Inhibition of neuronal Ca2+ channel currents by the funnel web spider toxin omega-Aga-IA

Ca2+ channel currents were recorded from cultured rat dorsal root ganglion neurons and cerebellar granule cells using the whole-cell recording variant of the patch clamp technique. omega-Aga-IA, a toxin purified from the venom of the American funnel web spider, Agelenopsis aperta, markedly inhibited high threshold barium currents (lBa) when applied at 10 nM concentration. The low threshold T-type current activated at Vc = -30 mV and the outward (Ca2+ channel) current activated at +120 mV were significantly less sensitive to omega-Aga-IA, omega-Conotoxin GVIA (1 microM) inhibited IBa irreversibly. In contrast, the action of omega-Aga-IA was partially reversed 5 min after its removal. The voltage-activated calcium current (ICa) was inhibited by omega-Aga-IA in a manner different from IBa. ICa measured at the end of a 100-msec voltage step command was reduced to a greater extent than the peak current. The residual ICa following application of omega-Aga-IA was a fast transient current. omega-Aga-IA did not inhibit voltage-activated sodium currents from dorsal root ganglion neurons in the absence of tetrodotoxin. omega-Aga-IA abolished the dihydropyridine (+)-202-791-sensitive L-type current component of IBa. We conclude that omega-Aga-IA is a very potent inhibitor of neuronal voltage-activated Ca2+ channel currents and that it may prove to be a useful tool in the characterization and isolation of Ca2+ channels.     

Riluzole inhibits spontaneous Ca2+ signaling in neuroendocrine cells by activation of K+ channels and inhibition of Na+ channels

The neuroprotective drug riluzole has multiple effects on cellular signaling. We found that riluzole rapidly and reversibly inhibited spontaneous Ca2+ oscillations in both immortalized GnRH-secreting hypothalamic neurons (GT1 cells) and in the prolactin and growth-hormone-secreting GH3 cell line. At lower concentrations (100 nm-5 microM), riluzole reduced the amplitude and frequency of spontaneous Ca2+ oscillations, whereas at higher concentrations it abolished spontaneous Ca2+ signaling. Whole-cell current clamp recordings in GH3 cells revealed that riluzole decreased the action potential frequency, amplitude, and duration. Riluzole inhibited voltage-gated Na+ currents, increased iberiotoxin-sensitive voltage-gated K+ currents, and had no effect on voltage-gated Ca2+ currents in GH3 cells. Riluzole also inhibited voltage-gated Na+ currents and increased voltage-gated K+ channels in GT1 cells. The inhibitory effects of riluzole on Ca2+ signaling were blocked by pretreatment with iberiotoxin in GH3 cells, but only partially reduced by iberiotoxin in GT1 cells. These results indicate that riluzole inhibits Ca2+ signaling primarily by activation of K+ channels in GH3 cells, and also by inhibition of Na+ channels in GT1 cells. Riluzole's inhibition of spontaneous excitability and Ca2+ signaling may be involved in its multiple effects on cellular function in the nervous system.     

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.     

Mefenamic acid as a novel activator of L-type voltage-dependent Ca2+ channels in smooth muscle cells from pig proximal urethra

The effects of mefenamic acid and Bay K 8644 on voltage-dependent nifedipine-sensitive inward Ba2+ currents in pig urethral myocytes were investigated by use of conventional whole-cell configuration patch clamp. Mefenamic acid increased the peak amplitude of voltage-dependent nifedipine-sensitive inward Ba2+ current without shifting the position of the current-voltage relationship. Mefenamic acid (300 microM) caused little shift in the activation curve although the voltage dependence of the steady-state inactivation was shifted to more positive potentials by 11 mV in the presence of mefenamic acid. Bay K 8644 (> or = 100 nM) enhanced voltage-dependent nifedipine-sensitive inward Ba2+ currents in a concentration- and voltage-dependent manner, shifting the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction. Bay K 8644 (1 microM) significantly shifted the voltage dependence of the activation curve to more negative potentials by approximately 9 mV although Bay K 8644 caused little shift in the steady-state inactivation curve. These results indicate that mefenamic acid increased voltage-dependent nifedipine-sensitive inward Ba2+ currents through the activation of L-type Ca2+ channels with different kinetics from those of Bay K 8644 in pig urethral myocytes.     

Modulation of voltage-gated Ca2+ current by 4-hydroxynonenal in dentate granule cells

Although recent studies have suggested that dentate granule cells play a key role in hippocampal functions, electrophysiological properties in these cells have not been sufficiently explored. In the present study, modification of voltage-gated Ca2+ channels by 4-hydroxynonenal (4HN), a major aldehydic product of membrane lipid peroxidation, in cultured dentate granule cells was examined using the whole-cell patch clamp technique. When whole-cell voltage clamp was applied, the cells exhibited a high-voltage-activated Ca2+ current, which was totally sensitive to 30 microM Cd2+ and partially sensitive to 2 microM nifedipine. 4HN enhanced the Ca2+ current in these cells. When L-type Ca2+ channels were blocked by application of nifedipine, the enhancement was completely canceled, whereas application of omega-conotoxin-GVIA or omega-agatoxin-IVA, blockers of N- and P/Q-type Ca2+ channels, respectively, had no effect. These results suggest that 4HN modulates L-type Ca2+ channels in the dentate granule cells, and thereby plays a role in the physiological and pathophysiological responses of these cells to oxidative stress.     

Forefront of Na+/Ca2+ exchanger studies: stoichiometry of cardiac Na+/Ca2+ exchanger; 3:1 or 4:1?

The stoichiometry of the Na+/Ca2+ exchanger (NCX) had been generally accepted as 3 Na+:1 Ca2+. However, recently a challenging stoichiometry of 4:1 was proposed. Therefore, using guinea pig ventricular cells, we re-examined the stoichiometry by measuring the reversal potential of the NCX current and intracellular Ca2+ concentrations under the whole-cell voltage clamp. We confirmed that the stoichiometry of NCX is 3:1 not 4:1. In addition, we explored the possible reasons for obtaining erroneous results of a 4:1 stoichiometry.     

Decrease in the Ca2+-activated K+ current of pulmonary arterial smooth muscle in pulmonary hypertension rats

Pulmonary hypertension exhibits acute elevation of vascular tone and hyperreactivity of pulmonary vasculature, which are closely related to patient mortality. In the present study, we investigated the characteristics of membrane currents of isolated pulmonary artery smooth muscle cells taken from rats with monocrotaline-induced pulmonary hypertension. Male Wistar rats were given a single subcutaneous injection of monocrotaline or saline, and then sacrificed between 18 to 21 days after the injection. The membrane currents in the smooth muscle cells from both groups of rats were compared using the whole-cell patch clamp technique. With 0.1 mM EGTA in the pipette, the densities of outward currents in monocrotaline-injected rats were smaller than those in control rats. When EGTA in patch pipettes was increased to 10 mM, the densities of the outward currents in monocrotaline-injected rats were equal to those of control rats. The Ca2+-activated K+ channel blockers (TEA, iberiotoxin) and nisoldipine were less effective on the outward currents of monocrotaline-injected rats. In the current clamp mode, a depolarization of membrane potential induced by 4-aminopyridine was greater in monocrotaline-injected rats than in control rats because of the reduced activity of the Ca2+-activated K+ channels. The Ca2+-activated K+ channels were decreased in pulmonary hypertension. The reduced activity of the currents may be related to the vascular hyperreactivity in pulmonary hypertension.     

Pacific ciguatoxin-1b effect over Na+ and K+ currents,inositol 1,4,5-triphosphate content and intracellular Ca2+ signals in cultured rat myotubes

1. The action of the main ciguatoxin involved in ciguatera fish poisoning in the Pacific region (P-CTX-1b) was studied in myotubes originated from rat skeletal muscle cells kept in primary culture. 2. The effect of P-CTX-1b on sodium currents at short times of exposure (up to 1 min) showed a moderate increase in peak Na+ current. During prolonged exposures, P-CTX-1b decreased the peak Na+ current. This action was always accompanied by an increase of leakage currents, tail currents and outward Na+ currents, resulting in an intracellular Na+ accumulation. This effect is blocked by prior exposure to tetrodotoxin (TTX) and becomes evident only after washout of TTX. 3. Low to moderate concentrations of P-CTX-1b (2-5 nM) partially blocked potassium currents in a manner that was dependent on the membrane potential. 4. P-CTX-1b (2-12 nM) caused a small membrane depolarization (3-5 mV) and an increase in the frequency of spontaneous action potential discharges that reached in general low frequencies (0.1-0.5 Hz). 5. P-CTX-1b (10 nM) caused a transient increase of intracellular inositol 1,4,5-trisphosphate (IP(3)) mass levels, which was blocked by TTX. 6. In the presence of P-CTX-1b (10 nM) and in the absence of external Ca2+, the intracellular Ca2+ levels show a transient increase in the cytoplasm as well as in the nuclei. The time course of this effect may reflect the action of IP(3) over internal stores activated by P-CTX-1b-induced membrane depolarization.     

High affinity block of myocardial L-type calcium channels by the spider toxin omega-Aga-toxin IIIA: advantages over 1,4-dihydropyridines.

The peptide omega-agatoxin IIIA (omega-Aga-IIIA) from venom of the funnel web spider Agelenopsis aperta blocks L-type Ca2+ channels in neurons and myocardial cells with high affinity. We report that omega-Aga-IIIA also blocks whole-cell Ca2+ channel currents in guinea pig atrial myocytes. Although other high affinity blockers of L-type Ca2+ channels are available (such as the 1,4-dihydropyridines), omega-Aga-IIIA is a valuable pharmacological tool; omega-Aga-IIIA is the only known ligand that blocks L-type Ca2+ channels with high affinity at all voltages (IC50 approximately 1 nM) and it causes little or no block of T-type Ca2+ channels, unlike the 1,4-dihydropyridines. We use omega-Aga-IIIA to selectively eliminate L-type Ca2+ currents and we show that felodipine blocks T-type Ca2+ currents. Consequently, the toxin is better than dihydropyridines for separating ionic currents through voltage-dependent Ca2+ channels and defining their physiological function.     

Effects of (+/-)-kavain on voltage-activated inward currents of dorsal root ganglion cells from neonatal rats.

Kava pyrones extracted from pepper Piper methysticum are pharmacologically active compounds. Since kava pyrones exhibit anticonvulsive, analgesic and centrally muscle relaxing properties, the influence of a synthetic kava pyrone, (+/-)-kavain, on voltage-dependent ion channel currents was studied. Effects of (+/-)-kavain on voltage-activated inward currents were analysed in cultured dorsal root ganglion cells derived from neonatal rats. Voltage-activated Ca2+ and Na+ currents were elicited in the whole-cell configuration of the patch clamp technique. Extracellularly applied (+/-)-kavain dissolved in hydrous salt solutions reduced voltage-activated Ca2+ and Na+ channel currents within 3-5 min. As the solubility of (+/-)-kavain in hydrous solutions is low, dimethyl sulfoxide (DMSO) was added to the saline as a solvent for the drug in most experiments. When (+/-)-kavain was dissolved in DMSO, the drug induced a fast and pronounced reduction of both Ca2+ and Na+ currents, which partly recovered within 2-5 min even in the presence of the drug. The present study indicates that (+/-)-kavain reduces currents through voltage-activated Na+ and Ca2+ channels.     

Effects of caffeine and 3-isobutyl-1-methylxanthine on voltage-activated potassium currents in vertebrate neurones and secretory cells.

1. The effects of caffeine and 3-isobutyl-l-methylxanthine (IBMX) on voltage-activated K+ currents were examined by use of patch clamp recording techniques in dissociated chick autonomic ganglion neurones, chick pineal cells and rat anterior pituitary cells. 2. In chick ciliary ganglion neurones, caffeine (0.1-10 mM) produced a robust blockade of delayed rectifier K+ currents (IDR). Blockade was rapid in onset and concentration- and voltage-dependent. Caffeine produced greater inhibition with larger depolarizing voltage pulses. Similar inhibition of IDR was observed in excised outside-out 'maxi-patches' indicating a direct effect on the K+ channels. Caffeine also inhibited IDR in chick sympathetic neurones, chick pineal cells and rat anterior pituitary cells. 3. Application of 10 mM caffeine caused inhibition of transient A-currents (IA) in chick ciliary ganglion neurones. Inhibition of IA was voltage-dependent with greater inhibition observed at more positive command potentials. Application of 1 mM caffeine did not cause inhibition of IA. 4. Application of 1 mM IBMX, a structural analogue of caffeine, caused inhibition of IDR and IA in chick ciliary ganglion neurones. The voltage-dependence of the inhibition of both currents was qualitatively different from that observed with caffeine. The inhibitory effects of 1 mM IBMX and 10 mM caffeine on IDR and IA were additive. 5. Direct inhibition of voltage-activated K+ currents can potentially produce significant secondary effects on intracellular free Ca2+. These results indicate that caution must be used in the design and interpretation of experiments in which millimolar concentrations of caffeine or IBMX are used in pharmacological studies of intracellular Ca2+ dynamics or other second messenger mechanisms.     

Properties of voltage-gated Na+ channels in the human rhabdomyosarcoma cell-line SJ-RH30: conventional and automated patch clamp analysis.

Conventional and automated patch clamp electrophysiology were used to characterise the Na+ current of the SJ-RH30 human rhabdomyosarcoma. In conventional recordings SJ-RH30 cells exhibited a fast activating, fast inactivating Na+ current at potentials positive to -40 mV; in full current-voltage curves maximum current occurred between -20 and -10 mV. Inactivation kinetics at 0 mV were biexponential with time constants of 0.5 and 3.7 ms. Deinactivation at -90 mV also exhibited two kinetic components. Tetrodotoxin (TTX) blocked the Na+ current completely at 1 microM. The NaV 1.4 selective toxin mu-CTx-GIIIB reversibly blocked the Na+ current approximately 60% at 10 microM. Very similar biophysical behaviour was observed in automated patch clamp and conventional recordings. For example, inactivation mid-point was -72+/-2 mV (slope factor 7.2+/-0.2) in automated patch clamp and -74+/-2 mV (slope factor 7.4+/-0.4) with conventional recording. The corresponding values for activation mid-point were -33.2+/-2.4 and -30.3+/-2.7 mV (slope 5.8+/-0.3 and 6.4+/-0.3, respectively). The throughput of the automated method was used to generate additional pharmacological data on inhibition of the Na+ current. TTX inhibited with an IC50 of 23 nM. Mu-CTx-GIIIB also inhibited the channel in a concentration-dependent manner. Inhibition produced by both tetracaine and amitriptyline were shown to be frequency-dependent. Our experiments indicate that the Na+ current of SJ-RH30 cells arises mainly from channels with a phenotype like recombinant NaV 1.4 channels. The suitability of these cells for automated patch clamp suggests they may be useful for higher throughput studies of the interaction of drugs with human skeletal muscle Na+ channels.     

Tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ channels differ in their sensitivity to Cd2+ and Zn2+

The sensitivity of Na+ channels to inhibition by Cd2+ and Zn2+ was studied in 22Na+ uptake experiments after stabilization of an open conformation of the Na+ channels with different neurotoxins and in voltage clamp experiments. Six different cell types of neuronal, cardiac or skeletal muscle origin were surveyed. Three cell types possess Na+ channels that are highly sensitive to tetrodotoxin (TTX) (Kd = 1-5 nM) and three possess Na+ channels that are resistant to TTX (Kd = 0.3-1 microM). The 22Na+ uptake experiments using veratridine or batrachotoxin to activate Na+ channels indicated that TTX-resistant Na+ channels are more sensitive to the inhibitory action of Cd2+ (IC50(Cd2+) = 0.2 mM) and of Zn2+ (IC50(Zn2+) = 50 microM) than TTX-sensitive Na+ channels (IC50(Cd2+) = 5 mM, IC50(Zn2+) = 2 mM). Electrophysiological experiments showed that high concentrations of Cd2+ (IC50 = 2 mM) are necessary to inhibit both TTX-sensitive and TTX-insensitive Na+ channels when the channels are activated by voltage steps. The results suggest that Cd2+ acts competitively with veratridine or batrachotoxin and that the difference in the effects of Cd2+ and Zn2+ on 22Na+ fluxes in TTX-sensitive and TTX-resistant cells is related to differences at the site of action of alkaloid neurotoxins.     

The effect of Huwentoxin-I on Ca(2+) channels in differentiated NG108-15 cells, a patch-clamp study.

Huwentoxin-I (HWTX-I), a 3.75 kDa peptide toxin isolated from the venom of the spider Selenocosmia huwena, was found to be a reversible presynaptic inhibitor by our previous work. Using whole-cell patch clamp methods, we found that HWTX-I had no significant effect on the TTX-sensitive Na(+) current or the delayed rectifier K(+) current (K(r)) in low-serum medium cultured NG108-15 cells, but High-Voltage-Activated Ca(2+) channel expressed in prostaglandin E(1) differentiated NG108-15 cells could be potently inhibited by HWTX-I (EC(50) approximately 100 nM), while it hardly affected low-voltage-activated Ca(2+) channel. Among types of high-voltage-activated Ca(2+) channel, HWTX-I selectively inhibited N-type Ca(2+) channel and had only very weak effect on L-type Ca(2+) channel in prostaglandin E(1) differentiated NG108-15 cells.     

Potassium currents in human freshly isolated bronchial smooth muscle cells.

1. K+ currents were studied in smooth muscle cells enzymatically dissociated from human bronchi, by use of the patch-clamp technique. 2. In whole-cell recordings a depolarization-induced, 4-aminopyridine (4-AP)-sensitive current was observed in only 26 of 155 cells, and in 20 of these 26 cells its amplitude at a test potential of 0 mV was less than 100 pA. 3. In the majority of cells depolarization to -40 mV or more positive potentials induced a noisy outward current which activated within milliseconds and showed almost no inactivation even during a 5 s depolarizing voltage step. This current was insensitive to 4-AP (up to 5 mM) but was strongly inhibited in the presence of tetraethylammonium (TEA, 1 mM), charybdotoxin (ChTX, 100 nM) or iberiotoxin (IbTX, 50 nM) in the bath. The same current was also recorded by the nystatin-perforated patch technique. 4. Single channels with a conductance of about 210 pS were recorded in cell-attached patch, inside-out patch, outside-out patch and whole-cell recording configurations. Channel open state probability in inside-out patches was 0.5 at a membrane potential of 4 +/- 14 mV (mean +/- s.d., n = 13) mV even with a free Ca2+ concentration on the cytosolic side of the patch of less than 0.1 nM. Open state probability increased with depolarization and internal Ca2+ concentration. Single channels could be reversibly blocked by externally applied TEA, ChTX and IbTX. 5. In current-clamp recordings with 100 nM free Ca2+ in the intracellular solution both TEA and ChTX caused substantial concentration-dependent depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

六肽FRCRSFa对大鼠心室肌Na^＋／Ca^＋交换的抑制

AIM: To study the effect of Phe-Arg-Cys-Arg-Ser-Phe-CONH2 (FRCRSFa) on Na+/Ca2+ exchange and its specificity in rat ventricular myocytes. METHODS: Na+/Ca2+ exchange current (INa+/Ca2+) and other currents were measured using whole-cell voltage clamp technique. RESULTS: A concentration-dependent inhibition of hexapeptide FRCRSFa on Na +/Ca2+ exchange was observed in rat ventricular myocytes. IC50 of inward and outward INa+/Ca2+ were 2 and 4 micromol/L, respectively. FRCRSFa 5 micromol/L did not affect L-type Ca2+ current, voltage-gated Na+ current, transient outward K+ current, and inward rectifier K+ current. CONCLUSION: These data indicate that FRCRSFa is an available inhibitor of Na+/Ca2+ exchange with relative selectivity and m ay be valuable for studies of the Na+/Ca2+ exchange in cardiac myocytes.     

Effects of excitatory neurotransmitters on Ca2+ channel current in smooth muscle cells isolated from guinea-pig urinary bladder.

1. A whole-cell voltage clamp technique was used to examine the effects of purinoceptor and muscarinic receptor agonists on voltage-sensitive Ca2+ channels in guinea-pig isolated urinary bladder cells. 2. When the cell membrane was clamped at the holding potential, rapid application of ATP elicited a large inward current in normal solution containing 2.5 mM Ca2+, and reduced the subsequent Ca2+ channel current evoked by a depolarizing pulse (0 mV). Carbachol (CCh) elicited little membrane current, but similarly reduced the Ca2+ current. 3. When purinoceptor agonists were rapidly applied during conditioning depolarizations at +80 mV, an outward current was elicited, and the Ca2+ channel current evoked by the subsequent test potential of 0 mV was not affected. Application of CCh at +80 mV also elicited an outward current, but it reduced the subsequently evoked Ca2+ current. 4. The inhibitory effect of muscarinic agonists on the Ca2+ channel current was attenuated by caffeine (10 mM). 5. In Ca(2+)-free, low-Mg2+ solution, a Na+ current flowing through voltage-dependent Ca2+ channels was evoked by depolarization. This current was not reduced by bath application of purinoceptor agonists (ATP and alpha,beta-methylene ATP). 6. These results suggest that the main effect of purinoceptor stimulation is opening of non-selective cation channels, and that muscarinic stimulation triggers Ca2+ release from intracellular stores. Voltage-sensitive Ca2+ channels are inactivated through an increase in intracellular Ca2+ induced by either activation of purinoceptor or muscarinic receptors.     

Characterization of the palytoxin-induced sodium conductance in frog skeletal muscle.

1. The effects of palytoxin (PTX) on transmembrane potentials and currents of frog skeletal muscle were analyzed by intracellular microelectrode techniques and the double sucrose-gap voltage clamp method. 2. PTX irreversibly depolarized the membrane. The depolarization was Na-sensitive. 3. Under voltage clamp, PTX induced an inward resting current which did not inactivate, was inhibited by external Na+ removal and was a function of external Na concentration. 4. This resting current could be carried either by Na+, Li+, K+ or by guanidinium according to the permeability sequence K+ less than Li+ less than Na+ less than Gua+. 5. The PTX-induced current was only weakly sensitive to tetrodotoxin. It was reversibly and dose-dependently inhibited by amiloride with a one to one stoichiometry and a KD of 0.3 mM. 6. Acidic pH partially inhibited the current induced by PTX which was also highly sensitive to external Cd2+ and La3+. The inhibitory sequence for divalent cations was: Mg2+ less than Ca2+ = Ba2+ = Mn2+ less than Cd2+; with La3+ greater than Cd2+. 7. The amplitude of the PTX-induced I(rest) was markedly reduced in the absence of external Ca2+. 8. PTX induced a Na+ resting conductance in frog skeletal muscle. The size of the channel induced by PTX is larger than the guanidinium ion. External membrane Ca2+ might be a cofactor involved in the mode of action of PTX.     

Caffeine-evoked, calcium-sensitive membrane currents in rabbit aortic endothelial cells.

1. Single cell photometry and whole-cell patch clamp recording were used to study caffeine-induced intracellular Ca2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2. Caffeine (5 mM) evoked a transient increase in [Ca2+]i in fura-2-loaded endothelial cells. Pretreatment of cells with 10 microM ryanodine did not alter resting [Ca2+]i but irreversibly inhibited the caffeine-induced rise in [Ca2+]i. The caffeine-induced increase in [Ca2+]i was not attenuated by the removal of extracellular Ca2+ and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3. Bath application of caffeine evoked a dose- and voltage-dependent outward current. The rate of onset and amplitude of the caffeine-evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine-evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage-sensitive. 4. In the absence of extracellular Ca2+, the amplitude of the caffeine-evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca(2+)-free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward currents (STOCs). 5. Inclusion of heparin (10 micrograms ml-1) in the patch pipette abolished the acetylcholine (ACh)-induced outward current but failed to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine-induced outward current.(ABSTRACT TRUNCATED AT 250 WORDS)     

大鼠酸感受离子通道亚基2a表达质粒的构建及生物学特性考察

目的构建大鼠酸感受离子通道亚基2a(acid-sensingion channel subunit 2a,ASIC2a)的表达质粒并研究其组成的同聚体离子通道的生物学特性。方法使用分子生物学技术构建大鼠ASIC2a亚基表达质粒;通过体外转录技术,使编码ASIC2a亚基的cRNA在爪蟾卵母细胞内表达并在膜表面形成同聚体离子通道;使用双电极电压钳技术研究ASIC2a的生物学特性。结果在注射ASIC2a亚基cRNA的爪蟾卵母细胞上,降低胞外液pH值可诱导出内向电流。H+诱发的ASIC2a内向电流具有稳态失活成分可被氨氯吡咪可逆性阻断,其pH50为5.12。提高胞外Ca2+浓度可降低H+诱发的电流幅度,其IC50为11.98 mmol.L-1。当细胞外液中无Na+时,H+基本上不能诱发出内向电流;当同时去除细胞外液中Na+和K+时,H+可诱发出外向电流。结论成功构建ASIC2a表达质粒;ASIC2a除了对Na+通透外,对K+也有一定的通透性,胞外Ca2+抑制ASIC2a孔道的开放。