二氧化硫衍生物对大鼠背根神经元瞬间外向钾电流和延迟整流钾电流的影响

运用全细胞膜片钳技术研究二氧化硫衍生物对大鼠背根神经元瞬间外向钾电流(IA和ID)和延迟整流钾电流(IK)的影响。结果发现二氧化硫衍生物剂量依赖性地增大钾通道的电导,电压依赖性地增大钾电流的幅度,且这种增大作用部分可逆。二氧化硫非常显著地使延迟整流钾电流的激活过程向超极化方向移动,使瞬间外向钾电流的失活过程向去极化方向移动。10μmol/L二氧化硫衍生物作用前后,延迟整流钾电流的半数激活电压分别是(20.3±2.1)mV和(15.0±1.5)mV;IA和ID的半数失活电压分别朝去极化方向移动了6mV和7.4mV。这些结果表明二氧化硫改变了钾通道的特性,改变了神经元的兴奋性。     

高效氯氰菊酯对大鼠海马CA3区神经元电压门控钾通道的影响

利用全细胞膜片钳技术,在急性分离的新生大鼠海马CA3区锥体细胞上研究高效氯氰菊酯的两种组分高顺氯氰菊酯和高反氯氰菊酯对瞬时外向钾电流（transient outward potassiumcurrent,IA）和延迟整流钾电流（delayed rectifier potassiumcurrent,Ik）的影响。高顺氯氰菊酯使IA增大,而高反氯氰菊酯则使IA减小。高顺和高反氯氰菊酯均使IA激活曲线左移,反式结构还可促进IA的失活。高顺和高反氯氰菊酯均使IK减小,并使其激活曲线左移,而对IK的失活过程无影响,高反氯氰菊酯可使IK失活后恢复过程延长。结果表明,瞬时外向钾通道和延迟整流钾通道同样是高效氯氰菊酯的作用靶点,这可能是高效氯氰菊酯对哺乳动物产生毒性作用的原因之一。     

SO2代谢衍生物对大鼠海马CA1区神经元瞬间外向钾电流和延迟整流钾电流的影响

本文利用全细胞膜片钳技术研究了SO2 代谢衍生物———NaHSO3 和Na2 SO3 (二者分子比为 1∶3)对大鼠海马CA1区神经元瞬间外向钾电流 (IA)和延迟整流钾电流 (IK)的影响。结果表明 ,SO2 代谢衍生物可显著增大IA 和IK,且呈剂量依赖性关系 ,使IA 和IK 增大 5 0 %的剂量分别为 2 6 19μmol/L和 14 5 0 μmol/L。此外还与电压呈依赖性关系 ,但不具有频率依赖性。结果还表明 ,10 μmol/LSO2 代谢衍生物不影响IA 的激活过程 ,而对IK 的激活过程有非常显著的影响 ,给药前后IK 的半数激活电压分别为 17 6 4± 7 31mV和 13 43± 2 0 0mV (n=10 ,P <0 0 1) ,但不改变其斜率因子。另外 ,10 μmol/LSO2 代谢衍生物还非常显著地影响IA 的失活过程 ,给药前后其半数失活电压分别为 - 6 5 93± 1 97mV和 - 5 9 2 2± 3 83mV (n =10 ,P <0 0 1) ,但不改变其斜率因子。由此推断 ,SO2 代谢衍生物增大大鼠海马CA1区神经元的IA 和IK,促进IK 的激活过程 ,并抑制IA 的失活过程 ,可导致胞内K 通过K 通道的外流增加 ,胞内K 浓度降低 ,造成中枢神经元功能紊乱 ,诱导神经细胞凋亡。这意味着SO2 代谢衍生物对中枢神经系统具有损伤作用 ,从而提示大气SO2 污染可能与一些中枢神经系统疾病的发生以及衰老有关 [动物学报 49(1) :73     

焦亚硫酸钠对大鼠海马CA1区神经元钾电流的影响

目的:探讨焦亚硫酸钠(SMB)、二氧化硫(SO2)及其体内衍生物(亚硫酸盐和亚硫酸氢盐)对中枢神经元钾通道的影响及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及谷胱甘肽过氧化物酶(GPx)相应的保护作用.方法:采用全细胞膜片钳技术研究了SMB对大鼠海马CA1区神经元瞬间外向钾电流(IA)和延迟整流钾电流(IK)的影响.结果:①焦亚硫酸钠可增大全细胞IA和IK,且具剂量依赖性和电压依赖性,使IA和IK增大50%的剂量分别为15.8 μmol/L和11.5μmol/L;②10 μmol/L的SMB均可显著影响IA和IK的激活过程,给药前后IA的半数激活电压分别为(-12.6±1.6)mV和(-7.0±1.3)mV(n=8,P＜0.01),IK的半数激活电压分别为(10.8±0.9)mV和(21.6±0.7)mV(n=8,P＜0.01),但不改变其斜率因子;③10μmol/L的SMB还非常显著地影响IA的失活过程,给药前后其半数失活电压分别为(-97.0±1.1)mV和(-84.4±3.3)mV(n=8,P＜0.01),但也不改变其斜率因子;④抗氧化酶SOD(1×106U/L)、CAT(2×106U/L)及GPx(105U/L)均可使SMB(10μmol/L)增大的IA和IK部分恢复.结论:SMB可显著增大IA和IK,抑制IA和IK的激活过程及IA的失活过程,从而导致胞内K 的外流增加,使胞内K 浓度降低,从而对中枢神经元功能产生不利影响.     

白细胞介素-6对新生大鼠海马神经元电压依赖离子通道和NMDA电流的影响

目的: 研究白细胞介素-6对海马神经元电压依赖离子通道和NMDA电流的影响.方法: 应用全细胞膜片钳技术观察IL-6对电压依赖性钠通道电流(INa),延迟整流性钾通道电流(IK),电压依赖性钙通道电流(ICa),NMDA(N-methyl-D-aspartate)受体通道电流的影响.结果: 50 ng/ml IL-6作用24 h后IK 和ICa明显减小,Cm明显增大.50,500 ng/ml时减小NMDA电流.结论: IL-6通过作用于电压依赖钾通道,钙离子通道及NMDA通道影响神经元功能.     

二氧化硫衍生物对大鼠海马CA3区神经元瞬间外向钾电流的影响

为研究二氧化硫(SO2)衍生物——NaHSO3和Na2SO3(二者分子比为1：3)对大鼠海马CA3区神经元瞬间外向钾电流(IA)的影响,利用全细胞膜片钳技术,根据动力学和药理学特性分离鉴定大鼠海马CA3区神经元IA,观察SO2衍生物对IA的效应。发现SO2代谢衍生物可浓度依赖性地增大IA,使IA增大50％的剂量为25μmol/L。此外还与电压呈依赖关系,但不具有频率依赖性。10μmol/L的SO2代谢衍生物不影响IA电流的激活过程,但升高了A-通道稳态失活电压,延长了A-电流失活时间。说明SO2代谢衍生物可增大大鼠海马CA3区神经元IA电流,延长A-电流的失活时间,从而影响海马神经元的膜生理感应,这可能是SO2影响神经细胞功能的机理之一。     

白细胞介素-6对新生大鼠海马神经元电压依赖离子通道和NMDA电流的影响

目的 :研究白细胞介素 6对海马神经元电压依赖离子通道和NMDA电流的影响。方法 :应用全细胞膜片钳技术观察IL 6对电压依赖性钠通道电流 (INa) ,延迟整流性钾通道电流 (IK) ,电压依赖性钙通道电流 (ICa) ,NMDA(N methyl D aspartate)受体通道电流的影响。结果 :5 0ng/mlIL 6作用 2 4h后IK和ICa明显减小 ,Cm明显增大。 5 0 ,5 0 0ng/ml时减小NMDA电流。结论 :IL 6通过作用于电压依赖钾通道 ,钙离子通道及NMDA通道影响神经元功能。     

大鼠海马神经干细胞体外培养分化过程中钾电流的变化

目的:探讨新生大鼠海马神经干细胞体外培养分化后的神经元样细胞钾电流的变化.方法:神经干细胞体外扩增培养并传代后,撤除有丝分裂原并加血清诱导分化,应用全细胞电压钳技术检测分化后培养1 d、7 d、14 d、21 d细胞的电压依赖性钾电流.结果:分化后培养1 d的细胞,未检测出钾电流;分化后培养7 d、14 d、21 d的细胞,在 50 mV电压水平下的钾电流幅值分别为(18.077±2.789)pA/pF, (13.099±2.742)pA/pF, (34.045±8.067)pA/pF.该电流为两种电流的混合,分别能被TEA和4-AP所阻断,可能为缓慢失活的延迟整流钾电流(IK)和快速失活的瞬时外向钾电流(IA).结论:新生大鼠海马神经干细胞诱导分化后,随着体外培养时间的延长,钾离子通道的功能逐渐成熟.     

成年蜜蜂脑神经细胞的培养和电生理特征

为了研究杀虫剂等对蜜蜂毒性作用的神经机制,需在体外建立成年蜜蜂脑神经细胞的分离培养和电生理记录技术并研究其正常电生理特征,而对成年蜜蜂脑神经细胞的分离培养和电生理特性的研究报道甚少。我们采用酶解和机械吹打相结合的方法获得了数量较多且活力较好的成年意大利蜜蜂Apis mellifera脑神经细胞,并用全细胞膜片钳技术研究了成年意大利蜜蜂脑神经细胞对电流和电压刺激的反应,获得了成年意蜂脑神经细胞的基本电生理特征以及钠电流和钾电流的特性。全细胞电流钳的记录结果表明,在体外培养条件下,细胞无自发放电发生,注射电流后仅引起细胞单次放电,引起细胞放电的阈电流平均为60.8±63 pA; 细胞动作电位产生的阈电位平均为−27.4±2.3 mV。用全细胞电压钳记录了神经细胞的钠电流和钾电流。钠电流的分离是在电压刺激下通过阻断钾通道和钙通道实现。细胞的内向钠电流在指令电压为−40～−30 mV左右激活,−10 mV达峰值,钠通道的稳态失活电压V_1/2为−58.4 mV; 外向钾电流成份至少包括较小的快速失活钾电流和和较大的缓慢失活钾电流（占总钾电流的80%）,其半激活膜电位V_1/2为3.86 mV,无明显的稳态失活。结果提示缓慢失活钾电流的特征可能是细胞单次放电的机制之一。     

白介素1β时间依赖性地抑制大鼠皮层神经元钾电流

白细胞介素1β（Interleukin-1β,IL-1β）是一种重要的促炎细胞因子,在中枢神经系统中发挥着广泛的生物学功能。大量研究表明,IL-1β的作用非常复杂,在不同的模型和条件下作用不同,包括神经损伤或者神经毒性作用。电压门控钾通道调节神经元电学性质,也参与多种中枢神经系统的病理学过程。虽然IL-1β和钾通道都在脑损伤和脑疾病过程中发挥重要作用,但目前还很少有它们之间关系的研究报导。文章作者以原代培养的大鼠皮层神经元为材料,使用全细胞膜片钳技术,研究了10 ng/mL的IL-1β在不同处理时间下对皮层神经元电压依赖性钾电流的影响。根据电流的性质,可以将记录到的钾电流分为瞬时外向电流（IA）为主的IA样电流和延迟整流电流（IK）为主的IK样电流两部分,结果显示：IL-1β处理8 h对二者没有作用;处理24 h可使IA样和IK样电流的幅度降低20%左右。以上结果提示IL-1β对大鼠皮层神经元电压门控钾电流具有抑制作用,并且这种抑制可能具有时间依赖性。     

Study of a neuronal potassium current in different culture conditions

The potassium current of neurons in explants cultured from quail mesencephalic neural crest were studied in voltage clamp, using the whole cell recording technique. Two voltage-dependent potassium currents were identified; they differed in their sensitivity to blocking agents and to sustained depolarizing voltages. The potassium current component most sensitive to 4-aminopyridine had fast activation kinetics and inactivated quickly at sustained depolarized voltages. By analogy with a current described in other preparations, this current was called IA. The current component most sensitive to tetraethylammonium had slower activation kinetics and inactivated more slowly than IA at sustained depolarized voltages. This current was called IK. The properties of IA and IK were examined in neurons cultured in a defined medium and in neurons co-cultured with striated muscle. The rate of inactivation of IA appeared to be increased when neural crest neurons were cultured in the presence of striated muscle. The change in the properties of IA could be due to a direct effect of the co-culture with muscle on the membrane current; another possibility could be that co-culture favors the survival of a neuronal population that does not survive well when cultured in a defined medium.     

Potassium currents in presynaptic hair cells of Hermissenda.

Apart from their primary function as balance sensors, Hermissenda hair cells are presynaptic neurons involved in the Ca(2+)-dependent neuronal plasticity in postsynaptic B photoreceptors that accompanies classical conditioning. With a view to beginning to understand presynaptic mechanisms of plasticity in the vestibulo-visual system, a locus for conditioning-induced neuronal plasticity, outward currents that may govern the excitability of hair cells were recorded by means of a whole-cell patch-clamp technique. Three K+ currents were characterized: a 4-aminopyridine-sensitive transient outward K+ current (IA), a tetraethyl ammonium-sensitive delayed rectifier K+ current (IK,V), and a Ca(2+)-activated K+ current (IK,Ca). IA activates and decays rapidly; the steady-state activation and inactivation curves of the current reveal a window current close to the apparent resting voltage of the hair cells, suggesting that the current is partially activated at rest. By modulating firing frequency and perhaps damping membrane oscillations, IA may regulate synaptic release at baseline. In contrast, IK,V and IK,Ca have slow onset and exhibit little or no inactivation. These two K+ currents may determine the duration of the repolarization phase of hair-cell action potentials and hence synaptic release via Ca2+ influx through voltage-gated Ca2+ channels. In addition, IK,Ca may be responsible for the afterhyperpolarization of hair cell membrane voltage following prolonged stimulation.     

Cell cycle-dependent expression of potassium channels and cell proliferation in rat mesenchymal stem cells from bone marrow

OBJECTIVE: Recently, our team has demonstrated that voltage-gated delayed rectifier K(+) current (IK(DR)) and Ca(2+)-activated K(+) current (I(KCa)) are present in rat bone marrow-derived mesenchymal stem cells; however, little is known of their physiological roles. The present study was designed to investigate whether functional expression of IK(DR) and I(KCa) would change with cell cycle progression, and whether they could regulate proliferation in undifferentiated rat mesenchymal stem cells (MSCs). MATERIALS AND METHODS: Membrane potentials and ionic currents were recorded using whole-cell patch clamp technique, cell cycling was analysed by flow cytometry, cell proliferation was assayed with DNA incorporation method and the related genes were down-regulated by RNA interference (RNAi) and examined using RT-PCR. RESULTS: It was found that membrane potential hyperpolarized, and cell size increased during the cell cycle. In addition, IK(DR) decreased, while I(KCa) increased during progress from G(1) to S phase. RT-PCR revealed that the mRNA levels of Kv1.2 and Kv2.1 (likely responsible for IK(DR)) reduced, whereas the mRNA level of KCa3.1 (responsible for intermediate-conductance I(KCa)) increased with the cell cycle progression. Down-regulation of Kv1.2, Kv2.1 or KCa3.1 with the specific RNAi, targeted to corresponding gene inhibited proliferation of rat MSCs. CONCLUSION: These results demonstrate that membrane potential, IK(DR) and I(KCa) channels change with cell cycle progression and corresponding alteration of gene expression. IK(DR) and intermediate-conductance I(KCa) play an important role in maintaining membrane potential and they participate in modulation of proliferation in rat MSCs.     

大鼠发育过程中下丘脑神经元钾离子通道温度效应变化的研究

为了探讨出生后钾离子通道在下丘脑神经元热敏感分化过程中的作用,采用膜片钳技术研究出生一个月内SD大鼠急性分离神经元的温度效应,结果表明IK电流密度在出生后一个月内变化不大(P>0.05),而IA电流密度则呈现为升高趋势(P<0.05).同时升高温度,不同出生日期的钾通道NPo都有不同程度的升高,但相较P1d的神经元来说,温度对P18d的电压依赖性影响更大一些.同时温度对IK和IA的影响是不一样的,IA的Q10>2,所有这些显示IA通道在神经元温度敏感性的发育分化过程中起着重要的作用.     

Regulation of Kv7.2/Kv7.3 channels by cholesterol: Relevance of an optimum plasma membrane cholesterol content

Kv7.2/Kv7.3 channels are the molecular correlate of the M-current, which stabilizes the membrane potential and controls neuronal excitability. Previous studies have shown the relevance of plasma membrane lipids on both M-currents and Kv7.2/Kv7.3 channels. Here, we report the sensitive modulation of Kv7.2/Kv7.3 channels by membrane cholesterol level. Kv7.2/Kv7.3 channels transiently expressed in HEK-293 cells were significantly inhibited by decreasing the cholesterol level in the plasma membrane by three different pharmacological strategies: methyl-β-cyclodextrin (MβCD), Filipin III, and cholesterol oxidase treatment. Surprisingly, Kv7.2/Kv7.3 channels were also inhibited by membrane cholesterol loading with the MβCD/cholesterol complex. Depletion or enrichment of plasma membrane cholesterol differentially affected the biophysical parameters of the macroscopic Kv7.2/Kv7.3 currents. These results indicate a complex mechanism of Kv7.2/Kv7.3 channels modulation by membrane cholesterol. We propose that inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol depletion involves a loss of a direct cholesterol-channel interaction. However, the inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol enrichment could include an additional direct cholesterol-channel interaction, or changes in the physical properties of the plasma membrane. In summary, our results indicate that an optimum cholesterol level in the plasma membrane is required for the proper functioning of Kv7.2/Kv7.3 channels.     

hSK4/hIK1, a calmodulin-binding KCa channel in human T lymphocytes. Roles in proliferation and volume regulation.

Human T lymphocytes express a Ca2+-activated K+ current (IK), whose roles and regulation are poorly understood. We amplified hSK4 cDNA from human T lymphoblasts, and we showed that its biophysical and pharmacological properties when stably expressed in Chinese hamster ovary cells were essentially identical to the native IK current. In activated lymphoblasts, hSK4 mRNA increased 14.6-fold (Kv1.3 mRNA increased 1.3-fold), with functional consequences. Proliferation was inhibited when Kv1.3 and IK were blocked in naive T cells, but IK block alone inhibited re-stimulated lymphoblasts. IK and Kv1.3 were involved in volume regulation, but IK was more important, particularly in lymphoblasts. hSK4 lacks known Ca2+-binding sites; however, we mapped a Ca2+-dependent calmodulin (CaM)-binding site to the proximal C terminus (Ct1) of hSK4. Full-length hSK4 produced a highly negative membrane potential (Vm) in Chinese hamster ovary cells, whereas the channels did not function when either Ct1 or the distal C terminus was deleted (Vm approximately 0 mV). Native IK (but not expressed hSK4) current was inhibited by CaM and CaM kinase antagonists at physiological Vm values, suggesting modulation by an accessory molecule in native cells. Our results provide evidence for increased roles for IK/hSK4 in activated T cell functions; thus hSK4 may be a promising therapeutic target for disorders involving the secondary immune response.     

Cholesterol influences voltage-gated calcium channels and BK-type potassium channels in auditory hair cells

The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitability are unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassium current by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (~30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.     

Ionic conductances in two types of sensory neurons in the leech, Macrobdella decora

1. Ion conductances were investigated in two kinds of leech sensory neurons (PM and N1 cells) which differ in their membrane excitability and action potential. 2. In the PM cell body excitable membrane behavior is dominated by only two currents, a sodium current (INa) and a delayed rectifier (IK). 3. In contrast, in the N1 cell INa and IK is supplemented with the presence of a transient potassium current, IA. 4. A comparison between INa and IK in the two cell types did not reveal any significant difference in activation and inactivation kinetics of either current between neurons. 5. Thus, the properties and presence of the A-current in the N1 cell and not in the PM cell may account for the functional difference in excitability of the two kinds of neurons.