短暂脑缺血大鼠海马CA1区锥体细胞大电导Ca2+依赖K+通道活动降低

短暂脑缺血可对随后的损伤性脑缺血表现出明显的耐受.有研究表明大电导Ca2+依赖K+(BKCa)通道活动增强参与了缺血性脑损伤.采用膜片钳的内面向外式,观察了3 min短暂脑缺血后6 h、24 h以及48 h大鼠海马CA1区锥体细胞上BKCa通道活动的动态变化.短暂脑缺血后BKCa通道的单通道电导和翻转电位均未见明显变化,但通道的开放概率则在缺血预处理后的前24 h内显著降低.通道动力学分析显示通道关闭时间变长是短暂脑缺血后通道活动降低的主要原因,因为通道的开放时间未发生明显变化.结果提示短暂脑缺血所致的BKCa通道活动降低可能与缺血耐受的产生有关.     

Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia

The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca(2+)](cyt)) is essential for pollen-tube growth. Local Ca(2+) influx mediated by Ca(2+)-permeable channels plays a key role in maintaining this [Ca(2+)](cyt) gradient. Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba(2+)) approximately Ca(2+) > magnesium (Mg(2+)) > strontium (Sr(2+)) > manganese (Mn(2+)). This channel conductance was selective for Ca(2+) over chlorine (Cl(-)) (relative permeability P(Ca)/P(Cl) = 14 in 10 mm extracellular Ca(2+)). We also showed that the channel was inhibited by the Ca(2+) channel blockers lanthanum (La(3+)) and gadolinium (Gd(3+)). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca(2+)-permeable channel is likely to play a role in mediating Ca(2+) influx into the growing pollen tubes to maintain the [Ca(2+)](cyt) gradient.     

小脑皮质神经元K^＋单通道电流的生理特性

用膜片钳技术的细胞贴附式和内面向外式,在分离培养的新生大鼠小脑皮质颗粒细胞膜上记录到钾离子通道电流,有以下的生理特性,最常见的通道电导为２５ｐＳ,单通道电流幅度、开放时间、开放概率均受膜电位控制,通道开放时间分布直方图可用双指数拟合,无时间依赖性失活,不依赖钙离子,能被ＴＥＡ阻断,表明此通道可能为延迟整流型Ｋ＋通道。提示,小脑皮质颗粒细胞存在有不失活的２５ｐＳ钾离子通道,不同于文献报道的,可能是一种新亚型的钾离子通道。     

Heterotrimeric G-protein participation in Arabidopsis pollen germination through modulation of a plasmamembrane hyperpolarization-activated Ca2+-permeable channel

The role of heterotrimeric G proteins in pollen germination and tube growth was investigated using Arabidopsis thaliana plants in which the gene (GPA) encoding the G-protein a subunit (Galpha) was null or overexpressed. Pollen germination, free cytosolic calcium concentration ([Ca(2+)](cyt)) and Ca(2+) channel activity in the plasma membrane (PM) of pollen cells were investigated. Results showed that, compared with pollen grains of the wild type (ecotype Wassilewskija, ws), in vitro germinated pollen of Galpha null mutants (gpa1-1 and gpa1-2) had lower germination percentages and shorter pollen tubes, while pollen from Galpha overexpression lines (wGalpha and cGalpha) had higher germination percentages and longer pollen tubes. Compared with ws pollen cells, [Ca(2+)](cyt) was lower in gpa1-1 and gpa1-2 and higher in wGalpha and cGalpha. In whole-cell patch clamp recordings, a hyperpolarization-activated Ca(2+)-permeable conductance was identified in the PM of pollen protoplasts. The conductance was suppressed by trivalent cations but insensitive to organic blockers; its permeability to divalent cations was Ba(2+) > Ca(2+) > Mg(2+) > Sr(2+) > Mn(2+). The activity of the Ca(2+)-permeable channel conductance was down-regulated in pollen protoplasts of gpa1-1 and gpa1-2, and up-regulated in wGalpha and cGalpha. The results suggest that Galpha may participate in pollen germination through modulation of the hyperpolarization-activated Ca(2+) channel in the PM of pollen cells.     

成年大鼠海马CA1区锥体细胞K_(ATP)通道的特性

为了解成年大鼠海马CA1区锥体细胞KATP 通道的特性 ,实验采用膜片钳技术的内面向外式记录法 ,在急性分离的CA1区锥体神经元上 ,研究了可被胞浆侧ATP所抑制的钾离子单通道的特性。当细胞膜内外两侧的K 浓度均为 14 0mmol/L时 ,通道的电导为 63pS ,翻转电位为 1 71mV ,通道呈弱内向整流性。在负钳制电位时 ,通道开放时常被短时程的关闭所打断 ,而在正钳制电位时 ,这种短时程的关闭状态明显少于负钳制电位时。但通道开放概率未见明显的电压依赖性。ATP对通道活动的抑制作用呈浓度依赖性 ,抑制通道活动 5 0 %的ATP浓度为 0 1mmol/L。KATP 通道的特异性阻断剂tolbutamide (甲糖宁 ,1mmol/L)可完全阻断通道的活动 ,而KATP 通道开放剂diazoxide (二氮嗪 ,1mmol/L)则不增强通道的活动。     

Patch-clamp studies in human macrophages: Single-channel and whole-cell characterization of two K+ conductances

Summary Human peripheral blood monocytes cultured for varying periods of time were studied using whole-cell and single-channel patch-clamp recording techniques. Whole-cell recordings revealed both an outward K current activating at potentials >20 mV and an inwardly rectifying K current present at potentials negative to –60 mV. Tail currents elicited by voltage steps that activated outward current reversed nearE _K, indicating that the outward current was due to a K conductance. TheI–V curve for the macroscopic outward current was similar to the mean single-channelI–V curve for the large conductance (240 pS in symmetrical K) calcium-activated K channel present in these cells. TEA and charybdotoxin blocked the whole-cell outward current and the single-channel current. Excised and cell-attached single-channel data showed that calcium-activated K channels were absent in freshly isolated monocytes but were present in >85% of patches from macrophages cultured for >7 days. Only 35% of the human macrophages cultured for >7 days exhibited whole-cell inward currents. The inward current was blocked by external barium and increased when [K] _o increased. Inward-rectifying single-channel currents with a conductance of 28 pS were present in cells exhibiting inward whole-cell currents. These single-channel currents are similar to those described in detail in J774.1 cells (L.C. McKinney & E.K. Gallin,J. Membrane Biol. 103:41–53, 1988).     

K+ Channels in the Plasma Membrane of Lily Pollen Protoplasts

Using the patch-clamp technique K⁺ channels could be observed in the plasma membrane of protoplasts from pollen grains of Lilium longiflorum. With depolarizing membrane potentials the open probability of the different K⁺ channels increased. Two K⁺ channel populations occurring occasionally had a single channel conductance of 120 pS and 42 pS, respectively. The most often observed K⁺ channel had a single channel conductance of 19 pS which showed an increase of channel activity with increasing free cytoplasmic Ca²⁺ concentration. This channel population might be involved in the pathway of endogenous transcellular K⁺ currents which are activated during pollen tube tip extension.     

Mechanism of asymmetric block of K channels by tetraalkylammonium ions in mouse neuroblastoma cells

Experiments were performed to compare the mechanism of block of voltage-dependent K channels by various short and long alkyl chain tetraalkylammonium (TAA) ions at internal and external sites. Current through single channels was recorded from excised membrane patches of cultured neuroblastoma cells using the patch-clamp technique. All of the TAA derivatives tested blocked the open channel when applied to either side of the membrane. Tetraethylammonium (TEA) reduced the amplitude of current through the open channel. Tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) reduced the open time as a function of the concentration. An additional nonconducting state was observed when TBA or TPeA was applied internally or externally, due to the presence of a drug-bound and blocked state of the channel. The closing rate under control conditions was similar to that in the presence of external tetramethylammonium (TMA), suggesting that channel closing is independent of external drug binding. The concentration for half maximal block of the channel by external TEA was 80 microM. The channel was less sensitive to internal TEA, which half blocked the channel at 27 mM. The dissociation rate of long alkyl chain TAA ions from the channel was slower when applied to the inside, compared to external application, suggesting the presence of distinct internal and external receptors. Long alkyl chain TAA derivatives, such as TBA had a faster association rate with the open channel when applied to the inside of the membrane than when applied to the outside.     

Unique inner pore properties of BK channels revealed by quaternary ammonium block

Potassium channels have a very wide distribution of single-channel conductance, with BK type Ca(2+)-activated K(+) channels having by far the largest. Even though crystallographic views of K(+) channel pores have become available, the structural basis underlying BK channels' large conductance has not been completely understood. In this study we use intracellularly applied quaternary ammonium compounds to probe the pore of BK channels. We show that molecules as large as decyltriethylammonium (C(10)) and tetrabutylammonium (TBA) have much faster block and unblock rates in BK channels when compared with any other tested K(+) channel types. Additionally, our results suggest that at repolarization large QA molecules may be trapped inside blocked BK channels without slowing the overall process of deactivation. Based on these findings we propose that BK channels may differ from other K(+) channels in its geometrical design at the inner mouth, with an enlarged cavity and inner pore providing less spatially restricted access to the cytoplasmic solution. These features could potentially contribute to the large conductance of BK channels.     

Coupling of activation and inactivation gate in a K+‐channel: potassium and ligand sensitivity

Potassium (K⁺)‐channel gating is choreographed by a complex interplay between external stimuli, K⁺ concentration and lipidic environment. We combined solid‐state NMR and electrophysiological experiments on a chimeric KcsA–Kv1.3 channel to delineate K⁺, pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH‐induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K⁺ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K⁺ ion on the inactivation gate modulate activation‐gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K⁺‐channel pore.     

下丘脑神经元钙激活钾通道的整流现象

目的研究SD乳鼠下丘脑神经元中钙激活钾通道的整流现象.方法采用膜片钳内面向外式记录方式.结果记录到一种大电导钙激活钾通道(KCa),在对称140mmol/L[K+]时内向电导为(171±12)pS,不随[Ca2+]变化而改变,而外向电导可受[Ca2+]调控,当[Ca2+]为500μmol/L时,外向电导为(76±14)pS.[Ca2+]越大,整流现象越明显,Mg2+对这种KCa的整流作用不明显.结论下丘脑神经元中KCa具有Ca2+依赖性整流现象,它可能与神经元的兴奋性和稳定性有关.     

Characterization of whole-cell k<Superscript>+</Superscript> currents across the plasma membrane of pollen grain and tube protoplasts of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>

Outward and inward currents, mainly carried by K⁺, were detected in protoplasts of pollen grains (PG) and pollen tubes (PT) of Lilium longiflorum Thunb. by using the whole-cell configuration of the patch-clamp technique. The outward K⁺ current (I_{K+ out}) was similar in both protoplast types, while the inward K⁺ current (I_{K+ in}) was higher in pollen tube protoplasts. In PT but not in PG protoplasts, inward K⁺ currents were already detectable at negative membrane voltages usually monitored in lily pollen. I_{K+ in} consisted of a slow and a fast current component, as revealed by fitting a sum of two exponential functions to the time-dependent current. The contribution of the fast component to the total inward current was higher in PT than in PG protoplasts, which was even more evident at acidic pH of the external medium. Therefore, based on the measured characteristics, the I_{K+ in} of PT protoplasts may contribute to the endogenous K⁺ currents surrounding a growing pollen tube. Abbreviations: BS, bath solution; BTP, bis-Tris-propane; MES, 2-N-morpholinoethane sulfonic acid; V_act, activation voltage; V_M, membrane voltage; E_rev, reversal potential; I_{K+ in}, inward K⁺ current; I_{K+ out}, outward K⁺ current; PG, pollen grain; PT, pollen tube; PM, pipette medium