Comparison of the effects of DC031050, a class III antiarrhythmic agent, on hERG channel and three neuronal potassium channels.

AIM: This study was conducted to test the selectivity of DC031050 on cardiac and neuronal potassium channels. METHODS: Human ether-à-go-go related gene (hERG), KCNQ and Kv1.2 channels were expressed in CHO cells. The delayed rectifier potassium current (I(K)) was recorded from dissociated hippocampal pyramidal neurons of neonatal rats. Whole-cell voltage patch clamp was used to record the voltage-activated potassium currents. Drug-containing solution was delivered using a RSC-100 Rapid Solution Changer. RESULTS: Both DC031050 and dofetilide potently inhibited hERG currents with IC(50) values of 2.3 ± 1.0 and 17.9 ± 1.2 nmol/L, respectively. DC031050 inhibited the I(K) current with an IC(50) value of 2.7 ± 1.5 µmol/L, which was >1000 times the concentration required to inhibit hERG current. DC031050 at 3 µmol/L did not significantly affect the voltage-dependence of the steady activation, steady inactivation of I(K), or the rate of I(K) from inactivation. Intracellular application of DC031050 (5 µmol/L) was insufficient to inhibit I(K). DC031050 up to 10 µmol/L had no effects on KCNQ2 and Kv1.2 channel currents. CONCLUSION: DC031050 is a highly selective hERG potassium channel blocker with a substantial safety margin of activity over neuronal potassium channels, thus holds significant potential for therapeutic application as a class III antiarrhythmic agent.

SKF83959 suppresses excitatory synaptic transmission in rat hippocampus via a dopamine receptor-independent mechanism.

Dopamine (DA) profoundly modulates excitatory synaptic transmission and synaptic plasticity in the brain. In the present study the effects of SKF83959, the selective agonist of phosphatidylinositol (PI)-linked D(1) -like receptor, on the excitatory synaptic transmission were investigated in rat hippocampus. SKF83959 (10-100 µM) reversibly suppressed the field excitatory postsynaptic potential (fEPSP) elicited by stimulating the Schaffer's collateral-commissural fibers in CA1 area of hippocampal slices. However, the inhibition was not blocked by the D(1) receptor antagonist SCH23390, the D(2) receptor antagonist raclopride, the 5-HT(2A/2C) receptor antagonist mesulergine, or the α(1) -adrenoceptor antagonist prazosin. In addition, SKF83959 inhibited the afferent volley and significantly reduced the paired-pulse facilitation ratios. In dissociated hippocampal CA1 pyramidal neurons, SKF83959 had no detectable effect on glutamate-induced currents but potently inhibited voltage-activated Na(+) current (IC50 value = 26.9 ± 1.0 µM), which was not blocked by SCH23390 or by intracellular dialysis of GDP-ß-S. These results demonstrate that SKF83959 suppressed the excitatory synaptic transmission in hippocampal CA1 area, which was independent of D(1) -like receptor. The mechanism underlying the effect could be mainly inhibition of Na(+) channel in the afferent fibers. The suppression of excitatory synaptic transmission and the Na(+) channel by SKF83959 may contribute to its therapeutic benefits in Parkinson's disease.

Electrophysiological effects of SKF83959 on hippocampal CA1 pyramidal neurons: potential mechanisms for the drug's neuroprotective effects.

Although the potent anti-parkinsonian action of the atypical D1-like receptor agonist SKF83959 has been attributed to the selective activation of phosphoinositol(PI)-linked D1 receptor, whereas the mechanism underlying its potent neuroprotective effect is not fully understood. In the present study, the actions of SKF83959 on neuronal membrane potential and neuronal excitability were investigated in CA1 pyramidal neurons of rat hippocampal slices. SKF83959 (10-100 µM) caused a concentration-dependent depolarization, associated with a reduction of input resistance in CA1 pyramidal neurons. The depolarization was blocked neither by antagonists for D1, D2, 5-HT(2A/2C) receptors and α1-adrenoceptor, nor by intracellular dialysis of GDP-ß-S. However, the specific HCN channel blocker ZD7288 (10 µM) antagonized both the depolarization and reduction of input resistance caused by SKF83959. In voltage-clamp experiments, SKF83959 (10-100 µM) caused a concentration-dependent increase of Ih current in CA1 pyramidal neurons, which was independent of D1 receptor activation. Moreover, SKF83959 (50 µM) caused a 6 mV positive shift in the activation curve of Ih and significantly accelerated the activation of Ih current. In addition, SKF83959 also reduced the neuronal excitability of CA1 pyramidal neurons, which was manifested by the decrease in the number and amplitude of action potentials evoked by depolarizing currents, and by the increase of firing threshold and rhoebase current. The above results suggest that SKF83959 increased Ih current through a D1 receptor-independent mechanism, which led to the depolarization of hippocampal CA1 pyramidal neurons. These findings provide a novel mechanism for the drug's neuroprotective effects, which may contributes to its therapeutic benefits in Parkinson's disease.

Magnesium lithospermate B dilates mesenteric arteries by activating BKCa currents and contracts arteries by inhibiting K(V) currents.

AIM: To examine the involvement of K(+) channels and endothelium in the vascular effects of magnesium lithospermate B (MLB), a hydrophilic active component of Salviae miltiorrhiza Radix. METHODS: Isolated rat mesenteric artery rings were employed to investigate the effects of MLB on KCl- or norepinephrine-induced contractions. Conventional whole-cell patch-clamp technique was used to study the effects of MLB on K(+) currents in single isolated mesenteric artery myocytes. RESULTS: MLB produced a concentration-dependent relaxation in mesenteric artery rings precontracted by norepinephrine (1 micromol/L) with an EC(50) of 111.3 micromol/L. MLB-induced relaxation was reduced in denuded artery rings with an EC(50) of 224.4 micromol/L. MLB caused contractions in KCl-precontracted artery rings in the presence of N-nitro-L-arginine methyl ester (L-NAME) with a maximal value of 130.3%. The vasodilatory effect of MLB was inhibited by tetraethylammonium (TEA) in both intact and denuded artery rings. In single smooth muscle cells, MLB activated BK(Ca) currents (EC(50) 156.3 micromol/L) but inhibited K(V) currents (IC(50) 26.1 micromol/L) in a voltage- and concentration-dependent manner. CONCLUSION: MLB dilated arteries by activating BK(Ca) channels in smooth muscle cells and increasing NO release from endothelium, but it also contracted arteries precontracted with KCl in the presence of L-NAME.

Activation of phosphatidylinositol-linked D1-like receptors increases spontaneous glutamate release in rat somatosensory cortical neurons in vitro.

Central dopaminergic system exerts profound modulation on spontaneous glutamate release in various brain regions mainly through D(1) receptor/cAMP/PKA pathway. It remains unclear whether the phosphatidylinositol (PI)-linked D(1)-like receptors are also involved in such modulatory actions. The identification of substituted phenylbenzazepine SKF83959 as the selective agonist for the atypical D(1)-like receptors has given impetus to study their influence on the spontaneous glutamate release in the brain. In the present study the effects of SKF83959 on the spontaneous excitatory postsynaptic currents (sEPSCs) were investigated through whole-cell recording from layer V-VI pyramidal neurons in rat somatosensory cortical slices. Perfusion with SKF83959 (10-100 microM) considerably increased the frequency of sEPSCs, while had no significant effect on the amplitude of sEPSCs. The increase of sEPSC frequency by SKF83959 was blocked by SCH23390, a D(1)-like receptor antagonist, but not by the antagonists for D(2) receptor, alpha(1)-adrenoceptor and 5-HT(2A/2C) receptor. U-73122 (PLCbeta inhibitor), 2-APB (IP(3) receptor antagonist), chelerythrine chloride (PKC inhibitor) and capsazepine (TRPV1 antagonist) could block the effects of SKF83959, whereas H-89 (PKA inhibitor) and forskolin (adenylyl cyclase activator) had no effect. Taken together, sensitization of TRPV1 channels by PKC after activation of D(1) receptor/PLCbeta signaling pathway mediated SKF83959-induced increase in the sEPSC frequency. To our knowledge, this is the first pharmacological evidence that PI-linked D(1)-like dopamine receptors do exist in presynaptic terminals of cortical neurons and play an important role in controlling the spontaneous glutamate release.

Arylbenzazepines are potent modulators for the delayed rectifier K+ channel: a potential mechanism for their neuroprotective effects.

(+/-) SKF83959, like many other arylbenzazepines, elicits powerful neuroprotection in vitro and in vivo. The neuroprotective action of the compound was found to partially depend on its D(1)-like dopamine receptor agonistic activity. The precise mechanism for the (+/-) SKF83959-mediated neuroprotection remains elusive. We report here that (+/-) SKF83959 is a potent blocker for delayed rectifier K(+) channel. (+/-) SKF83959 inhibited the delayed rectifier K(+) current (I(K)) dose-dependently in rat hippocampal neurons. The IC(50) value for inhibition of I(K) was 41.9+/-2.3 microM (Hill coefficient = 1.81+/-0.13, n = 6), whereas that for inhibition of I(A) was 307.9+/-38.5 microM (Hill coefficient = 1.37+/-0.08, n = 6). Thus, (+/-) SKF83959 is 7.3-fold more potent in suppressing I(K) than I(A). Moreover, the inhibition of I(K) by (+/-) SKF83959 was voltage-dependent and not related to dopamine receptors. The rapidly onset of inhibition and recovery suggests that the inhibition resulted from a direct interaction of (+/-) SKF83959 with the K(+) channel. The intracellular application of (+/-) SKF83959 had no effects of on I(K), indicating that the compound most likely acts at the outer mouth of the pore of K(+) channel. We also tested the enantiomers of (+/-) SKF83959, R-(+) SKF83959 (MCL-201), and S-(-) SKF83959 (MCL-202), as well as SKF38393; all these compounds inhibited I(K). However, (+/-) SKF83959, at either 0.1 or 1 mM, exhibited the strongest inhibition on the currents among all tested drug. The present findings not only revealed a new potent blocker of I(K) , but also provided a novel mechanism for the neuroprotective action of arylbenzazepines such as (+/-) SKF83959.

Electrophysiological characterization of a novel Kv channel blocker N,N'-[oxybis(2,1-ethanediyloxy-2,1-ethanediyl) ]bis(4-methyl)-benzenesulfonamide found in virtual screening.

AIM: N,No-[oxybis(2,1-ethanediyloxy-2,1-ethanediyl)]bis(4-methyl)- benzenesulfonamide (OMBSA) is a hit compound with potent voltage-gated K+ (Kv) channel-blocking activities that was found while searching the MDL Available Chemicals Directory with a virtual screening approach. In the present study, the blocking actions of OMBSA on Kv channels and relevant mechanisms were characterized. METHODS: Whole-cell voltage-clamp recording was made in acutely dissociated hippocampal CA1 pyramidal neurons of newborn rats. RESULTS: Superfusion of OMBSA reversibly inhibited both the delayed rectifier (I(K)) and fast transient K+ currents (I(A)) with IC50 values of 2.1+/-1.1 micromol/L and 27.8+/-1.5 micromol/L, respectively. The inhibition was voltage independent. OMBSA markedly accelerated the decay time course of IK, without a significant effect on that of I(A). OMBSA did not change the activation, steady-state inactivation of IK, and its recovery from inactivation, but the compound caused a significant hyperpolarizing shift of the voltage dependence of the steady-state inactivation of I(A) and slowed down its recovery from inactivation. Intracellular dialysis of OMBSA had no effect on both I(K) and I(A). CONCLUSION: The results demonstrate that OMBSA blocks both I(K) and I(A) through binding to the outer mouth of the channel pore, as predicted by the molecular docking model used in the virtual screening. In addition, the compound differentially moderates the inactivation kinetics of the K+ channels through allosteric mechanisms.

Electrophysiological actions of cyclosporin A and tacrolimus on rat hippocampal CA1 pyramidal neurons.

AIM: The aim of the present study was to investigate the electrophysiological actions of cyclosporin A (CsA) and tacrolimus (FK506) on neurons in the brain, and to elucidate the relevant mechanisms. METHODS: Whole-cell current-clamp recording was made in CA1 pyramidal neurons in rat hippocampal slices; whole-cell voltage-clamp recording was made in dissociated hippocampal CA1 pyramidal neurons of rats. RESULTS: CsA (100 micromol/L) and FK506 (50 micromol/L) did not significantly alter the passive electrical properties of hippocampal CA1 pyramidal neurons, but slowed down the repolarizing phase of the action potential. CsA (10-100 micromol/L) selectively inhibited the delayed rectifier K+ current (IK) in a concentration-dependent manner. CsA did not affect the kinetic properties of IK. Intracellular dialysis of CsA (100 micromol/L) had no effect on IK. The inhibition of IK by CsA (100 micromol/L) persisted under the low Ca2+ conditions that blocked the basal activity of calcineurin. CONCLUSION: CsA exerted calcineurin-independent inhibition on the IK in rat hippocampal pyramidal neurons. Taken together with our previous finding with FK506, it is conceivable that the spike broadening caused by the immunosuppressant drugs is due to direct inhibition on the IK.

Calcineurin-independent inhibition of the delayed rectifier K+ current by the immunosuppressant FK506 in rat hippocampal neurons.

The immunosuppressant drug FK506 was found to be a potent neuroprotective agent in animal models of brain ischemia. However, the mechanisms underlying the action remain to be elucidated. The delayed rectifier K(+) channel has been implicated in ischemic injury and neuronal death in the brain. The aim of the present study is to investigate whether the neuroprotective action of FK506 results from blocking the K(+) channel. In acutely dissociated CA1 pyramidal neurons of rat hippocampus, superfusion of FK506 (0.01-100 microM) selectively inhibited the delayed rectifier K(+) current (I(K)) with an IC(50) value of 13.2+/-4.9 microM. The inhibition of I(K) by FK506 (10 microM) had a rapid onset, and then gradually reached a steady-state level. The inhibition was voltage-dependent, became more potent when the currents were elicited by strong depolarization. Moreover, FK506 (10 microM) caused marked negative shifts of the steady-state activation and inactivation curves of I(K), and accelerated its recovery from inactivation. Intracellular dialysis of FK506 (30 microM) was ineffective. The inhibition of I(K) by FK506 (10 microM) persisted under the low-Ca(2+) conditions that blocked the basal activity of protein phosphatase 2B (calcineurin). Rapamycin did not antagonize FK506 but mimicked it. Cyclosporin A inhibited I(K) only at 30 and 100 microM. Taken together, the results suggest that FK506 exert a direct inhibition on the delayed rectifier K(+) channel without involvement of calcineurin.

Chlorahololides A and B, two potent and selective blockers of the potassium channel isolated from Chloranthus holostegius.

[structure: see text] Chlorahololides A(1) and B(2), two highly complex sesquiterpenoid dimers, were isolated from Chloranthus holostegius. Their structures and absolute configurations were established by NMR spectroscopy, X-ray crystallography, and CD. Chlorahololides A (1) and B (2) exhibited potent and selective inhibition on the delayed rectifier (IK) K+ current, with an IC50 of 10.9 and 18.6 microM, respectively.

Discovering potassium channel blockers from synthetic compound database by using structure-based virtual screening in conjunction with electrophysiological assay.

Potassium ion (K+) channels are attractive targets for drug discovery because of the essential roles played in biological systems. However, high-throughput screening (HTS) cannot be used to screen K+ channel blockers. To overcome this disadvantage of HTS, we have developed a virtual screening approach for discovering novel blockers of K+ channels. On the basis of a three-dimensional model of the eukaryotic K+ channels, molecular docking-based virtual screening was employed to search the chemical database MDL Available Chemicals Directory (ACD). Compounds were ranked according to their relative binding energy, favorable shape complementarity, and potential to form hydrogen bonds with the outer mouth of the K+ channel model. Twenty candidate compounds selected from the virtual screening were examined using the whole-cell voltage-clamp recording in rat dissociated hippocampal neurons. Among them, six compounds (5, 6, 8, 18-20) potently blocked both the delayed rectifier (IK) and fast transient K+ currents (IA). When applied externally, these six compounds preferentially blocked IK with potencies 2- to 500-fold higher than that of tetraethylammonium chloride. Intracellular application of the six compounds had no effect on both K+ currents. In addition, the interaction models and binding free energy calculations demonstrated that hydrophobic interaction and solvent effects play important roles in the inhibitory activities of these compounds. The results demonstrated that structure-based computer screening strategy could be used to identify novel, structurally diverse compounds targeting the pore binding pocket of the outer mouth of voltage-gated K+ channels. This study provides an alternative way of finding new blockers of voltage-gated K+ channels, while the techniques for high-throughput screening of K+ channel drugs remain in development.

Inhibition of excitatory synaptic transmission by trans-resveratrol in rat hippocampus.

The red wine polyphenol trans-resveratrol has been found to exert potent protective actions in a variety of cerebral ischemia models. The neuroprotection by trans-resveratrol thus far is mainly attributed to its intrinsic antioxidant properties. In the present study, the effects of the red wine polyphenol on excitatory synaptic transmission were investigated in the CA1 region of rat hippocampal slices. Perfusion with trans-resveratrol (10-100 microM) caused a concentration-dependent inhibition on the filed excitatory postsynaptic potentials (the field EPSPs) without detectable effect on the presynaptic volleys. The inhibition had a slow onset and was reversible. Trans-resveratrol (30 microM) did not change the ratios of paired-pulse facilitation of the field EPSPs tested at intervals of 20, 40 and 80 ms, nor did it alter the membrane properties of postsynaptic CA1 pyramidal neurons. However, trans-resveratrol (30 microM) significantly suppressed glutamate-induced currents in postsynaptic CA1 pyramidal neurons. In dissociated hippocampal neurons, the IC(50) value of trans-resveratrol in inhibition of glutamate-induced currents was 53.3+/-9.4 microM. Kainite and NMDA receptors were more sensitive to the red wine polyphenol than AMPA receptors. The present study for the first time demonstrates that trans-resveratrol inhibits the postsynaptic glutamate receptors, which probably works in concert with its antioxidant action for ameliorating the brain ischemic injury. The findings also support the future use of trans-resveratrol in the treatment of various neurodegenerative disorders.

Electrophysiological characterization of 14-benzoyltalatisamine, a selective blocker of the delayed rectifier K+ channel found in virtual screening.

14-Benzoyltalatisamine is a potent and selective blocker of the delayed rectifier K+ channel found in a computational virtual screening study. The compound was found to block the K+ channel from the extracellular side. However, it is unclear whether 14-benzoyltalatisamine shares the same block mechanism with tetraethylammonium (TEA). In order to elucidate how the hit compound found by the virtual screening interacts with the outer vestibule of the K+ channel, the effects of 14-benzoyltalatisamine and TEA on the delayed rectifier K+ current of rat dissociated hippocampal neurons were compared using whole-cell voltage-clamp recording. External application of 14-benzoyltalatisamine and TEA reversibly inhibited the current with IC50 values of 10.1+/-2.2 microM and 1.05+/-0.21 mM, respectively. 14-Benzoyltalatisamine exerted voltage-dependent inhibition, markedly accelerated the decay of the current, and caused a significant hyperpolarizing shift of the steady-state activation curve, whereas TEA caused voltage-independent inhibition, without affecting the kinetic parameters of the current. The blockade by 14-benzoyltalatisamine, but not by TEA, was significantly diminished in a high K+ (60 mM) external solution. The potency of 14-benzoyltalatisamine was markedly reduced in the presence of 15 mM TEA. The results suggest that 14-benzoyltalatisamine bind to the external pore entry of the delayed rectifier K+ channel with partial insertion into the selectivity filter, which is in conformity with that predicted by the molecular docking model in the virtual screening.

Selective modulation of L-type calcium current by magnesium lithospermate B in guinea-pig ventricular myocytes.

Magnesium lithospermate B (MLB) is the main water-soluble principle of Salviae Miltiorrhizae Radix (also called as 'Danshen' in the traditional Chinese medicine) for the treatment of cardiovascular diseases. MLB was found to possess a variety of pharmacological actions. However, it is unclear whether and how MLB affects the cardiac ion channels. In the present study, the effects of MLB on the voltage-activated ionic currents were investigated in single ventricular myocytes of adult guinea pigs. MLB reversibly inhibited L-type Ca(2+) current (I(Ca,L)). The inhibition was use-dependent and voltage-dependent (the IC(50) value of MLB was 30 microM and 393 microM, respectively, at the holding potential of -50 mV and -100 mV). In the presence of 100 microM MLB, both the activation and steady-state inactivation curves of I(Ca,L) were markedly shifted to hyperpolarizing membrane potentials, whereas the time course of recovery of I(Ca,L) from inactivation was not altered. MLB up to 300 microM had no significant effect on the fast-inactivating Na(+) current (I(Na)), delayed rectifier K(+) current (I(K)) and inward rectifier K(+) current (I(K1)). The results suggest that the voltage-dependent Ca(2+) antagonistic effect of MLB work in concert with its antioxidant action for attenuating heart ischemic injury.

Symbiotic abilities of Sinorhizobium fredii with modified expression of purL.

Previous reports showed that a transposon-induced PurL- mutant of Sinorhizobium fredii induced pseudonodules on Glycine max and the addition of 5-aminoimidazole-4-carboxamide-riboside or adenine to the plant could not restore the mutant to establish effective symbiosis. To gain a better understanding of the impact of the purL gene on symbiosis formation, we measured the effect of modified expression of this gene on the symbiotic abilities of S. fredii on soybean (G. max). A 1.98-kb in-frame deletion mutant in the purL gene of S. fredii was constructed. Transcriptional modification of the purL gene was conducted using several promoters such as those of lac, nifH, nifQ, and fixN. It was found that reduced expression of purL gene or suitable symbiotic expression of purL (such as with the promoter nifH or nifQ) can efficiently establish symbiosis of S. fredii on G. max without the exogenous supplementation of any adenine or purine precursor; at least a minimal level of expression of purL is essential for effective symbiosis with soybean.

Trans-resveratrol, a red wine ingredient, inhibits voltage-activated potassium currents in rat hippocampal neurons.

The red wine ingredient trans-resveratrol was found to exert potent neuroprotective effects in different in vivo and in vitro models. Thus far, the mechanisms underlying the neuroprotection were attributed mainly to its antioxidant properties. The aim of this study was to investigate the actions of trans-resveratrol on voltage-gated K(+) channels, which have been implicated in neuronal apoptosis. Superfusion of trans-resveratrol reversibly inhibited both the delayed rectifier (I(K)) and fast transient K(+) current (I(A)) in rat dissociated hippocampal neurons with IC(50) values of 13.6 +/- 1.0 microM and 45.7 +/- 7.5 microM, respectively. The inhibition on I(K) had a slow onset, was neither voltage dependent nor use dependent. Trans-resveratrol (30 microM) shifted the steady-state inactivation curve of I(K) to the hyperpolarizing direction by 20 mV and slowed down its recovery from inactivation. The inhibition on I(A) was similar to that on I(K), but voltage dependent. Superfusion of trans-resveratrol (30 microM) shifted the steady-state activation curve of I(A) to the depolarizing direction by 17 mV. Intracellular application of trans-resveratrol (30 microM) was ineffective. Based on the comparable effective concentrations, the inhibition of voltage-activated K(+) currents by trans-resveratrol may contribute to its neuroprotective effects.

Donepezil blocks voltage-gated ion channels in rat dissociated hippocampal neurons.

Donepezil (E2020) is a novel cholinesterase inhibitor for the treatment of Alzheimer's disease. Recent studies show that it may act on targets other than acetylcholinesterase in the brain. In the present study, the actions of donepezil on voltage-gated Na+ and K+ channels were investigated in rat dissociated hippocampal neurons. Donepezil reversibly inhibited voltage-activated Na+ current (I(Na)), delayed rectifier K+ current (I(K)) and fast transient K+ current (I(A)). The inhibition of donepezil on I(Na) was dependent on the holding potential. When neurons were held at -100, -80 and -60 mV, the IC50 value was 436+/-19, 291+/-26 and 3.8+/-0.3 microM, respectively. The drug did not affect the activation, fast inactivation of I(Na) and its recovery from fast inactivation. The inhibition of donepezil on I(K) (IC50=78+/-5 microM) was voltage-dependent, whereas that on I(A) (IC50=249+/-25 microM) was voltage-independent. Donepezil caused a significant hyperpolarizing shift of the voltage-dependence of the activation and steady-state inactivation of I(K), without affecting the kinetic properties of I(A). Due to the high concentrations used, the blocking effects of donepezil on the voltage-gated ion channels are unlikely to contribute to the clinical benefits in patients with Alzheimer's disease.

Tetrahydroacridine inhibits voltage-dependent Na(+) current in guinea-pig ventricular myocytes.

AIM: To study the effects of tetrahydroacridine (tacrine) on voltage-gated Na(+) channels in cardiac tissues. METHODS: Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na(+) current was recorded using whole cell voltage-clamp technique. RESULTS: (1) Tacrine reversibly inhibited Na(+) current with an IC(50) value of 120 micromol/L (95 % confidence range: 108-133 micromol/L). (2) The inhibitory effects of tacrine on Na(+) current exhibited both a tonic nature and use-dependence. (3) Tacrine at 100 micromol/L caused a negative shift (about 10 mV) in the voltage-dependence of steady-state inactivation of Na(+) current, and retarded its recovery from inactivation, but did not affect its activation curve. (4) Intracellular application of tacrine significantly inhibited Na(+) current. CONCLUSION: In addition to blocking other voltage-gated ion channels, tacrine blocked Na(+) channels in guinea-pig ventricular myocytes. Tacrine acted as inactivation stabilizer of Na(+) channels in cardiac tissues.

Purification and pharmacological characterization of BmKK2 (alpha-KTx 14.2), a novel potassium channel-blocking peptide, from the venom of Asian scorpion Buthus martensi Karsch.

BmKK2 (alpha-KTx 14.2) is one of the novel short-chain peptides found in molecular cloning of a venom gland cDNA library from Asian scorpion Buthus martensi Karsch. Based upon its amino acid sequence, the peptide was proposed to adopt a classical alpha/beta-scaffold for alpha-KTxs. In the present study, we purified BmKK2 from the venom of B. martensi Karsch, and investigated its action on voltage-dependent K+ currents in dissociated hippocampal neurons from neonatal rats. BmKK2 (10-100 microM) selectively inhibited the delayed rectifier K+ current, but did not affect the fast transient K+ current. The inhibition of BmKK2 on the delayed rectifier K+ current was reversible and voltage-independent. The peptide did not affect the steady-state activation of the current, but caused a depolarizing shift (about 9 mV) of its steady-state inactivation curve. The results demonstrate that BmKK2 is a novel K+ channel-blocking scorpion peptide.

BmTx3B, a novel scorpion toxin from Buthus martensi Karsch, inhibits delayed rectifier potassium current in rat hippocampal neurons.

AIM: To examine the effect of BmTx3B, a novel short-chain peptide isolated from the venom of Asian scorpion Buthus martensi Karsch, on voltage-gated potassium channels. METHODS: Two types of voltage-dependent potassium currents were recorded from dissociated hippocampal neurons of neonatal rat in whole-cell voltage-clamp mode, and separated based upon their kinetic properties. RESULTS: BmTx3B (10-100 micromol/L) selectively inhibited the delayed rectifier potassium current (I(K)), without affecting the fast transient potassium current (I(A)). The inhibition of the peptide on I(K) was reversible, concentration-dependent and voltage-independent. BmTx3B did not affect the steady-state activation and inactivation kinetics of the current. CONCLUSION: The short-chain scorpion peptide BmTx3B selectively blocked the delayed rectifier potassium channel.