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     

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。     

NMDA受体与中枢神经系统发育

中枢神经系统兴奋性氨基酸离子型受体－ＮＭＤＡ受体,是由ＮＭＤＡＲ１和ＮＭＤＡＲ２两个亚单位共同构成的受体通道复合体。ＮＭＤＡ受本激活后可引起神经元细胞对Ｎａ＾＋,Ｋ＾＋和Ｃａ＾２＋通透性增强,产生兴奋性突触后电位,在中枢神经发育的过程中,ＮＭＤＡ受体通过不同亚型的选择性表达,改变自身的结构和功能,进而影响ＮＭＤＡ受体介导的Ｃａ＾２＋内流,调节神经元内Ｃａ＾２＋依赖的第二信使系统,最终实现对中枢神经     

NO和CO加强海马活动依赖性长时程增强效应

ＮＯ和ＣＯ加强海马活动依赖性长时程增强效应长时程增强（ｌｏｎｇ－ｔｅｒｍｐｏｔｅｎｔｉａｔｉｏｎ,ＬＴＰ）效应是突触可塑性的一种形式,突触可塑性被认为与学习记忆密切相关。海马ＣＡｌ区ＬＴＰ的产生需要Ｃａ２＋通过ＮＭＤＡ受体离子通道进入细胞内;ＬＴＰ的...     

强啡肽对脊髓原生型与诱生型一氧化氮合成酶活性、分布和基因表达的影响及机制

蛛网膜下腔注射（ｉ．ｔ．）强啡肽Ａ１－１７（Ｄｙｎ）引起剂量依赖性后肢和尾部瘫痪及甩尾甩足抑制。脊髓背角（侧）ＮＭＤＡ受体和ＮＯＳ／ＮＯ功能活性下降可能与Ｄｙｎ镇痛作用有关,脊髓腹角（侧）ＮＭＤＡ受体－Ｃａ２＋－ＮＯＳ／ＮＯ通路过度激活及ｃ－ｆｏｓ高表达可能与Ｄｙｎ致脊髓损伤（ＳＣＩ）作用有关。在Ｄｙｎ致ＳＣＩ机制中,过量ＮＯ（细胞水平）具有神经毒性作用,脑源性ＮＯＳ主要在早期,诱生型ＮＯＳ主要在后期起作用,而内皮细胞源性ＮＯＳ和适量ＮＯ（血管水平）可能具有保护作用。在原代培养脊髓神经元中,高浓度Ｄｙｎ可通过ＮＭＤＡ受体和κ受体直接引起细胞内Ｃａ２＋超负荷,低浓度Ｄｙｎ只通过κ受体抑制高钾刺激性Ｃａ２＋内流     

强啡肽对脊髓原生型与诱生型一氧化氮合成酶活性,分布和基因表达的影响

蛛网膜下腔注射强啡肽Ａ１－１７引起剂量依赖性后肢和尾部瘫痪及甩尾甩足抑制。脊髓背角（侧）ＮＭＤＡ受体和ＮＯＳ／ＮＯ功能活性下降可能与Ｄｙｎ镇痛作用有关,脊髓腹角（）ＮＭＤＡ受体－Ｃａ＾２＋－ＮＯＳ／ＮＯ通路过度激活及ｃ－ｆｏｓ高表达可能与Ｄｙｎ致脊髓损伤作用有关。     

体外培养新生大鼠大脑皮层星形神经元钾通道的特性及膜外钾离子浓度对其影响

用膜片钳技术中的细胞贴附方式和内面向外方式,首次在新生大鼠大脑皮层星形神经元胞体膜上记录到一类电压依赖性钾通道。此通道可被２０ｍｍｏｌ／ＬＴＥＡ,５ｍｍｏｌ／ＬＢａ２＋,１４０ｍｍｏｌ／ＬＣｓ＋阻断,不受２０ｍｍｏｌ／Ｌ４—ＡＰ影响,其激活不依赖Ｃａ２＋。膜外钾离子浓度对通道的特性有显著的影响,逆转电位随［Ｋ＋］０的增大而增大,并表现出一定的饱和现象,两者的对数呈线性关系;同一驱动电位下,平均开放时间和开放概率随［Ｋ＋］０的增大而增大,平均关闭时间的变化则相反。     

大鼠脑发育不同时期学习记忆的变化及与NMDA受体通道动力 …

目的：研究大鼠脑发育不同时期学习记忆的变化及与ＮＭＤＡ受体通道动力学特性的关系。方法：采用学习记忆行为和离子通道动力学特性测定相结合的方法。结果：在爬杆主动回避反应中,发育早期大鼠习得和保持能力场明显强于成年大鼠。同时,发育早期大鼠训练后ＮＭ受体ｐＳ导电,而且３５ＰＳ通道开放时间和开放概率增加,３５ＰＳ通道长开放成份增多,有长ｃｌｕｓｔｅｒ开放而砀上大鼠２０Ｓ,３５ＰＳ通道关闭时间常数明显长于年龄     

海马神经元膜片与细胞分离前后nAChR通道特性的改变

目的和方法：在新生大鼠海马神经元上,用膜片钳技术研究了乙酰胆碱受体单通道特性,离子选择性及细胞内物质对通道活动的影响。结果：该通道对单价阳离子通透,对Ｎａ＾＋和Ｋ＾＋的通透性相近,但不通透Ｃｌ＾－。     

大鼠内侧隔核、斜角带中NOS与ChAT、NGF-R、AChE的共存神经元

用酶组织化学和免疫组织化学双标技术,观察了正常ＳＤ大鼠基底前脑内侧隔核（ＭＳ）、斜角带垂直支（ＶＤＢ）和水平支（ＨＤＢ）中ＮＯＳ阳性神经元的形态和分布及ＮＯＳ与胆碱能神经元标志物ＣｈＡＴ、ＮＧＦ受体（ＮＧＦ－Ｒ）和ＡＣｈＥ之间的共存关系。结果发现,ＭＳ、ＶＤＢ和ＨＤＢ的头端ＮＯＳ阳性神经元较多、胞体较大、突起多,尾端ＮＯＳ阳性神经元数目较少、胞体较小、突起少而短。ＮＯＳ＋ＣｈＡＴ双标神经元占ＮＯＳ阳性神经元总数的９０％,占ＣｈＡＴ阳性神经元总数的３９％;ＮＯＳ＋ＮＧＦ－Ｒ双标神经元占ＮＯＳ阳性神经元总数的８３％,占ＮＧＦ－Ｒ阳性神经元总数的４０％;ＮＯＳ＋ＡＣｈＥ双标神经元占ＮＯＳ阳性神经元总数的９６％,占ＡＣｈＥ阳性神经元总数的３９％。这些结果为研究Ａｌｚｈｅｉｍｅｒ＇ｓｄｉｓｅａｓｅ病理过程中基底前脑隔区胆碱能神经元退变与ＮＯ的关系提供了形态学依据。     

The actions of intermediate and long-chain n-alkanols on unitary NMDA currents in hippocampal neurons.

The actions of the n-alkanols butanol, pentanol, and octanol on unitary currents passing through N-methyl-D-aspartate (NMDA) ion channels have been studied in cultured CA1 hippocampal neurons. The cell-attached patch clamp method, with L-homocysteic acid included in the patch pipette, was used to record single channel NMDA currents at the cell resting potential or for hyperpolarizing patch potentials. With the n-alkanols added to the bath solution, the mean open times for the NMDA channel were diminished and the channel conductance was unchanged. A decrease in mean open time to about 70% of control value was found with butanol (3 mM), pentanol (1 mM), and octanol (0.02 mM). In addition the n-alkanols had small effects to decrease the frequency of channel openings and to increase the amplitude of the unitary currents. The effects of the alcohols on intracellular calcium levels, during NMDA applications, were also measured using the fluorescent dye FURA II.     

Gating kinetics of potassium channel in rat dorsal root ganglion neurons analyzed with fractal model

The kinetics of ion channels have been widely modeled as a Markov process. In these models it is assumed that the channel protein has a small number of discrete conformational states and kinetic rate constants connecting these states are constant. To study the gating kinetics of voltage-dependent K(+) channel in rat dorsal root ganglion neurons, K(+) channel current were recorded using cell-attached patch-clamp technique. The K(+) channel characteristic of kinetics were found to be statistically self-similar at different time scales as predicted by the fractal model. The fractal dimension D for the closed times and for the open times depend on the pipette potential. For the open and closed times of kinetic setpoint, it was found dependent on the applied pipette potential, which indicated that the ion channel gating kinetics had nonlinear kinetic properties. Thus, the open and closed durations, which had the voltage dependence of the gating of this ion channel, were well described by the fractal model.     

Single-channel recordings from two types of amiloride-sensitive epithelial Na+ channels

We report here the first evidence in intact epithelial cells of unit conductance events from amiloride-sensitive Na+ channels. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). Two types of channels were observed: one with a high selectivity to Na+ and one with relatively low selectivity. The characteristics of the low-selectivity channel are as follows: single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette) and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed times as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches. The characteristics of the high-selectivity channel are: a single-channel conductance of 1-3 pS (mean = 2.8 +/- 1.2), the current-voltage relationship is markedly nonlinear with a Na+/K+ selectivity greater than 20:1. The mean open and closed times for the two types of channels are quite different, the high-selectivity channel being open only about 10% of the time while the low-selectivity channel is open about 30% of the time.     

Open channel block of NMDA receptor responses evoked by tricyclic antidepressants

The action of desipramine (DMI) and promazine on the response of mouse hippocampal neurons to the excitatory amino acid N-methyl-D-aspartic acid (NMDA) was investigated using whole-cell and single-channel recording. DMI at 20-50 microM was a potent, selective antagonist of responses to NMDA but not kainate or quisqualate. At -60 mV, the Kd for DMI block of responses to NMDA was 10 microM. The potency of DMI as an NMDA antagonist was highly voltage-dependent and behaved as though the Kd increased e-fold per 36 mV depolarization, reflecting an increase in the dissociation rate constant. Prior block of NMDA receptors with Mg2+ prevented binding of DMI, suggesting an action in the open channel. Single-channel analysis showed a decrease in the open time and burst length distributions, consistent with binding of DMI to open channels. We suggest that the action of DMI on NMDA receptor channels is similar to that of MK-801 and does not reflect binding to other domains, such as the regulatory sites for Zn2+ and glycine.     

Properties of Mg(2+)-dependent cation channels in human leukemia K562 cells

The endogenous Mg(2+)-inhibited cation (MIC) current was recently described in different cells of hematopoietic lineage and was implicated in the regulation of Mg2+ homeostasis. Here we present a single channel study of endogenously expressed Mg(2+)-dependent cation channels in the human myeloid leukemia K562 cells. Inwardly directed unitary currents were activated in cell-attached experiments in the absence of Ca2+ and Mg2+ in the pipette solution. The current-voltage (I-V) relationships displayed strong inward rectification and yielded a single channel slope conductance of approximately 30 pS at negative potentials. The I-V relationships were not altered by patch excision into divalent-free solution. Channel open probability (P(o)) and mean closed time constant (tau(C)) were strongly voltage-dependent, indicating that gating mechanisms may underlie current inward rectification. Millimolar concentrations of Ca2+ or Mg2+ applied to the cytoplasmic side of the membrane produced slow irreversible inhibition of channel activity. The Mg(2+)-dependent cation channels described in this study differ from the MIC channels described in human T-cells, Jurkat, and rat basophilic leukemia (RBL) cells in their I-V relationships, kinetic parameters and dependence on intracellular divalent cations. Our results suggested that endogenously expressed Mg(2+)-dependent cation channels in K562 cells and the MIC channels in other hematopoietic cells might be formed by different channel proteins.     

Cryptdin 3 forms anion selective channels in cytoplasmic membranes of human embryonic kidney cells

Cryptdins are antimicrobial peptides secreted by Paneth cells located at the base of intestinal crypts. In addition to their antimicrobial function, cryptdins may also regulate salt and water secretion by intestinal epithelial cells. Recent work with short-circuit current measurements indicated that at least one cryptdin peptide, cryptdin 3, induces apical conductance(s) in Cl(-) secretory, including cystic fibrosis, epithelia. In the present study, we characterized the cryptdin 3-induced anion channel activity in human embryonic kidney (HEK) cells with single-channel patch-clamp techniques. The patch pipette was filled with solution containing different concentrations of cryptdin 3, and, after gigaseal formation, the channel activity was recorded with either cell-attached or inside-out patch modes. We found an anion selective channel with a conductance of 15 pS and open probability of 0.19, regardless of cryptdin 3 concentration. The mean open and closed times varied with the cryptdin 3 concentration. For cryptdin 3 concentrations of 10, 4, 1, and 0.5 microg/ml in the pipette, the corresponding mean open times were 1.2, 7.0, 9.0, and 17.4 ms and the corresponding mean closed times were 1.1, 1.6, 4.2, and 12.5 ms. These results suggest that cryptdin 3 forms anion-selective channels on the cytoplasmic membrane of HEK cells and that the kinetics of one such channel are affected by its interaction with other such channels.     

Veratridine modifies open sodium channels

The state dependence of Na channel modification by the alkaloid neurotoxin veratridine was investigated with single-channel and whole-cell voltage-clamp recording in neuroblastoma cells. Several tests of whole-cell Na current behavior in the presence of veratridine supported the hypothesis that Na channels must be open in order to undergo modification by the neurotoxin. Modification was use dependent and required depolarizing pulses, the voltage dependence of production of modified channels was similar to that of normal current activation, and prepulses that caused inactivation of normal current had a parallel effect on the generation of modified current. This hypothesis was then examined directly at the single-channel level. Modified channel openings were easily distinguished from normal openings by their smaller current amplitude and longer burst times. The modification event was often seen as a sudden, dramatic reduction of current through an open Na channel and produced a somewhat flickery channel event having a mean lifetime of 1.6 s at an estimated absolute membrane potential of -45 mV (23 degrees C). The modified channel had a slope conductance of 4 pS, which was 20-25% the size of the slope conductance of normal channels with the 300 mM NaCl pipette solution used. Most modified channel openings were initiated by depolarizing pulses, began within the first 10 ms of the depolarizing step, and were closely associated with the prior opening of single normal Na channels, which supports the hypothesis that modification occurs from the normal open state.     

Concanavalin A modulates a potassium channel in cultured Aplysia neurons

A novel 100 pS K(+)-selective ion channel is frequently observed in cell-attached membrane patches from cultured Aplysia neurons. The activity of this channel is moderately voltage-dependent, but channel openings are rare and brief even when the patch is strongly depolarized. However, the activity of the channel is increased dramatically by the addition of the lectin concanavalin A (Con A), to the patch pipette. The channel is also activated by Con A in the bathing medium, suggesting that the lectin's action is via an as yet unidentified intracellular second messenger. In the one single-channel patch studied, Con A had no effect on the channel mean open time; rather it decreased the average duration of the long closed times between bursts of openings. Thus Con A increases either the open probability of single channels, the number of functional channels in the patch, or both. The functional significance of the Con A-induced modulation of K+ channel activity remains to be determined.     

Conserved structural and functional control of N-methyl-D-aspartate receptor gating by transmembrane domain M3

The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-d-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn(2+)). However, channels remain fully sensitive to Mg(2+) blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.