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1.
Effects of changing cytosolic free Mg(2+) concentration on L-type Ca(2+) (I(Ca)) and Ba(2+) currents (I(Ba)) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8mM [Mg(2+)] ([Mg(2+)](p)) buffered with 30mM citrate and 10mM ATP. Increasing [Mg(2+)](p) from 0.2 to 1.8mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (I(T)) protocols, respectively. Increasing [Mg(2+)](p) shifted the V(M) for half inactivation by -20mV but dramatically decreased I(Ca) amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I(T) amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg(2+)](p) might also inhibit permeation through open Ca(2+) channels at positive V(M). 相似文献
2.
Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels
Riccardo Olcese Ramón Latorre Ligia Toro Francisco Bezanilla Enrico Stefani 《The Journal of general physiology》1997,110(5):579-589
Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K+ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation. 相似文献
3.
目的 :探讨柯萨奇B3病毒 (coxsackievirusB3,CVB3)对大鼠心肌细胞离子通道的影响 ,以了解病毒感染导致细胞电生理活动异常的机理。方法 :酶消化法获得单个心肌细胞后利用膜片钳全细胞电流记录技术观察CVB3对L型钙通道电流、钠通道电流、外向钾电流和内向整流性钾电流的影响。结果 :CVB3感染使L型钙通道电流、外向钾电流增加 ,内向整流性钾电流减小 ,对钠通道电流无明显影响。结论 :CVB3对L型钙通道电流、外向钾电流和内向整流性钾电流的影响可能是病毒感染后细胞损伤和产生异常电活动的原因。 相似文献
4.
Inactivation of L-type Ca channels (LTCC) is regulated by both Ca and voltage-dependent processes (CDI and VDI). To differentiate VDI and CDI, several experimental and theoretical studies have considered the inactivation of Ba current through LTCC (IBa) as a measure of VDI. However, there is evidence that Ba can weakly mimic Ca, such that IBa inactivation is still a mixture of CDI and VDI. To avoid this complication, some have used the monovalent cation current through LTCC (INS), which can be measured when divalent cation concentrations are very low. Notably, INS inactivation rate does not depend on current amplitude, and hence may reflect purely VDI. However, based on analysis of existent and new data, and modeling, we find that INS can inactivate more rapidly and completely than IBa, especially at physiological temperature. Thus VDI that occurs during IBa (or ICa) must differ intrinsically from VDI during INS. To account for this, we have extended a previously published LTCC mathematical model of VDI and CDI into an excitation-contraction coupling model, and assessed whether and how experimental IBa inactivation results (traditionally used in VDI experiments and models) could be recapitulated by modifying CDI to account for Ba-dependent inactivation. Thus, the view of a slow and incomplete INS inactivation should be revised, and INS inactivation is a poor measure of VDI during ICa or IBa. This complicates VDI analysis experimentally, but raises intriguing new questions about how the molecular mechanisms of VDI differ for divalent and monovalent currents through LTCCs. 相似文献
5.
Ming-Jai Su PhD Wen-Pin Chen Tase-Yueh Lo Tian-Shung Wu 《Journal of biomedical science》1999,6(6):376-386
We investigated the electrophysiological effect and antiarrhythmic potential of cinnamophilin (Cinn), a thromboxane A2 antagonist isolated fromCinnamomum philippinense, on rat cardiac tissues. Action potential and ionic currents in single rat ventricular cells were examined by current clamp or voltage clamp in a whole-cell configuration. In 9 episodes of ischemia-reperfusion arrhythmia, 10 µM Cinn converted 6 of them to normal sinus rhythm. Cinn suppressed the maximal rate of rise of the action potential upstroke (Vmax) and prolonged the action potential duration at 50% repolarization (APD50). Voltage clamp study showed that the suppression of Vmax by Cinn was associated with an inhibition of sodium inward current (INa, IC50=10.0 ± 0.4 µM). At 30 µM, V1/2 for the steady-state inactivation curve of INa was shifted from –84.1 ± 0.2 to –93.0 ± 0.5 mV. Cinn also reduced calcium inward current (ICa) dose-dependently with an IC50 value of 9.5 ± 0.3 µM. Cinn (10 µM) reduced the ICa with a negative shift of V1/2 for the steady-state inactivation curve of ICa from –32.2 ± 0.3 to –50.7 ± 0.4 mV. The prolongation of APD50 was associated with an inhibition of the integral of potassium outward current with IC50 values between 4.8 and 7.1 µM. At 10 µM, Cinn reduced INa without a negative shift of its voltage-dependent steady-state inactivation curves. The inhibition of transient outward current (Ito) by Cinn (3–30 µM) was associated with an acceleration of its time constant of inactivation and negative shift of its potential-dependent steady-state inactivation curves. The equilibrium dissociation constant (Kd) of Cinn to inhibit open state Ito channels, as calculated from the time constant of developing block, was 18.3 µM. The time constant of recovery of Ito from inactivation state was unaffected by Cinn. The rate constant for the relief from the depolarization-dependent block of Ito was calculated to be 23.9 ms. As compared with its effect on Ito, Cinn exerted about half the potency to block INa and ICa. These results indicate that the inhibition of INa, ICa and Ito may contribute to the antiarrhythmic activity of Cinn against ischemia-reperfusion arrhythmia. 相似文献
6.
Whole-cell patch recording techniques were used to analyze spontaneous electrical activity in cerebellar Purkinje cells acutely isolated from postnatal rats. Spontaneous activity was present in 65% of the cells examined, and it included simple and complex firing patterns which persisted under conditions that eliminated residual or reformed synaptic contacts. Under voltage clamp, both spontaneous and quiescent cells displayed similar voltage-dependent conductances. Inward current was carried by Na+ through tetrodotoxin (TTX)-sensitive channels and by Ca2+ through P-type and T-type Ca channels. P-type current was present in all cells examined. T-type current was found in <50%, and it did not correlate with spontaneous activity. We found no evidence of a transient (A-type) potassium current or hyperpolarization-activated cationic current in either spontaneous or quiescent cells. Spontaneous activity did correlate with a lower activation threshold of the Na current, resulting in substantial overlap of the activation and inactivation curves. TTX reduced the holding current of spontaneous cells clamped between −50 and −30 mV, consistent with the presence of a Na "window" current. We were unable, however, to measure a persistent component of the Na current using voltage steps, a result which may reflect the complex gating properties of Na channels. An Na window current could provide the driving force underlying spontaneous activity, as well as plateau potentials, in Purkinje cells. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 18–32, 1997 相似文献
7.
培养的新生大鼠交感神经元膜电学性质及电压门控通道的膜片箝研究 总被引:5,自引:0,他引:5
在培养的新生大鼠颈上神经节交感神经元标本上,用全细胞电流箝及电压箝技术观察记录了交感神经元膜电学参数及电压门控性通道电流。用全细胞电流箝测得下列平均值:静息电位,-51±6mV;输入阻抗,1432±389MΩ;时间常数,130±32ms;动作电位幅度,96±10mV,超射值,42±6mV。培养早期即可记录到自发或诱发的动作电位,大多数动作电位后跟随快或慢的后超极化电位。在电压箝状态下可分别记录到电压门控钠、钾、钙通道电流。钾电流以延迟整流型为主,钙电流只有高电压激活,缓慢失活的L或N型,未能记录到低电压激活,快速失活的T型钙电流。 相似文献
8.
Cerebellar Purkinje neurons in vivo exhibit high frequency and multi-spike action potentials with transient (INaT), resurgent (INaR) and persistent (INaP) Na+ currents arising from voltage-gated Na+ channels, which play important roles in shaping the action potentials and electrical activity of these cells. However, little is known about Na+ channel expression in cultured Purkinje neurons despite the use of in vitro approaches to study these cells. Therefore, GFP-expressing Purkinje neurons isolated from transgenic mice were analysed after four weeks in culture, when, coincident with distinct axonal and dendritic morphologies, cultured Purkinje neurons exhibited dendrite-specific MAP2 expression characteristic of polarized neurons. In cell-attached patch clamp recordings, Na+ currents occurred at significantly higher frequencies and amplitudes in patches from the soma and axon than from dendrites, similar to the polarized distribution observed in vivo. INaT, INaR and INaP Na+ currents with properties similar to those observed in acutely isolated Purkinje neurons were detected in nucleated outside-out patches from cultured Purkinje cells. RT-PCR analysis detected Nav1.1, Nav1.2 and Nav1.6, but not Nav1.3, Nav1.4, Nav 1.5 or Nav1.8 Na+ channel alpha subunit gene expression in cultured Purkinje neurons, as observed in vivo. Together, the results indicate that key aspects of Na+ channel expression in mature Purkinje neurons in vivo occur in vitro. 相似文献
9.
《Chronobiology international》2013,30(4):453-469
Rhythmic activity of single cells or multicellular networks is a common feature of all organisms. The oscillatory activity is characterized by time intervals of several seconds up to many hours. Cellular rhythms govern the beating of the heart, the swimming behavior of sperm, cycles of sleep and wakefulness, breathing, and the release of hormones. Many neurons in the brain and cardiac cells are characterized by endogenous rhythmic activity, which relies on a complex interplay between several distinct ion channels. In particular, one type of ion channel plays a prominent role in the control of rhythmic electrical activity since it determines the frequency of the oscillations. The activity of the channels is thus setting the “pace” of the oscillations; therefore, these channels are often referred to as “pacemaker” channels. Despite their obvious important physiological function, it was not until recently that genes encoding pacemaker channels have been identified. Because both hyperpolarization and cyclic nucleotides are key elements that control their activity, pacemaker channels have now been designated hyperpolarization-activated and cyclic nucleotide–gated (HCN) channels. The molecular identification of the channels and the upcoming studies on their properties in heterologous systems will certainly enhance our understanding of “pacemaking” in physiological systems. This review gives a brief insight into the physiological importance of these channels and sums up what we have learned since the first cloning of genes succeeded (for recent reviews, see also Clapham 1998; Luüthi and McCormick 1998a; Biel et al. 1999; Ludwig, Zong, Hofmann, et al. 1999; Santoro and Tibbs 1999). (Chronobiology International, 17(4), 453–469, 2000) 相似文献
10.
Shiji Wang Yu Wang Xin Guo Yingji Li Li Yan Chengxi Wei Ming Zhao 《Journal of cellular physiology》2019,234(3):2021-2030
Gram-negative bacteria-induced infections result in fever, arrhythmia, and even death. Lipopolysaccharide (LPS), a constituent of bacteria, leads to an inflammatory response under sepsis and increase arrhythmogenesis. This study analyzed the effects on human embryonic stem cell-differentiated cardiomyocytes (HIPSC-CMs) exposed to LPS. A whole cell patch clamp was used to record the action potential (AP) and ionic currents with or without different doses of LPS in HIPSC-CMs. Compared with the control, a different dose (0.04, 0.2, 1, and 5 µg/ml) of LPS-treated HIPSC-CMs resulted in a longer AP duration. The IC50 of sodium channel current was 1.254 µg/ml, L-type calcium channel current was 5 µg/ml, and Ik channel currents were 1.254 µg/ml. LPS-treated HIPSC-CMs showed a lower sodium channel current, L-type calcium channel current, and Ik channel currents. Furthermore, the expressions of Nav1.5 were decreased, and L-Ca, Kv11.1, and Kv7.1 were increased in LPS-treated HIPSC-CMs. LPS-induced arrhythmogenesis was related to the electrophysiological characteristics of sodium channel current, L-type calcium channel current, and Ik channel currents. These results suggest a potential mechanism of cardiomyocyte injury in endotoxemia. 相似文献
11.
Bett GC Morales MJ Strauss HC Rasmusson RL 《Biochemical and biophysical research communications》2006,340(4):1167-1177
Rapidly activating Kv4 voltage-gated ion channels are found in heart, brain, and diverse other tissues including colon and uterus. Kv4.3 can co-assemble with KChIP ancillary subunits, which modify kinetic behavior. We examined the affinity and use dependence of nifedipine block on Kv4.3 and its modulation by KChIP2b. Nifedipine (150 microM) reduced peak Kv4.3 current approximately 50%, but Kv4.3/KChIP2b current only approximately 27%. Nifedipine produced a very rapid component of open channel block in both Kv4.3 and Kv4.3/KChIP2b. However, recovery from the blocked/inactivated state was strongly sensitive to KChIP2b. Kv4.3 Thalf,recovery was slowed significantly by nifedipine (120.0+/-12.4 ms vs. 213.1+/-18.2 ms), whereas KChIP2b eliminated nifedipine's effect on recovery: Kv4.3/KChIP2b Thalf,recovery was 45.3+/-7.2 ms (control) and 47.8+/-8.2 ms (nifedipine). Consequently, Kv4.3 exhibited use-dependent nifedipine block in response to a series of depolarizing pulses which was abolished by KChIP2b. KChIPs alter drug affinity and use dependence of Kv4.3. 相似文献
12.
目的:观察人参皂甙Rd(ginsenoside Rd)对大鼠坐骨神经分支选择性损伤(spared sciatic nerve injury,SNI)引起的痛敏的影响及其作用机制。方法:坐骨神经分支选择性损伤术后7天,观察腹腔注射不同浓度人参皂甙Rd后大鼠后足的机械性缩足反应阈值(paw withdrawl mechanical threshold,PWMT)的变化;在术后7天,急性分离并取出大鼠腰4和腰5段背根节,对整节DRG上的中小型神经元运用全细胞膜片钳技术进行记录。结果:坐骨神经分支选择性损伤术后7天,大鼠出现明显的机械性痛敏,腹腔注射5 mg/ml和10 mg/ml的人参皂甙Rd能剂量依赖性的翻转大鼠机械性痛敏;坐骨神经分支选择性损伤能明显地增大SNI大鼠DRG中小型神经元上的钠电流以及减小电压依赖性钾电流,而100μM人参皂甙Rd能有效翻转该钠、钾电流的变化。结论:人参皂甙Rd能有效地改善坐骨神经分支选择性损伤引起的机械性痛敏,其机制可能与人参皂甙Rd明显地调节SNI大鼠DRG中小型神经元上的电压依赖性钠、钾电流有关。 相似文献
13.
Activation kinetics of the sodium and potassium conductances were re-examined in fresh axons of Loligo forbesi exhibiting very little if any potassium accumulation and a very small leak conductance, special attention being paid to the initial lag phase which precedes the turning-on of the conductances. The axons were kept intact and voltage-clamped at 2–3°C.In all cases, the rising phase of the currents could be fitted with very good accuracy using the Hodgkin-Huxley (1952) equations although, in most cases, the turning-on of the conductance did not coincide with the beginning of the depolarizing test pulse. The delay which separates the change in potential and the turning-on of current (the activation delay) was analyzed quantitatively for different prepulse and pulse potentials. The measured activation delay differed significantly from the delay predicted by the original HH equations. This difference (the non-HH delay) varied with prepulse and pulse potentials. For the potassium current, the relationship between the non-HH delay and pulse potential for a constant prepulse was bell shaped, the maximum value (0.7 ms for a prepulse to –80 mV) being reached for about 0 mV For this same current, the relationship between the non-HH delay and the prepulse potential for a constant pulse potential was sigmoidal, starting from a minimum value of around 0.5 ms at –100 mV and rising to 5 ms at –15 mV Essentially similar results were obtained for the sodium current although the non-HH delay was three to five times smaller and the dependency upon prepulse potential not significant. These results are in agreement with previous observations on squid axons and frog nodes of Ranvier and suggest that the opening of an ionic channel is preceded by a short but essential voltage-dependent conformational change of the channel protein.
Offprint requests to: Y. Pichon 相似文献
14.
目的: 建立新生大鼠大脑皮层、海马细胞及交感神经元细胞的培养方法及其钠、钾和钙通道的膜片钳全细胞记录技术.方法: 取出生1~3 d的大鼠大脑皮层、海马及交感神经节,用胰蛋白酶(0.125%)消化组织并分离出神经细胞,种植在涂有多聚赖氨酸的35 mm培养皿中,用高糖的DMEM培养液培养,一周后,镜下可见神经细胞壁光滑、完整,周围有明亮的光润,在高倍倒置显微镜下可见到完整的细胞核及均匀的胞浆,细胞间形成良好的突触连接,可用于细胞膜片钳记录.结果: 用胰蛋白酶消化分离培养的神经细胞,功能状态良好,在膜片钳全细胞记录中,易形成细胞与记录电极间的高阻抗封接,可分别记录到INa、IA、IK和ICa.结论:在神经系统电生理学研究中,此方法可应用于中枢神经系统不同脑区培养以及钠、钾和钙通道电流的记录. 相似文献
15.
16.
[3H]Ouabain binding in frog and toad urinary bladder was investigated by short-circuit current (SCC), scintillation counting and authoradiographic techniques. SCC data and analysis of tissue digests following serosal exposure to ouabain showed that ouabain binding and inhibition of Na+ transport was completely reversible in toad bladder whereas, in frog bladder, [3H] ouabain was tightly bound and Na+ transport remained suppressed even after a 60-min washout. Mucosal exposure of frog bladder to [3H]ouabain or serosal exposure after preincubation with unlabeled ouabain led to a marked reduction in binding. Specificity of binding was assessed further by adjusting the concentration of cecrtain (Na+?K+)-ATPase ligands (K+, ATP) to levels known to reduce ouabain binding. High K+ concentrations and depletion of endogenous ATP by incubation under anoxic conditions resulted in a significant drop in [3H]ouabain binding. Autoradiographic analysis showed that grains are localized primarily to the basolateral plasma membranes of the granular cells, providing direct morphological evidence for the location of Na+ pumps at these sites. Although autoradiographs did not provide sufficient resolution to rule out unequivocally ouabain binding to the mitochondria-rich cell, morphological evidence suggests that grain densities are significatly higher between adjacent granular cells than between granular cell-mitochondria-rich cell interfaces. 相似文献
17.
J. D. Angstadt 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1999,184(1):49-61
Current-clamp studies of cultured leech Retzius cells revealed inward rectification in the form of slow voltage sags in response
to membrane hyperpolarization. Sag responses were eliminated in Na+-free saline and blocked by Cs+, but not Ba2+. Voltage clamp experiments revealed a Cs+-sensitive inward current activated by hyperpolarization negative to −70 mV. Cs+ decreased the frequency of spontaneous impulses in Retzius cells of intact ganglia. Plateau potentials were evoked in Retzius
cells following block of Ca2+ influx with Ni2+ and suppression of K+ currents with internal tetraethylammonium. Plateau potentials continued to be expressed with Li+ as the charge carrier, but were eliminated when Na+ was replaced with N-methyl-d-glucamine. A persistent Na+ current with similar pharmacology that activated positive to −40 mV and reached its peak amplitude near −5 mV was identified
in voltage-clamp experiments. Inactivation of the persistent Na+ current was slow and incomplete. The current was revealed by slow voltage ramps and persisted for the duration of 5-s voltage
steps. Persistent Na+ current may underlie Na+-dependent bursting recorded in neurons of intact ganglia exposed to Ca2+-channel blockers.
Accepted: 22 September 1998 相似文献
18.
The conformational changes associated with activation gating in Shaker potassium channels are functionally characterized in patch-clamp recordings made from Xenopus laevis oocytes expressing Shaker channels with fast inactivation removed. Estimates of the forward and backward rates for transitions are obtained by fitting exponentials to macroscopic ionic and gating current relaxations at voltage extremes, where we assume that transitions are unidirectional. The assignment of different rates is facilitated by using voltage protocols that incorporate prepulses to preload channels into different distributions of states, yielding test currents that reflect different subsets of transitions. These data yield direct estimates of the rate constants and partial charges associated with three forward and three backward transitions, as well as estimates of the partial charges associated with other transitions. The partial charges correspond to an average charge movement of 0.5 e0 during each transition in the activation process. This value implies that activation gating involves a large number of transitions to account for the total gating charge displacement of 13 e0. The characterization of the gating transitions here forms the basis for constraining a detailed gating model to be described in a subsequent paper of this series. 相似文献
19.
Harakawa S Inoue N Saito A Doge F Nagasawa H Suzuki N Martin DE 《Bioelectromagnetics》2004,25(3):204-210
The effect of a 60 Hz electric field (EF) on alteration of cytosolic free Ca2+ level ([Ca2+]c) was examined in mouse splenocytes stimulated by lectins, namely concanavalin A (ConA) or phytohemagglutinin. In order to understand the role of EF on alterations in [Ca2+]c and to determine whether EF exposure increased cell mortality the splenocytes were cultured under the 60 Hz EFs producing current densities of 6 or 60 microA/cm2 for 30 min or 24 h. Cell mortality was less than 2% in experimental all conditions. [Ca2+]c in the splenocyte was not changed by the 6 microA/cm2 exposure alone, while a lectin-induced [Ca2+]c elevation in the EF exposed cells was significantly higher than that of the sham exposed cells (P <.05: ANOVA, P <.05: paired t-test). Moreover, the enhanced increase of [Ca2+]c in the EF exposed, lectin stimulated cells was only observed in the presence of extracellular Ca2+. The EF dependent upregulation of [Ca2+]c persisted after EF exposure (P <.05: paired t-test). The results clearly indicate that Ca2+ influx across the plasma membrane is responsible for the enhanced increase of [Ca2+]c in the EF exposed, lectin stimulated cells and that EF has persistent effect on the cells. Although the precise mechanisms of the EF dependent upregulation of [Ca2+]c is not fully elucidated, the present results demonstrate that the 60 Hz EF (6 microA/cm2) affects [Ca2+]c during cell activation via a Ca2+ influx pathway induced by lectin stimulation. 相似文献
20.
The Na(v)1.6 voltage-gated sodium channel has been implicated in the generation of resurgent currents in cerebellar Purkinje neurons. Our data show that resurgent sodium currents are produced by some large diameter dorsal root ganglion (DRG) neurons from wild-type mice, but not from Na(v)1.6-null mice; small DRG neurons do not produce resurgent currents. Many, but not all, DRG neurons transfected with Na(v)1.6 produce resurgent currents. These results demonstrate for the first time the intrinsic ability of Na(v)1.6 to produce a resurgent current, and also show that cell background is critical in permitting the generation of these currents. 相似文献