大脑皮层神经元NMDA受全的单通道特性

本文和膜片箝技术对机械分离培养的大鼠大脑皮层神经元胞ＮＭＤＡ受体的单通道特性进行了研究,实验用细胞贴附和内面向外两种形式记录单离子通道的活动。电极液内含有ＮＭＤＡ或Ｌ－门冬氨酸时,在皮层神经元上常见电导为３５ｐＳ的离子通道。通道对Ｎａ＾＋,Ｋ＾＋非选择必通透,对Ｃｌ＾－不通透,其平均开放时间和开放概率随超极化程度增大而降低。开放、关闭时间及ｂｕｒｓｔ时程的分布直方图均需双指数拟合。Ｍｇ＾２＋以电压     

大脑皮层神经元NMDA受体的单通道特性

本文用膜片箝技术对机械分离培养的大鼠大脑皮层神经元胞体上的ＮＭＤＡ受体的单通道特性进行了研究,实验用细胞贴附和内面向外两种形式记录单离子通道的活动。电极液内含有ＮＭＤＡ或Ｌ－门冬氨酸时,在皮层神经元上常见电导为３５ｐＳ的离子通道。通道对Ｎａ＋,Ｋ＋非选择性通透,对Ｃｌ－不通透,其平均开放时间和开放概率随超极化程度增大而降低。开放、关闭时间及ｂｕｒｓｔ时程的分布直方图均需双指数拟合。Ｍｇ２＋以电压和浓度依赖性的方式减小通道开放时间,ＡＰＶ能阻断通道活动,温度降低使通道开放时间延长及电流幅度减小。本文结果表明大脑皮层神经元上ＮＭＤＡ受体通道活动自身具有电压依赖性,因此提示ＮＭＤＡ受体通道的正常功能活动可能依赖于某些细胞内调控过程的存在。     

钙激活钾通道在黎芦碱致神经细胞损伤中的作用

目的:观察新生SD大鼠原代培养皮层神经元的钙激活钾通道(Kca)在黎芦碱致神经元损伤模型上的激活、抑制效应.方法:采用细胞贴附和内面向外两种膜片钳单通道记录方法记录新生SD大鼠原代培养皮层神经元的Kca电生理活动.结果:黎芦碱在胞外可激活Kca.在有钙浴液内,细胞贴附式,钳制膜电位 30 mV,加入不同浓度黎芦碱(μmol/L:15、25、50、75),通道开放概率由0.005分别增加为0.014±0.003、0.085±0.010、0.132±0.016、0.059±0.006(P＜0.01),在50μmol/L以内表现出浓度依赖性.无钙浴液内,细胞贴附式膜片上,钳制膜电位 50 mV,随药物浓度(μmol/L)增加为15、40、60、100时,通道开放概率由0.005分别增加为0.014±0.010、0.113±0.006、0.141±0.004、0 295±0.009(P＜0.05).6例内面向外式膜片上,钳制膜电位 40 mV,分别加入黎芦碱25 μmol/L、50μmol/L 3 min后,通道开放概率由0.011±0.008分别增加为0.010±0.010、0.012±0.007(P＞0.05).黎芦碱在胞内Kca开放概率,平均开放/关闭时间,电流幅值均无明显变化.结论:黎芦碱通过影响胞内游离钙水平间接调节Kca,在缺血缺氧早期,胞内游离钙增高激活Kca开放.     

新生大鼠下丘脑神经元L—Ca^2＋通道电流活动特征

目的研究新生大鼠下丘脑神经元L-Ca2+通道单通道特性;Ca2+通道激动剂BayK8644对Ca2+通道单通道特性的影响.方法采用神经元急性分离技术;用膜片钳细胞贴附式记录方式进行研究.结果大鼠下丘脑神经元L-Ca2+通道是一种电导相对较大的Ca2+通道,其电导为(29.5±3.1)pS,平均开放时间(τ0)为0.28ms,平均关闭时间的短关闭时间常数(τc1)为2.91ms,长关闭时间常数(τc2)为53.22ms.此通道几乎不存在时间依赖性失活.BayK8644显著增加通道的开放概率,通道平均开放时间增加为1.61ms.结论下丘脑神经元存在L-Ca2+通道,该通道具有明显电压依赖性,而无显著的时间依赖性.通道特征与文献报道的其它神经元上L-Ca2+通道相似,也有明显不同,显示下丘脑神经元L-Ca2+钙通道的独特性.     

新生大鼠下丘脑神经元L-Ca~(2 )通道电流活动特征

目的 :研究新生大鼠下丘脑神经元L Ca2 通道单通道特性 ;Ca2 通道激动剂BayK 86 44对Ca2 通道单通道特性的影响。方法 :采用神经元急性分离技术 ;用膜片钳细胞贴附式记录方式进行研究。结果 :大鼠下丘脑神经元L Ca2 通道是一种电导相对较大的Ca2 通道 ,其电导为 (2 9.5± 3.1)pS ,平均开放时间 (τ0 )为 0 .2 8ms,平均关闭时间的短关闭时间常数 (τc1)为 2 .91ms,长关闭时间常数 (τc2 )为 5 3.2 2ms。此通道几乎不存在时间依赖性失活。BayK86 44显著增加通道的开放概率 ,通道平均开放时间增加为 1.6 1ms。结论 :下丘脑神经元存在L Ca2 通道 ,该通道具有明显电压依赖性 ,而无显著的时间依赖性。通道特征与文献报道的其它神经元上L Ca2 通道相似 ,也有明显不同 ,显示下丘脑神经元L Ca2 钙通道的独特性     

新生大鼠大脑皮层神经元胞体ATP激活的离子通道及其特性

用膜片箝技术的细胞贴附式和内面向外式,在机械分离的新生ＳＤ大鼠的大脑皮层神经元上,记录到ＡＴＰ激活的离子通道。此通道的电导为３２ｐＳ,对Ｎａ~＋,Ｋ~＋和Ｃｓ~＋无选择性通透,而对Ｃｌ~－不通透。通道开放时间分布直方图多数需用双指数拟合,少数可用单指数拟合;通道关闭时间分布直方图均需用双指数拟合。通道的平均开放时间和开放概率均不依赖于膜电位;但通道的开放概率随着激动剂ＡＴＰ浓度的增加而增大。当电极内液无ＡＴＰ时,无通道电流。六烃季胶和美加明不能阻断此通道。上述结果表明,新生大鼠的大脑皮层神经元胞体可能存在ＡＴＰ激活的离子通道。     

Bay K8644及nifedipine对大鼠下丘脑神经元L-型Ca~(2 )通道特性的影响

采用神经元急性分离和膜片箝技术以及细胞贴附式方式记录通道活动 ,探讨DHP类Ca2 通道激动剂BayK8644及拮抗剂nifedipine对下丘脑神经元L 型Ca2 通道的影响。结果显示 ,在BayK8644作用下 ,通道开放形式发生变化 ,明显可见多级开放 ;通道平均开放时间、平均开放概率显著增加 ,但单通道电导无明显变化。nifedipine的作用与BayK8644相反。结果提示 ,BayK8644对下丘脑神经元L 型Ca2 通道有明显激动作用 ,nifedip ine有显著抑制作用     

Bay K8644及nifedipine对大鼠下丘脑神经元L—型Ca^2＋通道特性的影响

采用神经元急性分离和膜片箍技术以及细胞贴附式方式记录通道活动,探讨DHP类Ca^2 通道激动剂Bay K8644及拮抗剂nifedipine对下丘脑神经元L－型Ca^2 通道的影响,结果显示,在Bay K8644作用下,通道开放形式发生变化,明显可见多级开放;通道平均开放时间,平均开放概况显著增加,但单通道电导无明显变化。nifedipine的作用与Bay K8644相反。结果提示,Bay K8644对下丘脑神经元L－型Ca^2 通道有明显激动作用 nifedipine有显著抑制作用。     

重组人白细胞介素-1β对小鼠骨髓基质细胞K+通道的影响

本文用细胞贴附式和内面向外式的膜片箝单通道记录方式研究了小鼠骨髓基质细胞K 通道的动力学特性以及重组人白细胞介素1β(IL1β)对通道的影响,发现了骨髓基质细胞膜上存在一类性质类似于延迟整流的K 通道的电压依赖性K 通道,单通道电导为167±14pS,通道的动力学特性具显著的电压依赖性。1000U/mlIL1β使单通道电导增加到261±36pS,显著增加通道的开放概率,延长开放时间τo2,缩短关闭时间τc2,并诱导通道出现多级开放。研究表明K 通道的激活参与了细胞因子IL1的生物信号转导。     

过氧化氢诱发的小脑皮质凋亡神经元膜K+通道电流的变化

用新生ＳＤ大鼠小脑皮质细胞进行培养,用Ａｒａ－Ｃ抑制非神经元生长,以Ｈ２Ｏ２诱发神经元凋亡。用膜片细胞贴附式观察了凋亡神经元膜钾离子通道电流的变化,结果表明,凋亡小脑皮质神经元膜Ｋ通道在不同箍位电压下,通道电流（ＩＫ）幅度小于正常神经元的,单位电导小于正常神经元的,通道的平均开放时间,开放概率、短开放及长开放时间常数亦均小于正常神经元的。说明小脑皮质凋亡神经元Ｋ通道活动减弱     

Characterization of a calcium-activated potassium channel in human fibroblasts

The cell-attached and inside-out patch clamp techniques were used to record single-channel currents from human epidermal fibroblasts. A large-conductance channel (320 pS in symmetric 140 mM KCl) with high potassium selectivity was observed in many patches, particularly those located at the borders of the cells. The channel exhibited both voltage and calcium sensitivity and, therefore, was regarded as a variety of the large-conductance calcium-activated potassium channels reported in many preparations. Probability density functions, fitted to histograms of open and closed time durations at 35 degrees C, usually displayed a minimum of one open state and two closed states. However, kinetic analysis by the fractal method suggested more complicated behavior, particularly for the closed condition. It was not uncommon to observe several channels in one patch. This was distinguishable from the presence of subconductances, which were also observed. Although this channel could have many roles, it seems likely to mediate the calcium-activated conductance that underlies the hyperpolarizing response of fibroblasts to mechanical, electrical, or chemical stimuli.     

Single delayed rectifier channels in frog atrial cells. Effects of beta-adrenergic stimulation.

The patch-clamp technique with two pipettes was used to record single delayed K+ channels (cell-attached electrode) and to control the potential and the composition of the intracellular compartment (whole-cell electrode). With 30 microM cAMP in the cell and physiological potassium concentrations inside and outside the patch, a channel carrying an outward current was characterized. Its open probability was very low and the channel was recorded in only 5% of patches under control conditions. Increasing intracellular cAMP increased the probability of finding a channel in a patch 10-fold. The channel had the characteristics expected of a delayed rectifier channel. The time-course of its ensemble average resembled the whole-cell current in the same cell. The current-voltage relationship exhibited inward rectification, with a slope conductance of 20 pS in the linear portion and a reversal potential close to EK. Both the open- and the closed-time distributions were described by the sum of two exponentials, suggesting a complicated gating scheme involving two closed states and two open states. The beta-adrenergic stimulation did not change the conductance of the channel, but increased its probability of opening.     

组胺拮抗剂对小鼠ATP-敏感性钾离子通道的影响

目的观察比较3种组胺拮抗剂对缺血性心肌细胞的ATP-敏感性钾离子通道中的影响。方法利用急性酶解法分离小鼠心室肌细胞。结果组胺拮抗剂pyrilamine、chlorpheniramine及diphenhydramine均可抑制ATP-敏感性钾离子通道的活性,抑制程度为pyrilamine〉chlorpheniramine〉diphenhydramine。组胺对KATP通道活性无影响。结论第一代的组胺拮抗剂（pyrilamine、chlorpheniramine及diphenhydramine）对KATP通道活性有抑制作用,其抑制作用与膜上H1受体无关。     

Blocking kinetics of Cl- channels in colonic carcinoma cells (HT29) as revealed by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)

The blocking effect of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was investigated on single Cl- channels of the cultured human colon carcinoma cells, HT29. In the absence of NPPB, the open-time histogram yielded two time constants, with 0.9 ms and 33 ms, whereas the closed-time distribution could be fitted by a single exponential with a time constant of 0.7 ms. Addition of NPPB in the range 1-50 microM induced brief closing events of the single-channel current. This resulted in a decrease of the long open-time constant to 2.1 ms and in an increase of the closed-time constant to 1.8 ms at 50 microM NPPB concentration. The short open-time constant did not change at low blocker concentration (1 microM), but could no longer be resolved at higher concentrations. The open-state probability decreased from 0.9 (control conditions) to 0.5 at 50 microM NPPB. The Hill plot yielded a Hill coefficient of about 0.7, compatible with one NPPB molecule inhibiting one channel molecule. The kinetics of channel gating are described by a sequential model with one closed and two open states. Since in the presence of NPPB no additional time constant appeared in the time histograms, we assumed the same kinetic scheme as under control conditions, and hypothesize that NPPB has an influence on rate constants.     

A non-selective cation channel in the apical membrane of cultured A6 kidney cells.

The patch-voltage clamp technique was used to investigate the characteristics of a non-selective cation channel (NSCC) identified in the apical membrane of cultured A6 toad kidney cells. The NSCC was present in cell-attached and inside-out membrane patches. The characteristics of this NSCC are as follows: (a) linear current-voltage relationship with a channel conductance of 21 +/- 2 pS; (b) a low selectivity between Na+ and K+ (1.5:1); (c) a high selectivity of Na+ to Cl- (greater than 45:1); (d) this channel has a single open state and two closed states; (e) the open-time constant and the second closed-time constant of this channel are voltage dependent; and (f) this NSCC is insensitive to amiloride (10(-7) M). We conclude that the NSCC resembles previously described non-selective cation channels. The NSCC of the apical membrane of A6 cells may aid in the movement of Na+ and K+ in response to varying ionic concentrations across the apical membrane.     

Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.     

Permeation and Gating of an Inwardly Rectifying Potassium Channel : Evidence for a Variable Energy Well

Permeation, gating, and their interrelationship in an inwardly rectifying potassium (K⁺) channel, ROMK2, were studied using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode. The gating kinetics of ROMK2 were well described by a model having one open and two closed states. One closed state was short lived (∼1 ms) and the other was longer lived (∼40 ms) and less frequent (∼1%). The long closed state was abolished by EDTA, suggesting that it was due to block by divalent cations. These closures exhibit a biphasic voltage dependence, implying that the divalent blockers can permeate the channel. The short closures had a similar biphasic voltage dependence, suggesting that they could be due to block by monovalent, permeating cations. The rate of entering the short closed state varied with the K⁺ concentration and was proportional to current amplitude, suggesting that permeating K⁺ ions may be related to the short closures. To explain the results, we propose a variable intrapore energy well model in which a shallow well may change into a deep one, resulting in a normally permeant K⁺ ion becoming a blocker of its own channel.     

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.     

The gating of single calcium-dependent potassium channels is described by an activation/blockade mechanism

Single calcium dependent potassium channels from cultured rat myoballs have been studied with the patch clamp technique, and current records subjected to statistical analysis. From the dependence of the mean open state probability on the internal calcium concentration, two calcium ions are required to open the channel. The open state and closed state lifetime distributions reveal that the usual activation model is not applicable to these channels. They are consistent with a two step gating mechanism that involves both activation by calcium and blockade by a calcium-sensitive gate.