonstrated that HWTX-I was a peptide neurotoxin and the postsynaptic nicotinic acetylcholine receptor was its site of action.

Effect of paeonol on L-type calcium channel in rat ventricular myocytes.

In order to study the possible mechanisms of paeonol on the cardiovascular system, the effect of paeonol on L-type Ca(2+) channel current (I(Ca,L)) was studied in rat ventricular myocytes using the whole cell patch-clamp technique. Exposure to paeonol (approximately 10 to 1000 microm/l) resulted in a concentration-dependent inhibition of peak I(Ca,L), with a half maximum inhibition concentration, IC(50), of 561 microm/l. Paeonol 600 microm/l inhibited I(Ca,L) by 55.3%, shifted the steady state activation and inactivation curve of I(Ca,L) to more positive and negative potentials, respectively, tended to prolong the recovery of I(Ca,L) from inactivation and did not have a use-dependent effect. However, the current-voltage relationship and reversal potential of I(Ca,L) were not altered. These results suggest that the protection by paeonol against myocardial injury is due to its blocking effect on I(Ca,L).

[Properties of the GM-CSF-induced outward K+ current in murine peritoneal exudate macrophages].

Whole-cell patch-clamp technique was used to study the changes of ionic currents in murine peritoneal exudate macrophages (PEMs) prestimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF, 16 ng/ml) for periods from 0.5 up to 6 d. The GM-CSF-treated PEMs developed a GM-CSF-induced transient inactivating outward K+ current (IA). IA showed steady-state inactivation over the physiological voltage range and possessed frequency dependence of inactivation when depolarizing pulses were applied at a frequency of 0.5 Hz. IA was selectively inhibited by extracellular 4-AP (3 mmol/L). When [Ca2+]i was increased (from pCa 8 to 6), the amplitudes of IA were depressed significantly. When the PEMs were exposed to cycloheximide (0.3 microgram/ml), a protein synthesis inhibitor, for 12 h, IA expression was completely suppressed. It was notable that the changes of the current expression, activation behavior and kinetic properties occurred during GM-CSF treatment. When PEMs were pretreated for a 2-d period, the frequency of IA expression reached a peak value (55% in a total of 27 cells), PEMs exhibited the highest density of the corresponding channel proteins, half-maximal activation of IA was most easily achieved with a value of -27.55 mV, and the time course of activation and inactivation during depolarization proceeded rapidly. However, along with continuous incubation with GM-CSF, the number of PEMs expressing IA decreased, the channel proteins were down regulated constantly, the activation curve for IA shifted to positive potentials, and the activation time and inactivation time of IA slowed down. These results indicated that GM-CSF could induce a transient inactivating outward K+ current in PEMs, which may have a close relation to the state of functional activation of macrophages primed with GM-CSF.

[Effects of glutamate on transient outward potassium current of dissociated hippocampal neurons in ca1 sector in rats].

The effects of glutamate on the transient outward potassium current of dissociated hippocampal CA(1) neurons of 7-10 d rats were studied by the whole-cell patch clamp technique. It was found that glutamate could block the transient potassium current in a dose- and time-dependent manner. The effect was partly voltage-dependent. These results suggest that glutamate may decrease the membrane conductance of K(+) channels in hippocampal neurons.

[Bepridil inhibition on the delayed rectifier K+ currents in thyroxine induced hypertrophied guinea pig ventricular myocytes].

AIM: To study the effects of bepridil on the rapidly activating component (IKr), the slowly activating component (IKs) of the delayed rectifier potassium current and the inward rectifier potassium current (IK1) in hypertrophied guinea pig ventricular myocytes. METHODS: The whole cell patch clamp techniques were used. RESULTS: In hypertrophied guinea pig ventricular myocytes, bepridil 30 mumol.L-1 markedly inhibited IKr and IKs (by 20.9% and 27.2% at 0 mV and mV, respectively). The effect of bepridil on IKs was larger than on IKr. Bepridil 30 mumol.L-1 also significantly inhibited the inward component of IK1 (by 15.1% at +100 mV), but the reverse potential of IK1 was unaffected. Bepridil (1-100 mumol.L-1) was shown to inhibit IKr and IKs in a concentration-dependent manner. Their IC50 were 46.7 mumol.L-1 and 23.8 mumol.L-1, respectively. CONCLUSION: Bepridil inhibit IKr, IKs and IK1 in hypertrophied guinea pig ventricular myocytes, which may be important in understanding the antiarrhythmic effects of this drug.

Puerarin blocks transient outward K+ current and delayed rectifier K+ current in mice hippocampal CA1 neurons.

AIM: To study the effects of puerarin (Pue) on IA and IK in mouse hippocampal neurons. METHODS: The whole cell patch clamp techniques were used. RESULTS: Pue reduced the amplitude of IA and IK, in a concentration-dependent, but not rate- or use-dependent manner (IC50 were 461 micromol/L and 215 micromol/L, respectively). Pue (0.5 mmol/L) shifted the steady state activation curves of IA and IK to positive and negative potentials (Vh about 20.6 mV and 28.6 mV) respectively, but inactivation curves of IA were not affected by Pue. CONCLUSION: Pue inhibited IA and IK in mouse hippocampal CA1 neurons and its blocking effect on I(K) was much stronger than on IA.

Bepridil inhibition of sodium current in rat hippocampal CA1 neurons.

AIM: To study the effects of bepridil on sodium current in rat hippocampal neurons. METHODS: All experiments were performed on acutely isolated hippocampal pyramidal neurons by means of whole-cell patch-clamp techniques. Recording media contained ion channel blockers to allow the selective activation of voltage-dependent sodium currents. RESULTS: Bepridil reduced the amplitudes of sodium current in time- and concentration-dependent manners. The half-blocking time was about 10 min in bepridil 10 micromol.L-1, and IC50 was 2.6 (2.3-2.9) micromol.L-1. Bepridil 10 micromol.L-1 shifted the maximal activation of sodium current from -50 mV to -40 mV, and the characteristic voltage of inactivation from -71 mV to -89 mV without changing the slope factor. CONCLUSION: Bepridil blocked voltage-dependent sodium current of hippocampal CA1 neurons and might have therapeutic actions for ischemia-induced brain damage.

Chronic levothyroxin treatment is associated with ion channel abnormalities in cardiac and neuronal cells.

1. We investigated the ion currents responsible for repolarization of guinea-pig isolated myocytes (ICa) and rat hippocampal CA1 neurons (IA and IK) by means of the whole-cell holding technique. 2. The rat hypertrophied heart, induced by levothyroxin, caused exaggerated cardiac arrhythmias after coronary ischaemia-reperfusion. 3. We found an enhanced ICa in guinea-pig isolated myocytes and decreased IA and IK in rat hippocampal CA1 neurons following levothyroxin treatment. Blockade of the outward K+ current and an increment in inward Ca2+ current by chronic levothyroxin treatment contributed to the delayed repolarization and aggravated cardiac arrhythmias. 4. Animals treated with chronic levothyroxin may serve as pathological models for the investigation of the pattern of ion channel disorders with regard to impaired repolarization and aggravated cardiac arrhythmias.